Microvascular Research biology and pathology - part 7 pdf

The increase in the sensitivity of arteri-oles to vasoconstrictor stimuli in hypertension may have anumber of underlying causes, including intrinsic alterations in the electrophysiologic

C HAPTER 103

Microvascular Responses to

Hypercholesterolemia

Karen Y Stokes and D Neil Granger

Louisiana State University Health Sciences Center, Shreveport, Louisiana

(ADMA), the levels of which are increased during cholesterolemia The elevated ADMA levels likely resultfrom the diminished activity of dimethylarginine dimet-hylaminohydrolase (DDAH), which normally degradesADMA, that accompanies hypercholesterolemia Further-more, the NOS enzyme cofactor tetrahydrobiopterin (BH4)

hyper-is also reduced during hypercholesterolemia, which would further reduce the bioavailability of NO The neteffect of these changes in both animal and human subjectsduring hypercholesterolemia is a diminished capacity forarteriolar endothelium to produce NO and to mediateendothelium-dependent vasorelaxation It is important tonote that these vessels are capable of responding to directstimulation of the smooth muscle cells by NO donors, sup-porting the concept that there is a loss of NO bioavailabilityrather than a deterioration of smooth muscle cell responses

to NO However, the response of denuded coronary oles to vasoconstrictors is reduced in hypercholesterolemichumans, compared to their normocholesterolemic counter-parts, suggesting that smooth muscle–dependent alterations

arteri-do occur in this condition, albeit via an NO-independentmechanism

Concomitant with these changes in NO production is therelease of reactive oxygen species (ROS) Although severalROS-producing enzymes have been implicated in theendothelial dysfunction induced in large arteries by hyper-cholesterolemia, the source of excess ROS generation inarterioles remains poorly understood We have recentlydemonstrated that, as in large arteries exposed to hypercho-lesterolemia, enhanced superoxide (O2 -) production alsocontributes to the impaired relaxation responses observed inarterioles during acute hypercholesterolemia Furthermore,using gp91phox-deficient mice, we demonstrated thatNAD(P)H oxidase is a major source of the O2 -that mediates

Introduction

Hypercholesterolemia is an established risk factor for the

development of cardiovascular diseases The atherosclerotic

lesions that result from a sustained elevation in blood

cho-lesterol concentration are associated with an accumulation

of inflammatory cells and platelets that facilitate the

deposi-tion of lipids in the walls of lesion-prone arteries However,

long before these changes occur in large arteries,

inflamma-tory and prothrombogenic responses are observed in

arteri-oles and venules throughout the vascular system (Figure 1)

These responses are manifested as endothelial dysfunction

and the binding of leukocytes and platelets to the vessel

wall Although several mechanisms have been proposed to

explain the phenotypic changes that occur in the

microvas-culature during hypercholesterolemia, oxidative stress and a

diminished bioavailability of nitric oxide (NO) have gained

much attention in recent years This chapter describes the

responses of the microcirculation to hypercholesterolemia

and addresses the mechanism that underlies this systemic

inflammatory condition

Hypercholesterolemia and Arterioles

Under normal physiological conditions, basal NO

pro-duction by endothelial cells maintains vascular tone and

inhibits inflammation However, during

hypercholes-terolemia, several events occur that negatively influence the

vasodilatory role of NO in arterioles Although the

concen-tration of L-arginine, the substrate for NO synthase (NOS),

is not reduced during hypercholesterolemia, the interaction

between L-arginine and endothelial NOS may be blocked

by the endogenous inhibitor asymmetric dimethylarginine

Copyright © 2006, Elsevier Science (USA).

the impaired endothelium-dependent vasodilation exhibited

by arterioles during hypercholesterolemia

Unlike in venules and atherosclerosis-prone large

arter-ies, there is little evidence for the accumulation of adherent

leukocytes or platelets in arterioles in animal models of

diet-induced hypercholesterolemia However, there is some

evi-dence that oxidized low-density lipoprotein (oxLDL), which

is elevated in blood of hypercholesterolemic humans, can

induce the rolling and firm adhesion of leukocytes in

arteri-oles The leukocyte rolling response is mediated by

P-selectin, while the firm adhesion of leukocytes is supported

by b2-integrins

Hypercholesterolemia and Capillaries

In response to the changes in arteriolar tone that

accom-pany hypercholesterolemia, red blood cell velocity is

reduced This leads to erythrocyte aggregation and stasis in

smaller microvessels Humans with elevated blood terol levels are said to exhibit reduced capillary perfusion,which likely reflects a diminished red blood cell velocity incapillaries Recent work suggests that NO-dependent path-ways contribute to the impaired capillary perfusion duringhypercholesterolemia There is evidence that leukocyteaccumulation in downstream venules may also contribute

choles-to the impaired capillary perfusion during terolemia, possibly through the release of inflammatorymediators

hypercholes-Administration of oxLDL to otherwise normal animalspromotes the degradation of the endothelial glycocalyx incapillaries Platelets adhere to the endothelial cells of thesedamaged capillaries The glycocalyx breakdown and result-ant platelet adhesion can be inhibited by superoxide dis-mutase (SOD) and catalase It remains unclear whetherdiet-induced hypercholesterolemia induces a similar injuryresponse in capillaries However, it has been shown thathypercholesterolemia exacerbates the capillary leak that

Baseline

Hypercholesterolemia

IL-12 IFN-g

NAD(P)H ox

O2

-NAD(P)H ox

-Monocyte Platelet

Neutrophil

Lymphocyte Endothelial

Cells

AA PAF LTB 4

O2

-L-arg

eNOS

NO

Smooth Muscle Cells

Figure 1 Inflammatory alterations in arterioles (left) and venules (right) elicited by hypercholesterolemia Under baseline conditions (top portion of

ves-sels), the basal release of nitric oxide (NO) maintains arteriolar smooth muscle tone and prevents cell–cell interactions in venules Antioxidants such as lase and CuZn-SOD minimize the levels of proinflammatory oxidants During hypercholesterolemia (lower portions of vessels), NO bioavailability is reduced and oxidant production is enhanced This promotes smooth muscle contraction (constriction) in arterioles In the venular segment of the microcir- culation, a proinflammatory and prothrombogenic phenotype is assumed Adhesion molecule expression is increased resulting in the recruitment of platelets and leukocytes The adhesion response is induced by lipid mediators and cytokines L-arg, L -Arginine; eNOS, endothelial nitric oxide synthase; O2- , super- oxide; cat, catalase; H2O2, hydrogen peroxide; SOD, superoxide dismutase; DDAH, dimethylarginine dimethylaminohydrolase; ADMA, asymmetric dimethylarginine; ICAM-1, intercellular adhesion molecule-1; IL-12, interleukin-12; IFN- g, interferon-g; AA, arachidonic acid; PAF, platelet-activating factor; LTB4, leukotriene B4; NAD(P)H ox, NAD(P)H oxidase.

cata-C HAPTER 103 Microvascular Responses to Hypercholesterolemia 699

occurs in response to acute inflammatory stimuli, such as

ischemia–reperfusion (I/R) This likely reflects impaired

endothelial junction integrity and occurs in a

neutrophil-dependent manner, suggesting that leukocytes adherent

within venules may release inflammatory mediators such as

ROS that worsen the response to other stimuli

Hypercholesterolemia and Postcapillary Venules

Although large veins appear to be relatively unaffected

by acute or chronic elevations in blood cholesterol

concen-tration, postcapillary venules in the diameter range of 20 to

40mm exhibit profound changes in response in these

condi-tions Some of the alterations in signaling and inflammatory

pathways induced in arterioles by hypercholesterolemia are

also manifested in venules For example, the reduced NO

bioavailability and elevated ROS production are seen in

both segments of the microvasculature However, the

conse-quences of these changes vary between the vascular

seg-ments, with NO and ROS exerting minimal influence on the

diameter of venules while exerting a profound effect on

arte-rioles In venules, NO exerts a major influence on the

expression of cellular adhesion molecules and consequently

serves to minimize the adhesive interactions between

circu-lating blood cells and venular endothelium However,

dur-ing hypercholesterolemia the expression of several adhesion

molecules is increased on venular endothelium These

adhesion molecules support the leukocyte infiltration and

platelet–endothelial interactions that occur in postcapillary

venules when blood cholesterol concentration is elevated

Blood Cell–Endothelial Cell Interactions

The use of blocking antibodies and mutant mice has

revealed key roles for several adhesion molecules in the

pathogenesis of atherosclerosis The adhesion molecules

that contribute to the binding of leukocytes and platelets in

postcapillary venules during hypercholesterolemia have

been less well defined oxLDL also causes degradation of

the endothelial glycocalyx in venules It is noteworthy that

this leads to loss of heparin sulfate proteoglycans, which

would normally contribute to the negative charge and

anti-adhesive properties of the normal endothelial cell Hence,

the increased adhesion molecule expression (and possibly

the oxidatively modified surface of the endothelial cell)

causes the endothelial cells to assume a phenotype that

supports the adhesion of leukocytes and platelets during

hypercholesterolemia

Both intercellular adhesion molecule-1 (ICAM-1) and

P-selectin are upregulated on venular endothelium when

mice are placed on a cholesterol-enriched diet for 1 week

This protein expression coincides with the recruitment of

leukocytes In the early stages of leukocyte recruitment in

hypercholesterolemic venules, neutrophils appear to

repre-sent the major cell population that interacts with the vessel

wall Both CD4+ and CD8+ T-lymphocytes participate in

this response, but in an indirect manner, by producingcytokines that upregulate endothelial cell adhesion molecules

Platelets are also recruited into postcapillary venules ing acute hypercholesterolemia It has been demonstratedusing knockout mice that the platelets interact with thevenular wall via P-selectin that is expressed on the surface

dur-of circulating platelets, although P-selectin on venularendothelium also contributes but to a lesser extent The latter may participate by binding P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes, which in turn may serve

as a platform for platelet binding to the venular wall oxLDLpromotes the formation of leukocyte–platelet aggregatesthat can interact with the venular wall The formation ofthese aggregates can be inhibited using a P-selectin blockingantibody suggesting that platelet P-selectin is interactingwith PSGL-1 on the leukocytes

Nitric Oxide and Reactive Oxygen SpeciesAlthough there are very few mechanistic data available

on platelet accumulation in postcapillary venules, a largebody of evidence supports a role for an NO-ROS imbalance

in the hypercholesterolemia-induced leukocyte–endothelialcell interactions First, NO inhibitors fail to exacerbateleukocyte adhesion responses in postcapillary venules ofhypercholesterolemic mice, unlike the greatly enhancedresponses observed in normal mice This suggests that basal

NO release is impaired, possibly because of the augmentedcirculating levels of ADMA mentioned earlier Exposure ofnormal postcapillary venules to an analog of ADMA (at lev-els comparable to circulating levels during hypercholes-terolemia) elicits leukocyte adhesion in venules and impairsendothelial barrier function, supporting the proposal that theelevated ADMA levels during hypercholesterolemia areindeed proinflammatory Second, many NO donors (e.g.,sodium nitroprusside, spermine-NO, and L-arginine) havebeen successfully employed to reduce the inflammatoryresponses (adhesion molecule expression and leukocyteaccumulation) observed both in diet-induced hypercholes-terolemia and following exposure to oxLDL, supporting arole for decreased NO bioavailability in this leukocyterecruitment process

The importance of ROS in the venular responses tohypercholesterolemia is underscored by the observation that oxidative stress, measured using an oxidant-sensitivefluorescent probe, is elevated in postcapillary venules ofhypercholesterolemic mice when compared with their normocholesterolemic counterparts This coincides withincreases in leukocyte adhesion and emigration in venules.Furthermore, the leukocyte recruitment is profoundly atten-uated in CuZn–SOD-overexpressing mice, suggesting that

O2- is a major component of the ROS generated duringhypercholesterolemia Similarly, administration of CuZn-SOD has been shown to be effective in preventing oxLDL-induced venular responses The enzymes that contribute

to the increased ROS generation in hypercholesterolemic

venules have not been clearly defined However, mice that

are genetically deficient in the p47phoxsubunit of NAD(P)H

oxidase demonstrate a significantly lower level of leukocyte

recruitment in response to hypercholesterolemia

Interest-ingly, when bone marrow chimeras were made to separate

blood cell versus vessel wall sources of this enzyme, both

sources appeared to be equally important in the generation

of the inflammatory phenotype observed in postcapillary

venules after 2 weeks on a cholesterol-enriched diet

Other Inflammatory Mediators

Several mediators have been implicated in inflammatory

responses of venules to oxLDL- or diet-induced

hypercho-lesterolemia For example, arachidonic acid metabolism

appears to be important in oxLDL-induced leukocyte

recruitment Blocking leukotriene biosynthesis can prevent

the leukocyte adhesion elicited by oxLDL in both arterioles

and venules Platelet-activating factor (PAF) receptor

antag-onists are equally effective in attenuating the inflammatory

responses to oxLDL Although the contribution of these

lipid mediators to diet-induced microvascular alterations has

not been assessed, a role for these factors in diet-induced

atherosclerotic lesion formation is well established,

support-ing the possibility that they may also contribute to the early

inflammatory responses elicited in venules

The T-cell-derived cytokine interferon-g (IFN-g) has also

been implicated in vascular responses to

hypercholes-terolemia IFN-g can promote adhesion molecule

expres-sion, and it is a potent activator of NAD(P)H oxidase The

microvasculature of IFN-g-knockout mice exhibits reduced

leukocyte adhesion and significantly lower oxidant stress in

response to hypercholesterolemia, when compared with

wild-type mice This suggests that T-lymphocytes may be

mediating the inflammatory responses to

hypercholes-terolemia via IFN-g, and that this cytokine acts by

promot-ing ROS generation Another step in this inflammatory

pathway may be the release of IL-12, a cytokine that is

inti-mately linked to IFN-g production Like IFN-g, IL-12 is

expressed in atherosclerotic lesions and it has recently been

shown to contribute to the oxidative stress and leukocyte

recruitment induced in postcapillary venules by

hypercho-lesterolemia These observations suggest that IFN-g and

IL-12 act in concert to promote leukocyte adhesion and

emi-gration by enhancing the production of ROS

Exaggerated Inflammatory Responses

during Hypercholesterolemia

There is a growing body of evidence that

hypercholes-terolemia renders microvascular endothelium more

suscep-tible to the deleterious consequences of inflammatory

stimuli such as I/R I/R per se is known to elicit an oxidative

stress and promote leukocyte adhesion in postcapillary

venules Both of these responses are exacerbated during

hypercholesterolemia and can be blocked by pretreatmentwith either SOD or a xanthine oxidase inhibitor (allopuri-nol) This suggests that O2- generated from xanthine oxidase mediates the leukocyte accumulation elicited byhypercholesterolemic tissues exposed to I/R Hypercholes-terolemia also enhances the P-selectin upregulation that

is normally elicited by I/R In addition, the terolemia-induced exacerbation of inflammation is seenwhen tissues are challenged with either lipid mediators(leukotriene B4and PAF) or cytokines such as TNF-a

hypercholes-It has also been shown that hypercholesterolemia bates the protein extravasation in venules induced by vari-ous inflammatory stimuli, and that this may be due to theenhanced leukocyte recruitment However, administration

exacer-of oxLDL in the local arterial supply exacer-of tissues exposed toI/R promotes leukocyte adhesion and emigration, without anaccompanying increase in albumin extravasation AlthoughoxLDL is able to induce most of the microvascular alter-ations observed during diet-induced hypercholesterolemia,the underlying mechanisms appear to differ between thesetwo forms of microvascular dysfunction

Relevance of Hypercholesterolemia-Induced Microvascular Responses to Atherosclerosis

Many of the inflammatory responses and pathways thatare initiated in the microvasculature by hypercholes-terolemia have also been implicated in the development ofatherosclerotic plaques Whether the early inflammatoryresponses seen in venules influence the development oflesions in large vessels remains unclear Since the inflam-matory responses to hypercholesterolemia appear to beexperienced by all tissues in the body, it appears tenable thatthe large endothelial surface area (> 500 m2) within themicrovasculature may serve as a motor that drives the sys-temic immune response, ultimately leading to lesion devel-opment in large arteries It is clear, however, that this riskfactor, while rendering tissue more likely to experience anischemic episode through the development of atherosclero-sis, also predisposes organs to greater microvascular dys-function and more tissue injury following a given ischemicinsult Hence, an improved understanding of the mecha-nisms that underlie the inflammatory phenotype that isassumed by the microvasculature during hypercholes-terolemia may reduce the morbidity and mortality associ-ated with cardiovascular diseases

Glossary

Adhesion molecules: Molecules expressed on the endothelial cells,

leukocytes, and platelets, which bind their ligands on other cells, thereby mediating the interactions between the circulating cells and the vessel wall.

Blood cell recruitment: The adhesion of blood cells (leukocytes and

platelets) to the vascular endothelium at sites of inflammation.

I/R: Ischemia/reperfusion, or the cessation and restoration of blood

flow to an organ or tissue.

C HAPTER 103 Microvascular Responses to Hypercholesterolemia 701

Oxidant stress: This usually occurs as a result of an imbalance

between nitric oxide and oxidant-generating systems, resulting in an

over-all increase in the oxidative capacity of the tissue.

OxLDL: Low-density lipoprotein (normally responsible for carrying

cholesterol to tissues) that is oxidatively modified, thereby attaining

Granger, D N (2003) Risk factors for cardiovascular disease amplify

reperfusion-induced inflammation and microvascular dysfunction In

Molecular Basis for Microcirculatory Disorders, G W

Schmidt-Schönbein and D N Granger, eds., pp 333–342 Paris:

Springer-Ver-lag France.

Hansson, G K (2001) Immune mechanisms in atherosclerosis

Arte-rioscler Thromb Vasc Biol 21, 1876–1890.

Landmesser, U., Hornig, B., and Drexler, H (2000) Endothelial

dysfunc-tion in hypercholesterolemia: mechanisms, pathophysiological

impor-tance, and therapeutic interventions Semin Thromb Hemost 26,

529–537.

Laroia, S T., Ganti, A K., Laroia, A T., and Tendulkar, K K (2003).

Endothelium and the lipid metabolism: The current understanding Int.

J Cardiol 88, 1–9.

Napoli, C., and Lerman, L O (2001) Involvement of oxidation-sensitive

mechanisms in the cardiovascular effects of hypercholesterolemia.

Mayo Clin Proc 76, 619–631 This article focuses on the pathways

involved in the oxidant-mediated events that are associated with

hyper-cholesterolemia and examines potential therapeutic strategies in this context.

Ross, R (1999) Atherosclerosis—an inflammatory disease N Engl

J Med 340, 115–126 This is a comprehensive review of the

mecha-nisms involved in the development of atherosclerosis, many of which are now being addressed in the microvascular responses to hypercholesterolemia.

Scalia, R., Appel 3rd , J Z and Lefer, A M (1998) lium interaction during the early stages of hypercholesterolemia in the

Leukocyte–endothe-rabbit: Role of P-selectin, ICAM-1, and VCAM-1 Arterioscler.

Thromb Vasc Biol 18, 1093–1100.

Stokes, K Y., Clanton, E C., Clements, K P., and Granger, D N (2003) Role of interferon-gamma in hypercholesterolemia-induced leukocyte-

endothelial cell adhesion Circulation 107, 2140–2145.

Stokes, K Y., Cooper, D., Tailor, A., and Granger, D N (2002) cholesterolemia promotes inflammation and microvascular dysfunc-

Hyper-tion: Role of nitric oxide and superoxide Free Radic Biol Med 33,

1026–1036 This provides a more in-depth review of the topic discussed

here, with particular reference to the role of oxidative stress in the responses to hypercholesterolemia.

Capsule Biography

Karen Stokes earned her Ph.D in physiology from Trinity College, Dublin She is currently an instructor in the Department of Molecular and Cellular Physiology at Louisiana State University Health Sciences Center

in Shreveport Her research interests include the microvascular responses to ischemia–reperfusion and to hypercholesterolemia.

D Neil Granger, Ph.D., is Boyd Professor and Head of the Department

of Molecular and Cellular Physiology at Louisiana State University Health Sciences Center in Shreveport He has served as President of the Micro- circulatory Society, President of the American Physiological Societ, and

Editor-in-Chief of Microcirculation.

S ECTION I

Hypertension

C HAPTER 104

Arteriolar Responses to Arterial Hypertension

Julian H Lombard

Medical College of Wisconsin, Milwaukee, Wisconsin

including the adrenergic neurotransmitter norepinephrineand other vasoconstrictor agonists, elevations in intravascu-lar pressure (myogenic response), and an enhanced constric-tion in response to physiological stimuli such as increasedoxygen availability The increase in the sensitivity of arteri-oles to vasoconstrictor stimuli in hypertension may have anumber of underlying causes, including intrinsic alterations

in the electrophysiological responses of the vascular smoothmuscle (VSM) cell membrane, changes in the nature and/orproduction of chemical modulators of vascular tone pro-duced by the endothelium or by the vascular smooth musclecells themselves, alterations in intracellular Ca2+ homeo-stasis or in other second-messenger systems regulating contractile function, and the potential effects of increasedsympathetic nerve activity, humoral factors, and elevatedarterial pressure per se in enhancing the sensitivity of thevessels to other vasoconstrictor stimuli

Under normal physiological conditions, the

transmem-brane potential (Em) of the vascular smooth muscle (VSM)cells is a crucial regulator of their active contractile force(and therefore the diameter of arterioles and small resistancearteries) As a result, diameters of small arteries and arteri-oles are tightly regulated by the dynamic interactionbetween Ca2+ and K+ ion channels in the smooth musclecells Calcium influx through voltage-gated Ca2+ channels(activated by membrane depolarization) induces vasocon-striction, whereas the opening of K+ channels mediateshyperpolarization and vasodilation due to the inactivation ofvoltage-gated Ca2+channels There is a very steep relation-ship between VSM transmembrane potential and contractile

force generation in the Em range between approximately

-50 mV and -30 mV, so that small changes in Em producelarge changes in vascular tone Because of the tight coupling

Importance of the Arterioles in Hypertension

Arterioles and the small arteries that are located

immedi-ately upstream from the arterioles are the major sites of

vas-cular resistance in the peripheral circulation Thus, changes

in the structure and function of these vessels can play a

cru-cial role in the development and maintenance of

hyperten-sion, since an elevation in peripheral vascular resistance is a

common denominator in virtually all forms of this disease

In addition to controlling the resistance to blood flow in

peripheral vascular beds, arterioles play a crucial role in

determining the distribution of blood flow within the tissues

Therefore, changes in arteriolar structure, function, and

microvessel density can have important implications for the

supply of oxygen and nutrients to the tissues in hypertensive

individuals A variety of alterations in arteriolar structure

and function can lead to an elevated vascular resistance in

hypertension (Figure 1) These include increases in active

resting tone; an enhanced response to vasoconstrictor

uli; an impaired relaxation in response to vasodilator

stim-uli; a reduced number of arterioles and capillaries

(microvascular rarefaction), and structural alterations

lead-ing to reduced lumen diameter, increases in wall/lumen

ratio, and increases in vessel stiffness

Enhanced Sensitivity of Arterioles to Vasoconstrictor

Stimuli in Hypertension

One of the primary functional alterations that has been

reported in arterioles during hypertension is an increase in

their sensitivity to a variety of vasoconstrictor stimuli

Copyright © 2006, Elsevier Science (USA).

between transmembrane potential and active tone in the

smooth muscle cells, alterations in the electrophysiological

responses of the VSM cell membrane could contribute to

both an enhanced response of arterioles to vasoconstrictor

stimuli and to an impaired relaxation in response to

vasodilator stimuli in resistance vessels of hypertensive

individuals In this regard, it is important to note that resting

Emin the VSM cells of in situ arterioles and resistance

arter-ies larter-ies within the steep portion of the Em–active force

relationship, or very near the threshold for mechanical

activation of the smooth muscle Therefore, arteriolar tone is

very sensitive to activation by vasoconstrictor stimuli such

as norepinephrine and transmural pressure elevation, which

would result in depolarization and VSM contraction In

hypertension, enhanced sympathetic nerve activity, elevated

levels of intravascular pressure, and local or circulating

humoral factors not only could activate the vessels directly

via depolarization of the VSM cells, but also could bring

VSM Em closer to the threshold for mechanical activation,

which would increase the sensitivity of the arterioles to

other vasoconstrictor constrictor stimuli that act via

mem-brane depolarization

Several types of voltage-sensitive ion channels, including

L-type Ca2+ (CaL) channels, voltage-gated K+ (KV)

chan-nels, and high-conductance voltage- and Ca2+-sensitive K+

(BKCa) channels, play a crucial role in the regulation of

arte-riolar tone, and many studies suggest that high blood

pres-sure may trigger cellular signaling cascades that alter the

expression of different ion channels in arterial smooth

mus-cle, leading to further modifications of vascular tone There

is substantial evidence that calcium current through Ca2+L

channels is increased in blood vessels of hypertensive

ani-mals, which would contribute to an enhanced response to

vasoconstrictor stimuli There is also evidence that Kvnel current is reduced in vessels of hypertensive animals.This reduced Kvcurrent would tend to depolarize the VSMcell membrane, resulting in increased resting tone and anenhanced response to vasoconstrictor stimuli On the other hand, KCachannel current at physiological membranepotentials is significantly higher in cerebral arterial smoothmuscle cells from spontaneously hypertensive rats (SHRs)compared to those of normotensive Wistar-Kyoto (WKY)controls, apparently because of an increased density of the

chan-KCa channels in the VSM cell membrane These findingssuggest that elevated levels of blood pressure lead toincreased KCa channel expression in vascular smooth musclemembranes as a compensatory mechanism to offset theenhanced Ca2+current and reduced Kv current in the cells

In the absence of this compensatory increase in KCa channelexpression, the increased Ca2+current and the reduced Kv

current in the VSM cells could lead to a “vicious cycle” ofpositive feedback that would cause intense vasoconstrictionand severe hypertension

As noted earlier, alterations in the nature or release ofchemical modulators of vascular tone, including a variety ofarachidonic acid metabolites, could also contribute to anenhanced response of arterioles to vasoconstrictor stimuli.For example, there is evidence that the release of the vaso-constrictor substances endothelin, prostaglandin H2(PGH2),and/or thromboxane A2(TxA2) from the vascular endothe-lium contributes to the enhanced myogenic response topressure elevation in arterioles of spontaneously hyperten-sive rats Release of PGH2and/or TxA2also appears to con-tribute to an enhanced response to vasoconstrictor stimuli inSHRs and other forms of hypertension Studies of the role

of these compounds in the potentiation of the myogenic

Figure 1 Summary diagram of the mechanisms by which changes in arteriolar structure and tion may contribute to an elevated vascular resistance in hypertension (Adapted with thanks from an original drawing by Dr Francis A Sylvester.) (see color insert)

func-C HAPTER 104 Arteriolar Responses to Arterial Hypertension 707

response to transmural pressure elevation in hypertensive

animals suggest that these endothelium-derived constrictor

substances increase the Ca2 + sensitivity of the contractile

apparatus of arteriolar smooth muscle cells in hypertension,

so that similar increases in internal Ca2 +concentration in the

VSM cells during pressure elevation in the vessel cause an

enhanced myogenic constriction of arterioles The latter

observation suggests that intracellular signaling cascades

(in addition to changes in VSM Em) can participate in the

altered vascular responses to vasoconstrictor stimuli

occur-ring in arterioles of hypertensive animals There is also

evi-dence that elevations in intravascular pressure can increase

the formation of superoxide anion in arterioles, which could

then interfere with nitric oxide (NO)-dependent vascular

relaxation and potentiate myogenic responses indirectly

The enhanced myogenic response and the increased

con-striction of arterioles in response to elevated Po2that have

been demonstrated in many forms of hypertension could

also be due to an enhanced production of

20-hydroxye-icosatetraenoic acid (20-HETE) or to an increased

sensitiv-ity of the smooth muscle cells to the vasoconstrictor effects

of 20-HETE, a metabolite of the cytochrome P450 pathway

of arachidonic acid metabolism that has been implicated in

mediating myogenic responses to transmural pressure

eleva-tion and arteriolar constriceleva-tion in response to increased Po2

Impaired Relaxation of Arterioles to

Vasodilator Stimuli

In addition to an enhanced response to vasoconstrictor

stimuli, arterioles of hypertensive animals exhibit an

impaired relaxation in response to a variety of vasodilator

stimuli including hypoxia, shear stress, and

endothelium-dependent vasodilators, such as acetylcholine (ACh)

Impaired relaxation of arterioles to endothelium-dependent

vasodilator stimuli such as ACh has also been demonstrated

in human hypertensive patients The impaired vascular

relaxation in hypertensive individuals has been proposed to

be due to a reduced production of endothelium-derived

vasodilator compounds, such as nitric oxide (NO) or

vasodilator prostaglandins, and/or to an enhanced release of

vasoconstrictor factors (e.g., thromboxane or prostaglandin

H2) from the endothelium The reduction in

endothelium-dependent dilation mediated by NO also may result from

increased levels of oxidative stress in the tissue, which

would destroy NO and reduce its availability for mediating

vascular relaxation There is also evidence that fundamental

alterations in receptor–heterotrimeric G protein coupling

may contribute to impaired vasodilator responses in

hyper-tensive animals and in normohyper-tensive animals on a high-salt

diet In the latter case, arterioles and resistance arteries of

hypertensive animals and normotensive animals on high-salt

diet not only exhibit impaired relaxation in response to

vasoactive agonists acting through the cyclic AMP pathway

of vascular relaxation, but also fail to respond to direct

activation of the alpha subunit of the Gsprotein with choleratoxin Taken together, these observations suggest that hyper-tension and high salt diet may both be associated with fun-damental alterations of signaling pathways in the vascularsmooth muscle cells

Nitric oxide–dependent relaxation and mediated vasodilation are both impaired in skeletal musclearterioles of spontaneously hypertensive rats This appears

prostaglandin-to be due prostaglandin-to an impaired synthesis and/or action of nitricoxide (including reduced bioavailability of NO due toincreased oxidative stress) and alterations in the metabolism

of arachidonic acid to favor an enhanced production of thevasoconstrictor metabolite PGH2and a reduced production

of vasodilator prostaglandins in the arterioles Findings such

as these suggest that a simultaneous dysfunction of thesetwo major endothelium-dependent vasodilator pathwayscould make a significant contribution to the elevated vascu-lar resistance in hypertension Agonists such as norepineph-rine and acetylcholine also cause an increased release of the endothelium-dependent vasoconstrictors thromboxane

A2 and/or PGH2 in arterioles and resistance arteries

of hypertensive rats, leading to a reduced sensitivity toacetylcholine and to an enhanced vasoconstrictor response

It has been proposed that the elevated hemodynamicforces present in hypertension may initiate alterations of sig-naling pathways in the endothelium and smooth musclecells of arterioles that could, in turn, enhance the release ofreactive oxygen species such as superoxide Any reduction

in the availability of NO due to increased levels of ide released by high pressure in the arterioles (or in response

superox-to other pathophysiological alterations in hypertension)would likely cause an impaired dilation of arterioles inresponse to shear stress- and other NO-dependent vasodila-tor stimuli, leading to the maintained elevation of wall shearstress and peripheral vascular resistance that exists in hyper-tension It has also been proposed that alterations in themechanisms of functional vascular control in hypertensionmay eventually lead to the development of irreversiblestructural changes in the microcirculation The latterhypothesis is consistent with the increasing body of evi-dence that elevated levels of reactive oxygen metabolitesmay contribute to the vascular dysfunction commonlyobserved in hypertension

Structural Alterations in Arterioles

during Hypertension

In addition to the changes in functional arteriolar control

mechanisms in hypertension, structural alterations of

arteri-oles and small arteries can contribute to the elevated

vas-cular resistance in this disease These changes include

structural narrowing of the lumen, thickening of the

vascu-lar wall leading to an increased wall/lumen ratio, and altered

mechanical properties of the vessel, such as increased

stiff-ness and reduced distensibility of the vessel wall These

structural alterations of the vascular wall may be caused, at

least in part, by alterations in the composition of the wall,

such as changes in collagen and elastin content in the vessel

wall, or by alterations in the specific types of collagen

present in the vascular wall

Most reports indicate that wall thickening is not a

com-mon response to elevated blood pressure in the smaller

arte-rioles; however, wall thickening is prominent in the larger

arteries that lie upstream from the microcirculation There is

evidence that the nature of the structural alterations

occur-ring in the vascular wall in response to hypertension may be

determined by the response of individual vessels to the

increase in circumferential wall stress occurring during

ele-vations in arterial pressure In large arteries or in resistance

arteries that do not exhibit strong myogenic responses,

hypertrophy of the smooth muscle cells and the deposition

of extracellular matrix thicken the walls of the vessel during

the development of hypertension without reducing the size

of the lumen In contrast, small arteries and arterioles that

exhibit myogenic contractile activation in response to

ele-vated pressure show an inward remodeling that reduces

lumen diameter without thickening the vessel wall This

inward remodeling is mediated through the rearrangement

of the smooth muscle cells around a smaller lumen In this

case, the initial increase in circumferential wall stress that

occurs in response to increased pressure in the vessel can

account for inward remodeling because myogenically active

small arteries and arterioles can constrict in response to an

elevation of intravascular pressure, thus restoring

circumfer-ential wall stress toward control levels In contrast, larger

arteries have little or no myogenic response and respond to

the increase in wall stress by initiating growth processes in

the vascular wall There is substantial evidence that the

structural alterations in small arteries of hypertensive

indi-viduals reflect an adaptation to the elevated blood pressure,

rather than being the primary cause of increased vascular

resistance in hypertension However, structural narrowing

of the vessel lumen and thickening of the vascular wall may

play a crucial role in the maintenance and exacerbation of

the elevated vascular resistance in hypertension In this

respect, elevations in vascular resistance that arise from

structural alterations are less responsive to therapeutic

approaches than those that result from an elevated vascular

smooth muscle tone, which can be treated with drugs that

lead to vascular smooth muscle relaxation

Arteriolar Rarefaction

In addition to structural narrowing and increased wallthickness, a decrease in the number of arterioles and capil-laries (rarefaction) has been widely reported in many differ-ent animal models of hypertension and in hypertensivehumans Microvessel rarefaction has two components: func-tional rarefaction, mediated by active closure of arterioles,and anatomical or structural rarefaction, mediated by anactual reduction in the number of arterioles Several lines ofevidence suggest that functional rarefaction can eventuallyprogress to anatomical rarefaction Microvascular rarefac-tion involves both the capillaries and the smaller (third- andfourth-order) arterioles and is accompanied by structuralchanges in the microvessels Mathematical models suggestthat microvessel rarefaction can have substantial effects onthe microcirculation, including an elevation in vascularresistance (especially in conjunction with the constriction ofarterioles) and a reduction in tissue Po2 The latter changesmay be particularly significant in contributing to tissuedamage under conditions of reduced perfusion One inter-esting observation regarding microvessel rarefaction in salt-dependent forms of hypertension is that it has also beendemonstrated to occur in normotensive animals on a high-salt diet Arteriolar rarefaction in salt-dependent hyperten-sion forms of hypertension and with high-salt diet innormotensive animals develops very rapidly and appears to

be mediated by the angiotensin II (ANG II) suppression thatoccurs in response to elevated salt intake, since it can be prevented by continuous intravenous (i.v.) infusion of a low dose of ANG II to maintain normal circulating levels

of ANG II without increasing blood pressure

Oxidative Stress and Arteriolar Function

in Hypertension

Enhanced production of reactive oxygen species such assuperoxide anion may contribute to arteriolar dysfunction,elevated vascular resistance, and organ damage in hyper-tensive individuals Studies assessing the contribution ofenhanced oxidative stress to altered microvascular functionhave demonstrated that dihydroethidine fluorescence andtetranitroblue tetrazolium dye reduction (indicators ofoxidative stress) are significantly increased in arterioles andvenules of spontaneously hypertensive rats and Dahl salt-sensitive hypertensive rats on high-salt diet This findingsuggests that there is an enhanced production of oxygen freeradicals in the microvasculature of hypertensive animalsthat could contribute to impaired relaxation in response toNO-dependent vasodilator stimuli or other vascular relax-ation mechanisms, for example, prostacyclin-induced dila-tion It also appears that elevated dietary salt intake alonecan lead to an increase in oxidative stress in arterioles andresistance arteries of normotensive animals As discussednext, alterations in the function of arterioles and resistance

C HAPTER 104 Arteriolar Responses to Arterial Hypertension 709

arteries due to the effects of high-salt diet alone may have

important implications for the development of an elevated

vascular resistance in salt-sensitive forms of hypertension

Dietary Salt Intake and Arteriolar Function

Many individuals exhibit salt-sensitive forms of

hyper-tension, in which elevated dietary salt intake leads to an

increase in arterial blood pressure This elevation of blood

pressure is accompanied by an increase in peripheral

vascu-lar resistance A particuvascu-larly valuable genetic animal model

of salt-sensitive hypertension is the Dahl salt-sensitive

(Dahl S) rat, an inbred strain of rats in which elevation of

dietary salt intake leads to an elevated vascular resistance

and a substantial degree of hypertension In Dahl S rats, the

development of hypertension in response to elevated dietary

salt intake is accompanied by a uniform increase in

hemo-dynamic resistance throughout most of the peripheral

vas-culature In the spinotrapezius muscle, this increase in

resistance is largely due to an intense constriction of

proximal arterioles The mechanisms responsible for this

increased arteriolar tone include increased responsiveness to

oxygen and a loss of tonic nitric oxide (NO) availability

caused by reduced endothelial NO production and/or

accel-erated degradation of NO by reactive oxygen species

In recent years, it has become increasingly clear that

ele-vated dietary salt intake alone can lead to profound changes

in the structure and function of resistance vessels of

nor-motensive animals, as well as vessels of salt-sensitive

exper-imental models of hypertension such as the Dahl S rat

These changes include microvascular rarefaction and an

impaired relaxation of blood vessels in response to

vasodila-tor stimuli such as hypoxia, acetylcholine, and prostacyclin

In normotensive Dahl salt-resistant (Dahl R) rats, elevated

dietary salt intake also leads to an impaired dilation during

the elevated shear stress that occurs in response to increased

flow in the arteriole The impaired dilation in response to

increased flow in arterioles of Dahl R rats on high-salt diet

appears to be due to a salt-induced suppression of NO

activity in the absence of hypertension Emerging evidence

suggests that impaired vascular relaxation in

normoten-sive animals on a high-salt diet involves alterations in the

function of both the endothelium and the vascular smooth

muscle cells, and that increased levels of oxidative stress in

the vasculature can contribute to the impaired vascular

relaxation in animals on high-salt diet

The impaired relaxation of blood vessels of

normoten-sive animals on high-salt diet in response to vasodilator

stimuli such as hypoxia, acetylcholine, and prostacyclin

appears to be due to the suppression of angiotensin II (ANG

II) levels that occurs in response to high-salt diet, because

continuous i.v infusion of a low dose of ANG II to prevent

salt-induced ANG II suppression restores normal

vasodila-tor responses without raising blood pressure in

normoten-sive animals on a high-salt diet The direct effect of high-salt

diet itself in contributing to microvessel rarefaction and

impaired vascular relaxation in normotensive rats suggeststhat elevated dietary salt intake may be an important initialcontributor to the increased vascular resistance in salt-sensitive forms of hypertension, since it would tend to elevate vascular resistance even before the increase in arte-rial blood pressure In combination with other predisposingfactors for hypertension, such as impaired renal function,these changes not only could lead to the development ofsalt-sensitive hypertension, but also could play a major role

in the maintenance and progression of the elevated vascularresistance in salt-sensitive forms of this disease

Influence of Gender on Arteriolar Function

in Hypertension

Females prior to menopause are much less susceptible tohypertension and other cardiovascular diseases than males,indicating that gender has a protective effect in these disor-ders and that female sex hormones can offset some of thealterations in arteriolar function that may occur with hyper-tension in males For example, flow-induced arteriolar dilation is significantly reduced in male spontaneouslyhypertensive rats compared to females, because of the loss

of the nitric oxide (NO)-mediated portion of the response.This impairment of the NO-mediated component of flowinduced dilation results in a maintained elevation of wallshear stress in the male rats, suggesting that female sex hor-mones play an important role in maintaining NO-dependentvasodilator responses and in preserving the regulation ofarteriolar shear stress by nitric oxide Arteriolar dilation inresponse to increases in perfusate flow is also impaired inisolated gracilis muscle arterioles of ovariectomized femaleSHRs, compared with those of intact female SHRs andovariectomized female SHRs receiving estrogen replace-ment The impaired flow induced dilation in ovariectomizedfemale SHRs appears to be due to the loss of the NO-dependent component of shear stress–induced vascularrelaxation, providing additional evidence that estrogen preserves the NO-mediated portion of flow/shearstress–induced dilation in female hypertensive rats, result-ing in a lower maintained wall shear stress in the femaleSHRs, compared to their male counterparts The lower wallshear stress in the females may contribute to a lowering ofsystemic blood pressure and to the lower incidence of car-diovascular diseases in females In contrast, the maintainedelevation of shear stress in arterioles of the male rats couldtrigger other pathological alterations in the vascular wall, asdiscussed earlier

Norepinephrine-induced constrictions are also enhanced

in arterioles of ovariectomized female SHRs compared withthose of intact female SHRs and ovariectomized femaleSHRs receiving estrogen supplementation These differ-ences in norepinephrine-induced constriction of arteriolesare eliminated by inhibiting NO synthesis, suggesting thatestrogen also preserves the modulating effect of NO on

arteriolar responses to vasoconstrictor agonists in female

rats

Although female sex hormones may attenuate

endothe-lial dysfunction in hypertensive animals by preserving

endothelium-dependent vasodilation, less is known

regard-ing the influence of ovarian hormones on the generation of

contractile substances by the endothelium However, it

appears that female sex hormones attenuate the generation

of vasoconstrictor prostanoids and superoxide anion (O•

2 -)

by the endothelium of mesenteric microvessels from

spon-taneously hypertensive rats Microvessels from

ovariec-tomized female SHRs exhibit an increased sensitivity to

norepinephrine and a reduced sensitivity to acetylcholine,

compared to those from intact female SHRs Treatment with

estradiol or estradiol + progesterone restores normal

reac-tivity to norepinephrine and acetylcholine in vessels of

ovariectomized female SHRs Inhibition of cyclooxygenase

and scavenging of superoxide with superoxide dismutase

(SOD) also restore normal responses to norepinephrine and

acetylcholine in vessels of ovariectomized female SHRs

Norepinephrine-induced release of prostaglandin F2a

(PGF2a), a vasoconstrictor metabolite of the

cyclooxyge-nase pathway of arachidonic acid metabolism, is also greater

in endothelium-intact microvessels of ovariectomized

female SHRs compared to those of intact female SHRs This

response is normalized by treatment with estrogen or

estro-gen + progesterone Taken together, these findings suggest

that estrogen may protect female SHRs against severe

hypertension, not only by preserving NO-dependent

dila-tion, but also by decreasing the synthesis of endothelium

derived contracting factors such as PGH2, PGF2a, and O•

2 -

Glossary

Angiotensin II: Biologically active peptide formed from a precursor

peptide (angiotensin I) by angiotensin-converting enzyme (ACE).

Angiotensin II has numerous biological actions, including vasoconstriction,

stimulation of aldosterone release, stimulation of sodium reabsorption by

the kidney, and regulation of vessel structure, vessel funciton, and

microvessel density.

Arachidonic acid: Major lipid precursor to various eicosanoids,

which are fatty acid derivatives that act as signaling molecules to mediate

many biological functions Arachidonic acid is cleaved from membrane

phospholipids and converted into a variety of biologically active lipid

metabolites by various enzymes, such as cyclooxygenases, to form the

immediate precursor (PGH2) for various prostaglandins (e.g., prostacyclin,

prostaglandin E2, prostaglandin F2a) and thromboxane A2; lipoxygenases to

form leukotrienes; and cytochrome P450 enzymes to form vasodilator

com-pounds such as eicosatrienoic acids (EETs) and vasoconstrictor comcom-pounds

such as 20-hydroxyeicosatetraenoic acid (20-HETE).

Heterotrimeric G protein: Cell membrane spanning protein that

binds guanosine triphosphate (GTP) and mediates the functional coupling

of membrane receptors to downstream target enzymes or ion channels

involved in cellular signal transduction.

Reactive oxygen species (ROS): Reactive chemical derivatives of

oxygen, such as superoxide anion, hydrogen peroxide, hypochlorous acid,

and hydroxyl radical ROS can be formed by a variety of enzymes

includ-ing xanthine oxidase, nitric oxide synthase (NOS), NAD(P)H oxidase,

and cyclooxygenase Elevated levels of reactive oxygen species in blood

vessels cause increased oxidative stress and can contribute to vascular

dysfunction in hypertension.

Transmembrane potential (Em ): Electrical potential difference that

exists across the cell membrane The magnitude of Emdiffers among cell types, but generally ranges between -50 mV and -30 mV in vascular smooth muscle cells of in vivo microvessels and resistance arteries A

reduced magnitude of the Em(depolarization) is associated with contraction

of the smooth muscle due to increased Ca 2 + influx into the cells via voltage activated Ca 2 + (CaL) channels, while an increased magnitude of Em(hyper- polarization) is associated with reduced Ca 2 + influx into the cells, leading

to relaxation.

Bibliography

Dantas, A P., Scivoletto, R., Fortes, Z B., Nigro, D., and Carvalho, M H (1999) Influence of female sex hormones on endothelium-derived vasoconstrictor prostanoid generation in microvessels of spontaneously

hypertensive rats Hypertension 34, 914–919.

Huang, A., Sun, D., Kaley, G., and Koller, A (1998) Superoxide release to high intra-arteriolar pressure reduces nitric oxide-mediated shear

stress- and agonist-induced dilations Circ Res 83, 960–965 This

study demonstrated that elevated pressure in arterioles can cause increased superoxide production, which could subsequently lead to impaired dilation of arterioles in response to elevated shear stress and other NO-dependent vasodilator stimuli These findings may be directly relevant to the impairment of vascular relaxation that occurs in hyper- tensive individuals not exhibiting other alterations that may impair vas- cular relaxation mechanisms, such as diabetes or low circulating ANG

II levels.

Izzo, J L., Jr., and Black, H R (senior eds.) (2003) Hypertension Primer,

3rd ed Dallas, TX: American Heart Association.

Lee, R M K W (ed.) (1989) Blood Vessel Changes in Hypertension:

Structure and Function, Vols I and II Boca Raton, FL: CRC Press.

Liu, Y., Hudetz, A G., Knaus, H.-G., and Rusch, N J (1998) Increased expression of Ca 2 + -sensitive K+channels in the cerebral microcircula- tion of genetically hypertensive rats: Evidence for their protection from

cerebral vasospasm Circ Res 82, 729–737.

Lombard, J H., Sylvester, F A., Phillips, S A., and Frisbee, J C (2003) High salt diet impairs vascular relaxation mechanisms in rat middle

cerebral arteries Am J Physiol 284, H1124–H1133.

Special Topics Issue: Microcirculatory Adaptations to Hypertension.

(2002) Microcirculation 9(4), 221–328 This special issue of

Microcir-culation features a collection of recent reviews about various aspects

of microcirculatory adaptations to hypertension, including: (1)

“Microvascular structure and function in salt-sensitive hypertension,”

by M A Boegehold (pp 225–241); (2) “New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure,”

by R H Cox and N J Rusch (pp 243–257); (3) “The inflammatory aspect of the microcirculation in hypertension: Oxidative stress, leuko- cytes/endothelial interaction, apoptosis,” by M Suematsu, H Suzuki H,

F A Delano, and G W Schmid-Schönbein (pp 259–276); (4) ing pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension,” by A Koller (pp 277–294); (5)

“Signal-“Adaptation of resistance arteries to increases in pressure,” by R L Prewitt, D C Rice, and A D Dobrian (pp 295–304); (6) “Structural adaptation of microvascular networks and development of hyperten- sion,” by A R Pries and T W Secomb (pp 305–314); and (7) “Adap- tations of the renal microcirculation to hypertension,” by J D Imig and

E W Inscho (pp 315–328).

Stekiel, W J., Contney, S J., and Rusch, N J (1993) Altered b-receptor

control of in situ membrane potential in hypertensive rats Hypertension

21, 1005–1009 This study demonstrated that receptor-G-protein

cou-pling is impaired in arterioles and venules of rats with reduced renal mass hypertension VSM transmembrane potential (E m ) was measured with glass microelectrodes in first order arterioles and venules of the in situ cremaster muscle of hypertensive and normotensive rats Arterioles

of hypertensive rats failed to hyperpolarize in response either to the cAMP-dependent beta adrenergic agonist isoproterenol or to cholera toxin, a direct activator of the alpha subunit of the G s protein coupling

C HAPTER 104 Arteriolar Responses to Arterial Hypertension 711

the receptor to downstream signaling events In contrast, arterioles of

normotensive controls hyperpolarized in response to both isoproterenol

and cholera toxin Subsequent studies by other laboratories

demon-strated that G protein coupling is impaired in other forms of

hyperten-sion such as SHRs, and in animals on high salt diet Winner (W J.

Stekiel) of the 1993 Harry Goldblatt Award in Cardiovascular

Research, awarded by the publications committee of the American

Heart Association Council for High Blood Pressure Research, to

rec-ognize the most significant new contribution to the understanding of the

causes and/or consequences of hypertension.

Ungvari, Z., and Koller, A (2000) Endothelin and prostaglandin H2

/throm-boxane A2enhance myogenic constriction in hypertension by

increas-ing Ca 2 + sensitivity of arteriolar smooth muscle Hypertension 36,

856–861.

Zweifach, B W (1983) The microcirculation in experimental

hyperten-sion: State of the art review Hypertension 5, I-10–I-16.

Capsule Biography

Dr Lombard is currently Professor of Physiology at the Medical College of Wisconsin He is a former President of the Microcirculatory Society and is a fellow of the Cardiovascular Section of the American Physiological Society, the Council for High Blood Pressure Research of the American Heart Association, and the Council on Basic Cardiovascular Sci- ences of the American Heart Association His laboratory focuses on micro- circulatory control under normal conditions and during pathological conditions such as hypertension His work is currently supported by several grants from the National Institutes of Health.

otherwise, these models serve as the main basis for currentknowledge at the microvascular level.

Microcirculation in Hypertension

The Microvascular Network Pattern and

Cell MorphologyThe microvascular network topology in hypertensive andnormotensive animals is overall the same but differs inquantitative terms For example, in skeletal muscle the

microvascular branching pattern formed by feed arterioles,

by arcade arterioles, and by their regular side branches, the

terminal (previously designated also as transverse) oles, is the same The terminal arterioles form asymmetric

arteri-dichotomous trees, which give rise to the capillary network.

The SHR has a higher density of arcade arterioles withsmaller trees forming the terminal arterioles The capillaries

form bundles with a modular pattern of alternating terminal arterioles and collecting venules, which in turn feed into the

arcade venules and discharge into the central circulation

through the draining veins Compared to WKY rats, the

SHR exhibits on average a lower capillary network densityalthough individual capillaries have on average greaterlength (between bifurcations) and diameter Apart from thefact that collecting venules of SHR are narrower whilearcade venules are wider in lumen diameter than in theWKY rats, the two strains exhibit no differences in venularnetwork topology

The innervation of microvessels in skeletal muscleextends to the terminal arterioles in form of adrenergic

Introduction

Since arterial hypertension is diagnosed by elevated

blood pressure in central arteries, it is frequently regarded as

a condition that is almost exclusively affected by

hemody-namic resistance in the small arteries and arterioles This

point of view has lead for several decades to a focus on

arteries and arterioles as mediators of the syndrome and as

focus for therapeutic targets But microvascular studies

indi-cate that several forms of arterial hypertension may be

affected by more general mechanisms, which not only

involve the arteries and arterioles, but a range of

pathophy-siological mechanisms

This chapter will be focused on the manifestations of

hypertension in capillaries and venules, microvessels

that are not exposed to blood pressure elevation Capillaries

and venules are involved in blood flow regulation and

exchange functions They are also an integral part of the

inflammatory cascade, a potentially important aspect of

hypertension as a vascular disease with cell and organ

damage We will summarize an array of pathophysiological

phenomena in hypertension for which there is still no

conclusive evidence for a pressure-mediated mechanism

and which instead point towards a more general metabolic

and regulatory defect

The majority of the evidence cited here has been obtained

in the spontaneously hypertensive rat (SHR) and its

nor-motensive control, the Wistar Kyoto (WKY) rat, as well as

in the salt dependent Dahl-S hypertensive rat with its

nor-motensive control, the salt resistant Dahl-R strain Both of

these models have a strong genetic linkage Unless indicated

Copyright © 2006, Elsevier Science (USA).

fibers The nerve fibers are positioned at the interface

between smooth muscle media and adventitia down to the

endings of the terminal arterioles The density of adrenergic

fibers in the SHR is significantly higher compared to that of

the WYK rats Capillaries or venules have no adrenergic

innervation

The walls of capillaries in hypertensives and

normoten-sives consist of endothelial cells with pericytes Venules

have a thinner wall structure than their arteriolar

counter-part, with attenuated endothelial thickness, pericytes and

smooth muscle cells in the media, and fibroblast in the

adventitia Ultrastructural examination of adult capillaries

and venules in hypertensives often reveals morphological

damage not found in normotensives, e.g in form of

mem-brane bleb formation

Microvascular PressureThe elevated blood pressure in arteries of hypertensives

is reduced in arterioles and in terminal arterioles to values

which are similar to those in normotensive animals (Figure

1) Apart from the fact that the pressure drop on the venular

side is small in both normotensives and hypertensives, there

are no significant differences in blood pressure values in

venules

Microvascular FlowBoth the cardiac output and the average local flow rates

in different hierarchies of microvessels in hypertensive and

normotensive microvascular networks are almost

indistin-guishable (Zweifach et al., 1981) However, within each

microvessel hierarchy the hypertensives have larger

varia-tions of flow rates among individual vessels

Hemodynamic Resistance

Estimates of the average hemodynamic resistance

derived from micro-pressure and flow measurements

indi-cate a higher resistance in arcade and terminal arterioles of

the hypertensives without such significant differences in the

venular counterparts (Boegehold, 1991)

The control of the hemodynamic resistance involves

smooth muscle contraction and restructuring of the

arteri-oles In addition, also blood rheological mechanisms serve

to control the hemodynamic resistance in capillaries and

venules In spite of the relatively small number of

circulat-ing leukocytes compared to significantly faster movcirculat-ing

ery-throcytes, in capillaries with single file of blood cells the

larger and stiffer leukocytes have an important influence on

apparent viscosity and capillary resistance The mechanism

is due to hydrodynamic interaction of slower moving

leuko-cytes with more flexible erythroleuko-cytes, which in capillaries

displaces the erythrocytes away from their center-line

posi-tion and leads to an elevated apparent viscosity The effect

is sensitive with respect to the exact erythrocyte and

leuko-cyte counts, and does not require membrane attachment tothe endothelium (Helmke et al., 1997)

Reactive Oxygen Species Production in Microvessels

of Hypertensives

Oxygen Free Radical SpeciesEvidence derived from direct measurements in themicrocirculation and in blood samples, experimental results

SHR WKY

SHR WKY

no differences in average flow rates.

C HAPTER 105 Capillary and Venular Responses to Arterial Hypertension 715

obtained by use of scavengers, and observations on isolated

cells of hypertensives indicate an alteration in oxygen

metabolism and overproduction of biologically active

oxy-gen species in hypertension Reactive oxyoxy-gen species (ROS)

(superoxide anion (O2-), hydrogen peroxide (H2O2), nitric

oxide (NO), carbon monoxide (CO) and their derivatives)

serve to regulate vascular functions but may also be toxic In

the mesentery and skeletal muscle microcirculation,

reduc-tion of nitroblue tetrazolium and fluorescent labeling of

hydroethidine (superoxide dependent probes) show

enhanced levels of ROS in the endothelium not only in

arte-rioles, but also in capillaries and especially in venules The

rise of the NBT reduction in venules exceeds any

enhance-ment on the arteriolar side in all hypertensive models

inves-tigated so far (Figure 2).

Superoxide anion, a primary radical product generated by

one electron donation to molecular oxygen, is generated

through xanthine oxidase and nicotinamide adenine

dinu-cleotide/nicotinamide adenine dinucleotide phosphate

(NADH/NADPH) oxidase The superoxide anion has the

ability to react with NO and with guanylate

cyclase-dependent vasorelaxation, may activate platelets,

leuko-cytes, and endothelial cells After cancellation of

NO-mediated relaxation and elevation of tone in the

hyperten-sive rats, superoxide generation promotes overexpression of

NO synthase mRNA Superoxide anion inhibits soluble

guanylate cyclase, the major target of NO

Chronic overproduction of superoxide and related ROS

triggers alterations in the expression of genes encoding

pro-teins that control the tissue inflammatory responses, such

as endothelial adhesion molecules (intracellular adhesion

molecule-1 (ICAM-1), and P- and E-selectins) as well as

superoxide dismutase, NO synthase and heme oxygenase-1

Nitric oxide depression is by itself a pro-inflammatory

stim-ulator (Kubes et al., 1991) and a driving mechanism for

superoxide mediated injury in hypertensives

The significance of the ROS is further highlighted by the

fact that the enhanced peroxide production is detectable

before the blood pressure is elevated

Enzymatic Sources of Oxygen Free RadicalsNADP/NADPH oxidase and xanthine oxidase in neu-trophils, the monocyte/macrophage system and vascularendothelial cells, smooth muscle cells, as well as parenchy-mal cells, are involved in superoxide generation in vivo.Recent evidence derived from immuno-histochemistry indicates that both enzymes are expressed in almost all cells

of the microcirculation The population of generating neutrophils in the circulation is greater in SHRthan in WKY rats over their entire lifetime

superoxide-XANTHINEOXIDASEThe dehydrogenase (XD) oxidizes hypoxanthine to yielduric acid and is coupled with a reduction of NAD intoNADH Once the enzyme is converted to the oxidase form(XO), the same reaction utilizes molecular oxygen as anelectron acceptor and serves as a superoxide-generating sys-tem Endothelial cells in microvessels, but less those inlarger vessels, serve as a major source of the XD/XO sys-tem In the mesentery, where the vasculature constitutes amajor source of the enzyme, both XD + XO and XO activi-

ties are elevated in SHR compared to WKY rats (Suzuki

et al., 1998)

NADPH OXIDASE

In addition to the role of NADH oxidase in regulation ofproliferative responses in vascular smooth muscle cells, thisenzyme may also be a major player in the hypertensive syn-drome Phox22, a subunit necessary for the enzyme activity,exhibits a distinct feature that is characteristic of the super-oxide production from phagocytic NADPH oxidase In vas-cular smooth muscle cells, this enzyme can increasesuperoxide generation in response to angiotensin II and reg-ulate vascular hypertrophy

CYTOCHROMEP450 MONOOXYGENASEThis set of enzymes is responsible for elevation of vas-cular tone and tissue inflammatory responses in hyperten-

Figure 2 Microimages of the mesentery microcirculation in mature Wistar Kyoto (WKY, left) and age- and gender- matched spontaneously hypertensive rats (SHR, right) The tissue was labeled in the living state under identical conditions with nitro-blue tetrazolium (NBT), a superoxide sensitive indi- cator Note the enhanced labeling in microvessels compared with the interstitial state and the strong labeling in the venules (V) compared to arterioles (A) in both animals The SHR exhibits widespread enhancement of NBT labeling in almost all microvessels Both rat strains have reduced labeling in capillaries (see color insert)

sives Cytochrome P450-derived adrenocortical hormones

such as corticosterone and aldosterone play a role in the

for-mation of hypertensive states The metabolism of

arachi-donic acid by cytochrome P450 epoxygenases leads to the

formation of biologically active eicosanoids for regulation

of local inflammatory responses, such as

epoxye-icosatrienoic acids (EETs), dihydroxyeepoxye-icosatrienoic acids

(DHETs), and hydroxyeicosatetraenoic acids (HETEs)

HEMEOXYGENASE ANDCO

Heme oxygenase may also serve as a modulator of

vas-cular tone and smooth muscle cell hypertrophy due to

the biological actions of CO, a vasorelaxing mediator

(Imai et al., 2001) The potency of CO is less than that of

NO CO modestly activates cyclase when local NO levels

are low

ROS SCAVENGERDEPLETION

The enhanced oxidative stress in hypertensives may also

be due to the suppression of scavenger mechanisms for

oxy-gen radicals In the SHR, mRNA levels and enzyme activity

of superoxide dismutase and catalase are reduced in most

but not all tissues Treatment with superoxide dismutase or

analogues serves in part to control blood pressure

Treat-ment is hampered by the limited ability to transport current

scavenging agents, such as superoxide dismutase, to

rele-vant sites in the microcirculation, including the endothelial

or smooth muscle cells

The evidence for enhanced ROS formation supports the

hypothesis that the SHR may suffer from a genetic shift of

the glycolytic metabolism into oxidative metabolism, in line

with the close linkage of hypertension with insulin

resist-ance in this model

Microvascular Rarefaction

Rarefaction in the microcirculation of hypertensives has

been encounntered in patients and in experimental models

with hypertension (Hutchins and Darnell, 1974) Functional

rarefaction and structural rarefaction can be observed

(Pre-witt et al., 1982) Functional rarefaction refers to a condition

in which no or few blood cells are present in a microvessel

due to a state of high vascular tone, but blood cells can be

readily reintroduced by application of a vasodilator

Struc-tural rarefaction refers to the physical loss of intact

microvessel

Functional Rarefaction and Blood Cell Distribution

Capillary networks, which are supplied by arterioles

under high levels of vascular tone, exhibit functional

rar-efaction The tone in arterioles is due in large part to smooth

muscle contraction, which in small arterioles is also

associ-ated with deformation and folding of the endothelial cell In

terminal arterioles, a high tone may reach the point of

com-plete lumen closure Vascular tone is defined as

{steady state diameter minus the dilated diameter}/

{dilated diameter}

The dilated diameter is measured after application of

a saturating dose of vasodilators, such as papaverine oradenosine

The average tone in arterioles of hypertensives isenhanced, leading to a reduction of blood flow from thearcade arterioles into terminal arterioles At a divergentbifurcation from an arcade to a terminal arteriole, erythro-cytes and leukocytes enter preferentially into the vesselswith the higher flow rates, i.e terminal arterioles withenhanced microvascular tone receive a lower fraction ofblood flow and therefore also a lower fraction of the bloodcells Therefore, if a terminal arteriole is constricted to thepoint at which no erythrocytes or leukocytes and onlyplasma and sporadic platelets enter, its downstream capillar-ies are filled mostly with plasma and exhibit functional rar-efaction Dilation of the terminal arteriole raises the bloodflow and restores the flow of blood cells back into the capil-lary network Thus functional rarefaction represents a redis-tribution of the microhematocit in the smallest microvesselsand is reversible

Structural Rarefaction and Endothelial Apoptosis

In contrast, structural rarefaction is not readily reversible

by application of a vasodilator Recent evidence in severalforms of hypertension suggests that loss of capillaries is due

to endothelial cell apoptosis (Vogt and Schmid-Schönbein,2001)

Endothelial apoptosis may be detected in most segments

of the circulation in hypertensives In larger arterioles orvenules with multiple endothelial cells along the wallperimeter, apoptosis of individual endothelial cells leads to

a temporary shift in local endothelial permeability In trast, apoptosis of endothelial cells in true capillaries leads

con-to actual loss of the microvessels since their wall is made up

of single endothelial cells The mesentery microcirculation

of the SHR and WKY rats is subject to a non-uniform tern of cell death, and is enhanced in selected microvascularsegments by a glucocorticoid driven mechanism Apoptosis

pat-is present in arterioles but also in capillaries and venuleswithout elevated blood pressure Enhanced apoptotic activ-ity has been reported in every hypertensive model investi-gated to date, including the SHR, glucocorticoid-mediatedhypertensives, and one kidney/one clip Goldblatt hyperten-sives Apoptotic activity is observed in the kidney, heart,smooth and skeletal muscle, mesentery, and in the thymusand can be detected before blood pressure is elevated

Leukocyte-Endothelial Adhesion in Venules

P-selectin SuppressionP-selectin mediates the rolling interaction between neu-trophils and endothelial cells But in SHR the adhesion of

C HAPTER 105 Capillary and Venular Responses to Arterial Hypertension 717

leukocytes to microvascular endothelium induced by

inflammatory mediators under physiologic blood shear rates

is reduced (Suematsu et al., 1995) The downregulation of

leukocyte adhesion appears to involve both leukocyte- and

endothelial cell-dependent mechanisms The SHR has

reduced levels of P-selectin on the endothelial membrane of

post-capillary venules There is also a reduction of the sialyl

Lewis X-like carbohydrate structure on the leukocytes

Attenuation of leukocyte rolling has two important

consequences

• The SHR has a chronically elevated count of circulating

leukocytes with enhanced levels of free radical production

and cytoplasmic degranulation The elevation in the

num-ber of circulating neutrophils and monocytes may result

from demargination of these cells in postcapillary venules

by suppression of the selectin-dependent membrane

inter-action A similar leukocytosis is encountered in P-selectin

gene knockout mice with diminished ability for leukocyte

rolling on venular endothelium

• The SHR exhibits a diminished sensitivity to

inflamma-tory stimuli relative to WKY rats and consequently enjoys

a surprising protection against inflammatory mediators

The suppression of a P-selectin mediated adhesion

path-way may compromise normal leukocyte response under

physiological fluid shear conditions and early steps in

tis-sue and lesion repair

I-CAM UpregulationEndothelial ICAM-1 expression under both constitutive

and induced conditions is upregulated in SHR in splanchnic

organs but not necessarily in heart or skeletal muscle

Cir-culating leukocytes adhere to and spread on endothelium

with ICAM-1 overexpression under conditions of reduced

fluid shear stress and thereby cause a selectin-independent

margination of leukocytes

In contrast to the resistance to inflammation, the SHR is

more vulnerable than the WKY rat to hemorrhagic

hypoten-sion or acute ischemia and reperfuhypoten-sion (Cerwinka and

Granger, 2001) Enhanced numbers of activated leukocytes

trapped in the microcirculation during ischemia are

associ-ated with increased organ injury and reduced survival Once

exposed to hemorrhagic shock, activated neutrophils in the

circulation are trapped in microvessels of the SHR and

expose the tissue to greater oxidative stress than in the WKY

rat The SHRs display a greater extent of microvascular

pro-tein leakage upon ischemia-reperfusion than WKY

Lymphocyte Apoptosis

The SHR has an atrophied thymus and suppressed

indices of immune function with extensive lymphocyte

apoptosis Adrenalectomy in the SHR reduces apoptotic

death rates of lymphocytes in the thymus Supplementation

with a glucocorticoid enhances the apoptosis in the thymus

and several other organs The process may involve DNAbinding of the glucocorticoid receptor Glucocorticoids dis-rupt mitochondrial transmembrane potentials, depletenonoxidized glutathione levels, increase the production ofreactive oxygen species, elevate cytosolic free Ca2 +levelsand produce nuclear and cytoplasmic shape changes There

is evidence for oxygen free radical involvement in earlyapoptosis of dexamethasone-treated splenocytes and SHRsmooth muscle cells

The Glucocorticoid Pathway in the Spontaneously

Hypertensive Rat

We have seen that several diverse microvascular defects

in the SHR model of hypertension depend on coids These defects include capillary apoptosis andmicrovascular rarefaction, apoptosis of SHR thymocytesand lymphocytes, impaired leukocyte-endothelial interac-tion in post-capillary venules with central leukopenia,enhanced levels of xanthine oxidase and reactive oxygenspecies, and last not least increased blood pressure with ele-vated arterial tone Adrenalectomy serves to normalize theblood pressure in the SHR and attenuates most of themicrovascular abnormalities encountered in the SHR whilesupplementation with glucocorticoids restores the hyperten-sive state The response in the adrenalectomized WKY rats

glucocorti-at equal levels of glucocorticoids is significantly lower than

in the SHR The SHR suffers from a greatly increasedresponse to adrenal glucocorticoids as well as mineralocor-ticoids There is currently no conclusive evidence that theadult SHR has increased levels of glucocorticoids, although

it has in the mesentery microcirculation significantly vated density of glucocorticoid and mineralocorticoid recep-tors Glucocorticoids modulate phosphorylation of theinsulin receptor, and may be involved in the insulin resist-ance of the SHR, forming one of the links between hyper-tension and diabetes

ele-Conclusion

Hypertensives have a number of microvascular defects,which are independent of the arterioles, and affect the capil-lary and venular segment of the microcirculation, vesselsthat are not exposed to elevated blood pressure Thesedefects expose hypertensives to an enhanced risk for organinjury We still have little evidence to suggest that the arte-rial blood pressure elevation per se induces vascular lesionssimilar to those encountered in hypertension It appears that

a co-factor exists that serves to enhance cell activation in thecirculation In the SHR the glucocorticoid pathway may beinvolved in production of reactive oxygen in endothelium,apoptosis and capillary rarefaction, inhibition of leukocyteadhesion to postcapillary venules, and suppression of theglucose receptor These are indications of a microvascular

inflammatory response, which compared to that in

nor-motensives is blunted by the suppression of leukocyte

rolling and adhesion to the postcapillary venules The

enhanced organ injury in hypertension may be associated

with microvascular apoptosis

Acknowledgements

Supported by NIH Grant HL-10881 I thank Drs Makoto Suematsu,

Hidekazu Suzuki, Fred Lacy, Allan Swei, Camille Vogt, and Dale Parks for

numerous discussions and inspirations Special thanks to Frank A DeLano

for the assistance with Figure 2.

References

Boegehold, M A (1991) Effect of salt-induced hypertension on

microvas-cular pressures in skeletal muscle of Dahl rats American Journal of

Physiology 260, H1819–H1825.

Cerwinka, W H., and Granger, D N (2001) Influence of

hypercholes-terolemia, and hypertension on ischemia-reperfusion induced

P-selectin expression Atherosclerosis 154, 337–344.

Helmke, B P., Bremner, S N., Zweifach, B W., Skalak, R., and

Schmid-Schönbein, G W (1997) Mechanisms for increased blood flow

resist-ance due to leukocytes Am J Physiol 273, H2884–H2890.

Hutchins, P M., and Darnell, A E (1974) Observations of a decreased

number of small arterioles in spontaneously hypertensive rats Circ.

Res 34–35, 161–165.

The report contains the first quantitative assessment of microvascular

rar-efaction in arterial hypertension after repeated qualitative descriptions of

the phenomenon in hypertensive patients.

Imai, T., Morita, T., Shindo, T., Nagai, R., Yazaki, Y., Kurihara, H.,

Suematsu, M., and Katayama, S (2001) Vascular smooth muscle

cell-directed overexpression of heme oxygenase-1 elevates blood pressure

through attenuation of nitric oxide-induced vasodilation in mice Circ.

Res 89, 55–62.

Kubes, P., Suzuki, M., and Granger, D N (1991) Nitric oxide: An

endoge-nous modulator of leukocyte adhesion Proc Natl Acad Sci USA 88,

4651–4655.

The report summarizes the first detailed documentation of the inflammatory reaction after blockade of NO synthesis in the microcirculation.

Prewitt, R L., Chen, I I H., and Dowell, R F (1982) Development of

microvascular rarefaction in the spontaneously hypertensive rat Am J.

Physiol 243, H243-H251.

Suematsu, M., Suzuki, H., Tamatani, T., Iigou, Y., DeLano, F A., Miyasaka, M., Forrest, M J., Kannagi, R., Zweifach, B W., Ishimura, Y., and Schmid-Schönbein, G W (1995) Impairment of selectin-mediated leukocyte adhesion to venular endothelium in spontaneously hyperten-

sive rats J Clin Invest 96, 2009–2016.

An examination of the molecular mechanisms for attenuation of leukocyte adhesion to the endothelium in postcapillary venules of the SHR Suzuki, H., DeLano, F A., Parks, D A., Jamshidi, N., Granger, D N., Ishii, H., Suematsu, M., Zweifach, B W., and Schmid-Schönbein, G W (1998) Xanthine oxidase activity associated with arterial blood pres-

sure in spontaneously hypertensive rats Proc Natl Acad Sci USA 95,

4754–4759.

Vogt, C J., and Schmid-Schönbein, G W (2001) Microvascular lial cell death, and rarefaction in the glucocorticoid-induced hyperten-

endothe-sive rat Microcirculation 8, 129–139.

Zweifach, B W., Kovalcheck, S., DeLano, F A., and Chen, P (1981) Micropressure-flow relationship in a skeletal muscle of spontaneously

hypertensive rats Hypertension 3, 601–614.

This report summarizes a comprehensive set of micro-hemodynamic urements in young, and old SHR skeletal muscle.

meas-Capsule Biography

Dr Schmid-Schönbein has headed the Microcirculation Laboratory at the University of California San Diego since 1979 President of the Micro- circulatory Society in 2003, his laboratory primarily focuses on cell mechanics, cell activation, mechanisms of inflammation, and tissue injury His work is supported by grants from the NIH and NSF.

S ECTION J

Inflammation

C HAPTER 106

Free Radicals and Lipid Signaling

in Microvascular Endothelial Cells

Peter B Anning and Valerie B O’Donnell

Department of Medical Biochemistry, Cardiff University, Heath Park, Cardiff, Wales

tutive (PGHS-1: stomach, gut, kidney, platelets) andinducible (PGHS-2: fibroblasts, macrophages) isoforms.Synthesis involves a two-step conversion of arachidonicacid First, the enzyme oxidizes arachidonic acid to a cyclicendoperoxide, prostaglandin-G2(PGG2), by a cyclooxyge-nase activity; then a peroxidase reduces the peroxide to ahydroxide, yielding the endoperoxide, prostaglandin-H2(PGH2)

Biochemically, PGHS-1 and -2 are very similar, with 60percent sequence homology, identical reaction mechanisms,superimposable x-ray crystal structures, and the same sub-cellular localization at the endoplasmic reticulum andnuclear membrane However, PGHS isoforms function astwo independent prostaglandin synthesis systems utilizingdifferent cellular arachidonate pools in the same cell type,and with very different patterns of expression control

PGHS in Vascular Disease

In the vasculature, PGHS isoforms regulate vascularhomeostasis through generation of PGH2, the precursor forprostacyclin (PGI2, endothelial) or thromboxane (TXA2,platelets) PGHS is transiently activated in platelets orendothelial cells by agonists, such as thrombin, collagen(platelets), bradykinin, or acetylcholine (endothelium) Fol-lowing this, the PGH2 is rapidly converted into PGI2 orTXA2by the CYP enzymes, prostacyclin synthase or throm-boxane synthase, respectively Platelet PGHS-1 is the pri-mary source of plasma TXA2 in both healthy humans andpatients with vascular disease, whereas endothelial PGHS-2

is the major source of PGI2 These eicosanoids have ing effects, with PGI2being vasodilatory and an inhibitor ofplatelet activation via elevating cAMP, and TXA2causingvasoconstriction and platelet activation (Figure 2)

oppos-Lipoxygenases, Prostaglandin H Synthases and

Cytochrome P450s in endothelium

Endothelial cells (ECs) express several enzymes that

oxi-dize unsaturated lipid to signaling mediators These include

both constitutive and inducible isoforms of prostaglandin H

synthases (PGHS), lipoxygenases (LOX), and cytochrome

P450 (CYP), with the levels of expression and isoform type

being dependent on the tissue of origin and inflammatory

state of the cells The healthy endothelium generates a

num-ber of oxidized lipid mediators including prostacyclin

(PGI2) and epoxyeicosatetraenoic acids (EET) Following

an inflammatory challenge, the properties of the

endothe-lium alter with a switch from generation of vasoprotective

mediators, to formation of factors that can potentiate the

inflammatory response, including cysteinyl leukotrienes and

hydroxyeicosatetraenoic acids (HETEs) (Figure 1) The

pre-dominant substrate utilized by all these pathways is

arachi-donate, hydrolyzed from the sn2 position of phospholipids

by phospholipase A2, in response to agonist activation

Following release, it undergoes enzymatic oxidation and

isomerization forming a complex variety of signaling

medi-ators that either are released to signal in adjacent cells or

sig-nal intracellularly in the endothelium itself The following

sections will describe each signaling pathway focusing in

particular on their expression and function in the

microvas-cular endothelium and biological actions of their lipid

prod-ucts on EC themselves

Prostaglandin H Synthases-1 and -2 in ECs

Enzymology of PGHS-1 and -2

Prostaglandins (PGs) are predominantly generated

through the action of PGHS, of which there are both

consti-Copyright © 2006, Elsevier Science (USA).

The formation of PGHS-derived prostaglandins,

includ-ing TXA2, PGI2, and isoprostanes, is markedly elevated in

vascular disease For example, urinary 8-epi-prostaglandin

F2ais increased 130 percent in hypercholesterolemia Also,

isoprostanes are present in human atherosclerotic lesions

along with PGHS-1 and -2

Endothelial Expression of PGHS Isoforms

It has long been considered that PGI2 is the main

prostanoid synthesized by ECs, and TXA2 the main

prostanoid from platelets However, cultured human

umbil-ical vein endothelial cells (HUVECs) and lung

microvascu-lar and cerebral ECs express PGHS-1 constitutively, with

this enzyme being the major source of EC-derived PGH2

precursor for low-level TXA2synthesis in HUVECs Basal

expression of PGHS-2 is low or absent in most ECs, but

following stimulation with a number of mediators [including laminar flow, HIV-infected monocytes, platelet-derived TXA2, hypoxia, interleukin (IL)-1b, tumor necrosis

factor-a (TNFa), fibroblast growth factor, phorbol ester,

lipopolysaccharide (LPS) or vascular endothelial growthfactor (VEGF)], its upregulation through an immediate earlygene leads to generation of PGI2and PGE2in a number ofmicrovascular EC types (including human pulmonary, cere-bral, and atherosclerotic) Interestingly, IL-1b induces PGI

synthase and PGE synthase in tandem with PGHS-2, but not TX synthase It is therefore likely that the PGHS-2-dependent generation of PGI2in vivo in both healthy peopleand patients with vascular disease requires continuous stim-ulation of gene expression, for example by laminar flow orproinflammatory cytokines In contrast to HUVECs, PGHS-

2 is a significant source of TXA2 generated by humanmicrovascular endothelial cells, which can inhibit migration

Figure 1 Generation of bioactive eicosanoids by healthy and inflammatory-activated endothelium lowing its hydrolysis from the membrane by phospholipase A2(PLA2), arachidonate is oxidized to prostacy- clin (PGI2) or epoxyeicosatetraenoic acid (EET) by prostaglandin H synthase (PGHS) (see color insert)

Fol-Figure 2 Localization of PGHS isoforms in vascular cells Platelets contain PGHS-1, which forms prostaglandin H2(PGH2) and is subsequently metabolized by thromboxane synthase (TXS) to thromboxane A2(TXA2) Endothelial cells contain PGHS-1 and -2, both of which are responsible for providing PGH2for PGI2synthesis by PGI synthase (see color insert)

C HAPTER 106 Free Radicals and Lipid Signaling in Microvascular Endothelial Cells 723

and angiogenesis in vitro The in vivo importance of this is

unclear, however, since platelet PGHS-1 is the major source

of TXA2in healthy people PGHS-2 is also negatively

reg-ulated at the transcriptional level in ECs For example,

aspirin, sodium salicylate, or nitric oxide inhibits IL-1b-,

phorbol-, or LPS-induced PGHS-2 expression in HUVECs

and bovine pulmonary artery endothelial cells

Although PGHS-1 is expressed constitutively by a

num-ber of EC types, its expression is also controlled by

tran-scriptional regulation For example, upregulation of PGI2

synthesis in intrapulmonary vessels rises markedly during

late fetal life, because of a developmental increase in

PGHS-1 expression that occurs via estrogen stimulation of the

estrogen receptor This may also have implications for

PGHS-1 expression in pre- and postmenopausal women

where risk of vascular disease increases with decreased

estrogen levels, and estrogen replacement is associated with

decreased cardiovascular risk

Regulation of EC Function by PGHS Products

Endothelial cell function is regulated in several ways

through PGHS signaling (Figure 3) In particular, recent

data have implicated the prostaglandin 15-deoxy-

d(12,14)-prostaglandin J2 (15d-PGJ2) in mediating multiple

responses through activating peroxisome

proliferator–acti-vated receptors (PPARs) These are members of the nuclear

receptor superfamily of transcription factors that are

impor-tant mediators of the inflammatory response Through this

pathway, 15d-PGJ2 activation of endothelial PPARs inhibits

leukocyte–endothelial interactions, IFNg-induced

expres-sion of CXC chemokines, and TNF-induced oxidized LDL

receptor (LOX-1) and induces stress proteins including

heme oxygenase and plasminogen activator inhibitor type-1

(PAI-1) in a number of ECs (including brain microvascular)

15d-PGJ2 also signals in a PPAR-independent manner in

ECs, inducing apoptosis and synthesis of GSH and IL-8

In addition to 15d-PGJ2, additional prostaglandins thatsignal in ECs include PGE2, which induces expression of P-selectin, VEGF, and endothelial nitric oxide synthase(eNOS) through activation of ERK/JNK2 signaling path-ways, and PGD2, which can relax vessels through stimula-tion of eNOS activity in bovine coronary arteries (Figure 3)

In summary, PGHS isoforms expressed in ECs regulatenormal vascular function and participate in the pathophysi-ology of vascular disease In addition, PGHS products gen-erated by adjacent cells are important in regulatingnumerous microvascular EC functions, including apoptosis,integrin expression, and eNOS activity

Lipoxygenases in ECs

Enzymology of LipoxygenasesLipoxygenases (LOX) are nonheme iron-containingenzymes that catalyze arachidonate or linoleate oxidation toform a series of lipid hydroperoxides In mammalian cells,several isoforms are known, named by their position of oxy-gen insertion into arachidonate Lipoxygenases contain asingle nonheme iron that alternates between Fe2+and Fe3+during catalysis Resting enzyme predominantly exists asthe reduced form, requiring oxidation by hydroperoxidesbefore dioxygenation can occur

Vascular and Endothelial Expression of

LOX IsoformsLOX enzymes are predominantly expressed by leuko-cytes (5- and 15-LOX in humans, rabbits, 12/15-LOX inmice, rats, pigs) and platelets (12-LOX) Under basal condi-tions, ECs do not appear to express significant LOX protein.However, increased protein expression of 5-LOX has beenreported in pulmonary artery ECs of patients with primarypulmonary hypertension, in hypoxic rats, and in antigen-

Figure 3 Effects of PGHS products on endothelial cell function A series of PGHS metabolites have potent biological effects on endothelial cell function PPAR, peroxisomal proliferator activating receptor; HO, heme oxygenase; VEGF, vascular endothelial growth factor; 15 d-PGJ2, 15-deoxy- d (12,14)-prostaglandin J2; PAI-1, plasminogen activator inhibitor type-1 (see color insert)

challenged mice This suggests 5-LOX may be upregulated

in inflammatory-activated endothelium, although the

mech-anisms involved are unknown Finally, although functional

5-LOX protein is not expressed in ECs, leukotrienes can be

generated by HUVECs and pig aortic ECs following

in-tercellular transfer of LOX products from associated

granulocytes It is unknown whether similar generation of

leukotrienes can occur in microvascular ECs following

granulocyte transfer of precursors

Regulation of EC Function by LOX Products

LOX products stimulate a variety of EC functions In

particular an important role for platelet 12-LOX expressed

by tumor cells in regulating ECs in cancer microvessels is

emerging since 12-LOX can stimulate proliferation,

migra-tion, and tube differentiation in vitro and angiogenesis in

vivo In addition, 12-HETE upregulates expression of av b3

integrin on microvascular ECs, which is required for

angio-genesis of breast cancer, whereas biosynthesis of 12-HETE

by B16 melanoma cells is a determinant of their metastatic

potential Finally, 12-HETE can stimulate monocyte

endothelial interactions following incubation of ECs with

high glucose or minimally oxidized low-density lipoprotein,

suggesting a role for LOX activation of EC in inflammatory

vascular disease (Figure 4)

In summary, expression of LOX in most EC is low or

absent under normal conditions Upregulation in vivo

fol-lowing inflammatory challenge may result in generation of

low amounts of HETEs, but it is unclear whether this is of

biological significance In contrast, generation of LOX

products by adjacent cells including leukocytes and tumor

cells is centrally involved in regulating microvascular EC

function under pathophysiological conditions

CYP Enzymes in ECs

CYP enzymes are a ubiquitously expressed family of

heme enzymes that play central roles in xenobiotic

metabo-lism and lipid oxidation CYP-dependent arachidonate dation occurs through three pathways, allylic oxidation,omega hydroxylation, and olefin epoxidation These result

oxi-in a series of oxygenated metabolites, oxi-includoxi-ing epoxidesand fatty acid alcohols

Nonhepatic cytochrome P450 arachidonate metabolitesact as intracellular signaling molecules in vascular tissue(Figure 5) The major EC CYP isoforms are prostacyclinsynthase (PGI synthase) and thromboxane synthase (TXS),which generate prostacyclin (PGI2) or thromboxane A2(TXA2), respectively, from PGHS-derived PGH2(describedearlier) Both enzymes are controlled through transcrip-tional regulation, although the pathways are not well char-acterized For example, TXS is inducible in pig aortic ECs

by xenoreactive antibodies, whereas IL-1b elevates PGI

synthase in tandem with PGHS-2 in HUVECs

Additional EC-derived CYP products include the ides, 11,12-epoxyeicosatetraenoic acid (EET) and 5,6-EET,and dihydroxyeicosatrienoic acids (DHET) 11,12-EET isavidly esterified into endothelial phospholipid pools andmediates vascular relaxation, possibly accounting for acomponent of endothelial-derived hyperpolarizing factor(EDHF) activity Preformed EETs in endothelial membranescan influence vascular function by altering membrane char-acteristics, ion transport, or lipid-dependent signaling path-ways For example, 5,6-EET mediates vasodilation by eitherincreasing nitric oxide production through stimulating Ca2 +influx into ECs, including rat cerebral microvessels, or bydirectly activating smooth muscle Kca channels A finalimportant vasoactive CYP product, 20-HETE is generated

epox-by CYP 4A and promotes renal vasoconstriction However,this is generated by smooth muscle, rather than ECs

Generation of Free Radical Species by PGHS or LOX and CYP

Lipid peroxidation enzymes generate free radical mediates during catalysis For example, both PGHS andLOX form enzyme-bound lipid alkyl (L•) and peroxyl

inter-Figure 4 Signaling properties of 12-HETE in endothelial cells The LOX product 12-HETE induces multiple biological effects in endothelium (see color insert)

C HAPTER 106 Free Radicals and Lipid Signaling in Microvascular Endothelial Cells 725

(LOO•) radicals that are ultimately converted into

hydroper-oxides (LOOH) before release from the active site At low

O2tension, a small proportion of lipid radicals (up to 10%)

escape the active site These react with O2 at

diffusion-controlled rates to form free LOO•, which can then

propa-gate secondary nonenzymatic lipid peroxidation During

this, a proportion of racemic products is formed This

reac-tion may be a significant source of LOOH in late

athero-sclerosis where lipid peroxidation product specificity is lost

Although they do not directly bind or activate O2, PGHS

and LOX can generate O2• - through secondary side

reac-tions involving oxidation of certain peroxidase substrates In

these reactions, substrates including NAD(P)H and GSH are

oxidized to radicals [i.e., NAD(P)• and GS•, respectively]

that can ultimately react with O2either directly, or indirectly

forming O2• - To date, these reactions have only been

observed using purified enzyme and it is unknown whether

they contribute to free radical levels in intact cells or tissue

Finally, it has been suggested that CYP2C9 is a significant

source of reactive oxygen species in porcine coronary

arter-ies that may play a role in regulating vascular tone

Regulation of PGHS, LOX, and CYP by

Reactive Oxygen and Nitrogen Species

Lipid oxidation enzymes are regulated in several ways

through the action of reactive oxygen and nitrogen species

In general, enzyme turnover is activated by oxidation [e.g.,

for LOX or PGHS by LOOH, H2O2, or peroxynitrite

(ONOO-)] and inhibited by reduction (e.g.,

nordihy-droguairetic acid and baicalein as LOX inhibitors, or

removal of LOOH or H2O2 by glutathione peroxidase or

catalase-dependent reduction)

Nitric oxide (NO) inhibits LOX turnover through

scav-enging the enzyme-bound LOO•, but exerts no direct effect

on PGHS turnover in vitro The lack of effect on PGHS

turnover is intriguing since NO can interact with this enzyme

in multiple ways including scavenging of the catalytic syl radical and acting as a reducing peroxidase substrate Incontrast to its lack of effect on purified PGHS, NO has mul-tiple and often contradictory effects on PGHS expressionand activity in intact cells In several systems (includingpurified recombinant COX-2, intact platelets, endothelialcells, RAW-264.7 cells, an ex vivo model of renal inflam-mation, and following in vivo administration of •NO donors

tyro-to rats), •NO highly stimulates PG production However,other investigators have found •NO either to be inhibitorytowards PGHS, or to have no effect on either PGHS activity(platelets) or LPS-induced expression in RAW-264.7 cells

In some cell types however (rat microglial cells and toneal macrophages), •NO suppresses LPS-induced COX-2expression, resulting in apparent enzyme inhibition Finally,

peri-•NO inhibits CYP through formation of an iron–nitrosylcomplex, and perhaps additional uncharacterized mecha-nisms In rat renal microvessels, this attenuates EET-depend-ent dilation, but conversely inhibits 20-HETE-dependentvasoconstriction through inhibition of CYP4A

Conclusions

Oxidized lipid mediators generated by PGHS, LOX, orCYP are of central importance in the normal physiology ofthe endothelium, with their aberrant generation playing amajor role in the pathogenesis of inflammatory vascular dis-ease In addition, these enzymes generate a small amount oflipid radicals that may propagate nonenzymatic lipid perox-idation, a hallmark of atherosclerotic lesions Althoughmuch is known regarding function and control of these path-ways in ECs (especially PGHS and LOX), others, especiallythe CYP enzymes, are less studied Studying the biologicalroles and signaling pathways of CYP in EC is becoming

a major focus of research in vascular biology and willundoubtedly lead to a fuller understanding of their roles

in both normal homeostasis and vascular pathophysiology

Figure 5 Localization of CYP isoforms in vascular tissue Endothelium contains a number of CYP enzymes that generate bioactive lipid products An additional important isoform is CYP4A, in smooth muscle that generates 20-HETE TXS, thromboxane synthase; EET, epoxyeicosatetraenoic acid; DHET, dihydroxyeicosate- traenoic acid (see color insert)

Finally, although much is known regarding the biological

chemistry and cell biology of these pathways, their relative

importance in vessels of different origin is not clear In

par-ticular, the role of PGHS, LOX, or CYP in control of

vascu-lar tone through regulating vascuvascu-lar function in vascu-large

vessels, resistance vessels, and capillary beds may vary

tremendously Elucidation of tissue-specific functions and

control mechanisms for lipid oxidation pathways in

sub-types of EC is becoming an area of active and fruitful

inves-tigation that will yield major insights into their role in

regulating vascular biology in health and disease

Glossary

Lipoxygenases: Lipid oxidizing enzymes that play important roles in

vascular function and immune regulation There are several mammalian

isoforms, with one in particular (12/15-lipoxygenase) being involved

in vascular dysfunction associated with hypertension, diabetes, and

atherogenesis.

Nitric oxide: Free radical signaling molecule generated by oxidation

of L -arginine by nitric oxide synthases (NOS), which causes smooth

mus-cle relaxation and inhibits platelet and leukocyte activation.

Prostaglandin H synthases: Lipid oxidizing enzymes that generate

prostaglandins, signaling mediators that regulate vessel tone (e.g.,

prosta-cyclin) and platelet aggregation (e.g., thromboxane).

Acknowledgments

Research funding from the Wellcome Trust and British Heart

Founda-tion is gratefully acknowledged.

Bibliography

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syn-thase: Structure and catalysis Biochim Biophys Acta 1083, 1–17 This

is an excellent review on PGHS isoforms, focusing mainly on the chemistry and enzymology of these pathways.

bio-LIPOXYGENASES Feinmark, S J., and Cannon, P J (1986) Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthe-

sised by polymorphonuclear leukocytes J Biol Chem 261,

16466–16472.

Kühn, H., Belkner, J., Zaiss, S., Fahrenklemper, T., and Wohlfeil, S (1994).

Involvement of 15-lipoxygenase in early stages of atherogenesis J.

Exp Med 179, 1903–1911.

Liu, B., Marnett, L J., Chaudhary, A., Ji, C., Blair, I A., Johnson, C R.,

Diglio, C A., and Honn, K V (1994) Biosynthesis of

12(S)hydroxye-icosatetraenoic acid by B16 amelanotic melanoma cells is a

determi-nant of their metastatic potential Lab Invest 70, 314.

Zhang, Y Y., Walker, J L., Huang, A., Keaney, J F., Clish, C B., Serhan,

C N., and Loscalzo, J (2002) Expression of 5-lipoxygenase in

pul-monary artery endothelial cells Biochem J 361, 267–276.

CYTOCHROMEP450

Campbell, W B., Gebremedhin, S., Pratt, P F., and Harder, D R (1996) Identification of epoxyeicosatrienoic acids as endothelium-derived

hyperpolarizing factors Circ Res 78, 415–423.

Capdevila, J H., Falck, J R., and Estabrook, R W (1992) Cytochrome

P450 and the arachidonate cascade FASEB J 6, 731–736 This is an

excellent review on the enzymology of CYP oxidation of arachidonate

to bioactive mediators Although it may be a little out-of-date, it still is

a very useful starting point for the reader new to the area.

Rosolowski, M., and Campbell, W B (1996) Synthesis of icosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cul-

hydroxye-tured bovine artery endothelial cells Biochim Biophys Acta 1299,

267–277.

Capsule Biography

Drs Anning and O’Donnell are based at Cardiff University, UK Their work focuses on lipoxygenase and nitric oxide signaling in the vasculature and is funded by the British Heart Foundation and Wellcome Trust.

C HAPTER 107

Eicosanoids

Monica M Bertagnolli

Brigham and Women’s Hospital, Department of Surgery, Boston, Massachusetts

Converting Membrane Components into

Signaling Molecules

IntroductionUpon cell stimulation, arachidonic acid is released fromthe plasma membrane through the activation of phospholi-pase A2 (PLA2) Further metabolism of arachidonic acid,and the eventual composition of resulting eicosanoids,depends upon the availability of enzymes responsible forarachidonic acid metabolism within a specific cell Theseenzymes may be classified into three major groups, includ-ing the cyclooxygenases, lipoxygenases, and P450-monooxygenases (Figure 1)

The products of arachidonic acid metabolism exert a vastrange of downstream effects on cell signaling pathways Theprimary mode of eicosanoid action is through specific Gprotein—coupled receptors In the highly complex network

of cell signaling, these mediators influence many differentsystems, including those governing cell proliferation anddifferentiation (e.g., MAP kinase and PPARs), cytoskeletaldynamics (e.g., Rho GTPases), apoptosis (e.g., Akt and

PI3K), ion transport (e.g., Ca2 +channels), and many others.Some of the eicosanoid downstream signaling pathways,such as those involved in inflammation, have been exten-sively studied Others, such as the effects of metabolic path-ways mediated by different cyclooxygenase isoforms, haveonly recently come under intense scrutiny

The Cyclooxygenase PathwayProstaglandins and thromboxanes are bioactive sub-stances that result from the metabolism of arachidonic acid

by cyclooxygenases These molecules are produced by mostcells in the body and act as autocrine and paracrine media-tors of a diverse range of cell functions, including pain

Introduction: History and Definitions

Eicosanoids (from the Greek eicosa, “twenty”) are a

large family of inter- and intracellular signaling molecules

derived from arachidonic acid, a fourfold unsaturated C20

fatty acid sequestered in membrane phospholipids

Eicosanoid production is tightly controlled by mediators

of membrane lipid mobilization, and by the cellular

con-centrations and activities of the enzymes involved in

their metabolism Eicosanoids are capable of mediating a

great variety of cellular functions, including processes as

seemingly diverse as vascular contractility, inflammatory

response, protection of the gastric mucosa, and renal

func-tion Eicosanoids are rapid responders to external stimuli,

and in keeping with this role, they are not stored within the

cell, but are rapidly synthesized and then quickly degraded

as a result of spontaneous hydrolysis or enzyme-mediated

inactivation

The first eicosanoids identified were members of a

cate-gory known as prostaglandins Prostaglandins were

discov-ered in the 1930s when a reproductive physiologist, von

Euler, observed that a substance in human semen induced

contraction of uterine smooth muscle Because he assumed

that the active agent was produced by the prostate gland,

he named this substance “prostaglandin.” Members of the

leukotriene family were first identified in the early 1940s as

a result of their effects as mediators of anaphylaxis In 1969,

Piper and Vane showed that aspirin inhibited vasoactive

substances produced by rabbit aorta, and in 1971, Vane

dis-covered that prostaglandins were the target of nonsteroidal

anti-inflammatory drug (NSAID) activity Samuelsson

iden-tified thromboxanes as distinct products in 1979 In the

lat-ter half of the 20th century, the recognition of eicosanoids as

important mediators of both normal and pathologic

physio-logical responses has added a wealth of data to this

increas-ingly complex field

Copyright © 2006, Elsevier Science (USA).

generation, vasomotor tone, vascular permeability, febrile

response, and uterine contractility Stimulation of the cell by

growth factors, cytokines, or mechanical trauma leads to

mobilization of arachidonic acid from the phospholipid

membrane, followed by cyclooxygenase-mediated

conver-sion of arachidonic acid to a short-lived intermediate,

prostaglandin H2 (PGH2) This molecule is then modified

by specific enzymes to produce a variety of bioactive

sub-stances (Table I) that share a similar chemical structure

(Figure 2) Cell-specific expression of arachidonic

metabo-lites exists as a result of differential expression of both

downstream metabolizing enzymes and receptor isoforms

For example, epithelial cells contain prostaglandin

syn-thetase, leading to the production of prostaglandin E2

(PGE2), platelets contain thromboxane synthetase and

there-fore produce thromboxane A2(TxA2), and endothelial cells

produce prostaglandin I2 (PGI2), also known as

prostacy-clin, through the activity of prostacyclin synthase There are

at least nine known prostaglandin receptor forms, conveying

an additional level of tissue specificity to

prostaglandin-mediated activities Four of the receptor subtypes bind PGE2

(EP1-EP4), two bind PDG2(DP1, DP2), and separate

recep-tors bind PGF2 a (FP), PGI2 (IP) and TxA2 (TP) Thesereceptors are transmembrane G protein—coupled proteinslinked to a number of different signaling pathways In com-plex tissues, receptors for a wide variety of prostaglandinsare present on the surface of various components, such asepithelial cells, stromal fibroblasts, stromal endothelialcells, and inflammatory cells

Until 1991, only one form of cyclooxygenase was nized This family of enzymes is now known to contain atleast two forms, each with distinct roles in tissue regulation(Table II) Cyclooxygenase-1 (Cox-1) is constitutivelyexpressed in the gastrointestinal mucosa, kidneys, platelets,and vascular endothelium and is responsible for mainte-nance of normal physiologic function of these tissues.Cyclooxygenase-2 (Cox-2) was identified in the early 1990s

recog-as a distinct enzyme recog-associated primarily with tion Cox-2 is the product of an intermediate-early responsegene whose tissue expression is increased 20-fold in re-sponse to growth factors, cytokines, and tumor promoters.Cox-2 is not found in significant quantities in the absence ofstimulation, which explains why it remained undetected as adistinct molecule for 20 years

inflamma-The Lipoxygenase PathwayLipoxygenases convert arachidonic, linoleic, and otherpolyunsaturated fatty acids into biologically activehydroperoxy derivatives that modulate cell signaling Inmammals, lipoxygenases are classified according to theirpositional specificity for fatty acid oxygenation, and aretherefore designated as 5-, 8-, 12-, or 15-lipoxygenase The12- and 15-lipoxygenases are further differentiated accord-

ing to whether they are derived from platelets (12-S-LOX), epidermis (12-R-LOX and 15-LOX-2), or reticulocytes

(12-LOX-1)

The physiologic effects of lipoxygenase metabolites havenot been characterized as extensively as those produced bycyclooxygenase activity The 5-, 8-, and 12-LOX isoformsresult in production of hydroxyeicosatetraenoic acids

(HETEs), including 5-S-HETE, LTB4, 8-S-HETE, and

12-HETE (Figure 3) In general, these metabolites increase cell proliferation, contribute to inflammatory changes, and

Plasma membrane phospholipids

Phospholipase A 2

Epoxy-eicosanoids Hydroxy-eicosanoids

HETEs, leukotrienes Prostaglandins

PGE2 Vasodilatation, bronchodilatation, inhibition of gastric acid

secretion, gastric mucosal protection, hyperalgesia, pyrexia,

increased uterine contractility

PGD2 Vasodilatation, regulation of renal blood flow, pulmonary

artery constriction, bronchoconstriction

PGF2a Pulmonary artery constriction, bronchoconstriction, increased

uterine contractility

PGI2 Vasodilatation, inhibition of platelet aggregation

TxA2 Vasoconstriction, bronchoconstriction, promotion of platelet

aggregation, increased membrane permeability, neutrophil

activation

C HAPTER 107 Eicosanoids 729

promote angiogenesis The 15-LOX isoenzymes

(15-LOX-1, 15-LOX-2) convert arachidonic acid to 15-S-HETE and

linoleic acid to 13-S-hydroxy-9,11-octadecadienoic acid

(13-S-HODE) These substances may have effects opposite

to those of the 5-, 8-, and 12-LOX, as they induce epithelial

cell differentiation and promote both growth inhibition and

cell apoptosis

Leukotrienes are a family of paracrine mediators derived

from oxidative metabolism of arachidonic acid by 5-LOX

Leukotriene B4 (LTB4) is a powerful chemoattractant,

responsible for the recruitment of leukocytes to sites of

inflammation 5-LOX is found primarily in

inflam-matory cells, such as granulocytes, monocytes, and mast

cells Leukotriene receptors include B-LT1, a high-affinity

receptor present on leukocytes, and B-LT2, a

moderate-affinity receptor that has widespread tissue distribution

A cysteinyl leukotriene receptor, CysLT1, is found on

smooth muscle cells of the bronchioles and on vascular

endothelial cells

Products of P450 Monooxygenases

Cytochrome (CYP) P450s are a large family of enzymes

present in virtually all mammalian tissues These enzymes

have a variety of roles and have been most extensively

stud-ied for their ability to metabolize various exogenous

sub-stances such as xenobiotics, as well as a vast variety of

drugs A number of CYP P450s employ arachidonic acid

and other fatty acids as substrates, resulting in the

genera-tion of eicosanoids The only fatty acid—utilizing CYP to be

extensively studied is the CYP4A subfamily The CYP4Aepoxygenase metabolizes arachidonic acid and linoleic acid

to a set of compounds known as epoxyeicosatrienoic acids(EETs) The EETs are further metabolized by CYP4A to 19-and 20-hydroxylepoxyeicosatrienoic acids (HEETs) Thesemediators are vasodilators and modulators of intracellular

Ca2 +, Na+, and K+transport

Keeping the Balance: Implications for

Health and Disease

Vascular Effects

REGULATION OFBLOODFLOWBecause of their ability to modulate the balance betweenvasoconstriction and vasodilatation, eicosanoids provide ahighly responsive mechanism for regulating organ and tis-sue blood flow An excellent example of this is the effect ofPGE2on the ductus arteriosus Fetuses have high circulatinglevels of PGE2, and in the 1970s, experiments on fetal lambsshowed that the vasodilatatory effects of PGE2are responsi-ble for the maintenance of ductus arteriosus patency inutero After birth, PGE2 levels decrease dramatically, aresponse associated with closure of the ductus arteriosus andestablishment of postnatal patterns of pulmonary arteryblood flow Because they inhibit PGE2production, NSAIDssuch as indomethacin are used to induce ductus closure inlow-birth-weight infants who have persistent patency of theductus arteriosus Conversely, the synthetic agent, PGE1, isadministered to infants when maintenance of a patent ductusarteriosus is beneficial This situation occurs in newbornswith cardiopulmonary anomalies whose systemic or pul-monary blood flow depends upon shunting between theaorta and the pulmonary artery

ANGIOGENESISAngiogenesis is a process whereby new blood vessels arecreated in response to inducible stimuli This feature of themicrovasculature occurs in a variety of settings, both physi-ologic and pathologic, including chronic inflammation,embryogenesis, parturition, and tumorigenesis Products ofcyclooxygenase activity, including TxA2, PGE2, and PGI2,directly stimulate endothelial cell migration and angiogene-sis in vivo and may result in increased endothelial cell sur-

vival In addition, the product of 12-LOX, 12-S-HETE,

15-LOX-2 12-S-LOX 12-R-LOX 8-LOX 5-LOX

15-S-HETE 12-S-HETE 12-R-HETE 8-S-HETE LTB4

Figure 3 Products of lipoxygenase metabolism (see color insert)

Table II Cyclooxygenases.

Constitutive expression Inducible expression Effects Inhibited by

Cox-1 Gastric epithelium, platelets Rare or none under physiological Pain, platelet activation, Most NSAIDs, including aspirin; with

conditions protection of gastric minimal or no inhibition by Cox-2

Cox-2 Kidney, brain Induced in most tissues by growth Pain, inflammation, fever, Most NSAIDs

factors, inflammatory cytokines, angiogenesis, neurotransmitters, oxidative stress tumorigenesis

possesses activities contributing to angiogenesis, as it

mod-ulates both endothelial cell adhesion and motility In in vivo

studies, selective Cox-2 inhibitors effectively suppressed

formation of new blood vessels in response to basic

fibro-blast growth factor In in vitro model systems employing

coculture of endothelial cells with epithelial tumors,

cyclooxygenase inhibition reduced production of

pro-staglandins and proangiogenic factors and inhibited both

endothelial cell migration and in vitro angiogenesis Related

data also suggested that NSAIDs have antiangiogenic

prop-erties that are independent of cyclooxygenase inhibition As

a result, NSAIDs are currently under study as both

chemo-preventive and cancer therapeutic agents

RENALFUNCTION

In the kidney, eicosanoids are important regulators of

blood flow and glomerular filtration rate Consistent with

this, the predominant eicosanoids produced in the kidney

are PGE2, PGI2, and TxA2 As a component of the body’s

response to stress, the synthesis of eicosanoids by renal

parenchyma and endothelial cells is increased in response to

vasoconstrictive stimuli such as angiotensin, vasopressin, or

catecholamines PGE2 causes vasodilatation of the renal

vasculature, and production of PGE2 in the kidney is an

important compensatory response in patients with shock,

congestive heart failure, or ureteral obstruction

Administra-tion of NSAIDs to patients with these condiAdministra-tions reduces

prostaglandin production and is frequently associated with

impairment of renal function This failure is not associated

with structural damage to the renal parenchyma and is

reversible when the drugs are discontinued In normal

indi-viduals, NSAIDs only rarely cause changes in renal blood

flow or glomerular filtration rate

HEMOSTASIS ANDCOAGULATION

When the endothelium is injured, the resulting exposure

of collagen and thrombin lead to platelet activation and

adhesion to the site of injury Following adhesion, a number

of active substances are released by platelets, including

TxA2 TxA2 contributes to hemostasis by causing local

vasoconstriction, enhancing platelet aggregation, and

mediating further release of TxA2from platelets Although

beneficial following trauma, the vasoconstrictive and

platelet-aggregating effects of TxA2 are highly detrimental

in the setting of atheromatous narrowing of coronary

and cerebral arteries NSAIDs, because of their ability

to inhibit Cox-1, decrease TxA2 synthesis and reduce

platelet function As a result, NSAIDs, particularly aspirin,

are beneficial preventive agents for patients at high risk

of coronary artery and cerebral vascular disease An

overview of randomized trials of aspirin for the prevention

of occlusive vascular disease concluded that 81 to 325 mg

of aspirin daily provided protection against myocardial

infarction, stroke, and death due to cardiovascular disease

This benefit was achieved at a small risk of increased

hem-orrhage and gastrointestinal tract ulceration due to

long-term aspirin use

InflammationThe tissue response to inflammation is characterized byvasodilatation, increased vascular permeability, and earlyneutrophil accumulation This response is largely produced

by the local activity of eicosanoids that are produced byboth damaged tissues and inflammatory cells Cellularrelease of PGI2and PGE2causes vasodilatation, and TxA2,leukotrienes, and histamine all increase vascular permeabil-ity PGE2, together with histamine and bradykinin, producespain at the site of inflammation Neutrophil chemoattractionand activation are caused by TxA2and LTB4, as well as bycomplement activation LTB4 stimulates the synthesis andrelease of inflammatory cytokines, such as tumor necrosisfactor and IL-1, thereby potentiating the inflammatoryresponse

By virtue of their ability to inhibit prostaglandin, boxane, and leukotriene synthesis, NSAIDs markedly attenuate the inflammatory process As a result, naturallyoccurring salicylates have been used for centuries to treatpain and fever The term “nonsteroidal anti-inflammatorydrug” was coined by rheumatologists in 1949 to distinguishthe activity of phenylbutazone from that of glucocorticoids,whose anti-inflammatory properties in the treatment ofarthritis had recently been identified This term came toapply to all “aspirin-like drugs” that were used clinically asantipyretics, analgesics, and anti-inflammatory agents.Recently, Cox-2 has been identified as the inducible isoen-zyme responsible for inflammation Because the beneficialeffects of NSAIDs on the gastric mucosa and kidney aremediated by Cox-1, selective Cox-2 inhibitors were devel-oped to minimize the side effects of NSAIDs while preserv-ing their anti-inflammatory efficacy

throm-Protection of the Gastroduodenal MucosaProstaglandins produced by constitutive activity of Cox-

1 in the upper gastrointestinal tract exert important tive effects in gastroduodenal tissue In the harsh chemicalenvironment of the stomach and duodenum, prostaglandinsare responsible for protection of the mucosa through pro-motion of mucus production, bicarbonate secretion, andmucosal blood flow PGE2also inhibits both basal and stim-ulated gastric acid release This effect may be particularlyimportant in individuals with duodenal ulcer disease

protec-The use of nonselective NSAIDs (i.e., NSAIDs able toinhibit both Cox-1 and Cox-2) can produce damage to themucosa of the stomach and duodenum and increase thecomplication rate of preexisting peptic ulcers Some degree

of gastrointestinal upset is present in approximately 30 cent of patients using nonselective NSAIDs on a regularbasis In addition, endoscopic surveillance of patients usingNSAIDs regularly demonstrates a 20 percent prevalence

per-of gastric ulceration, per-often not associated with dyspepsia.Patients with a prior history of gastroduodenal ulcers are atparticular risk for serious complications, including uppergastrointestinal hemorrhage and perforation Because of

C HAPTER 107 Eicosanoids 731

their specificity for the inducible isoenzyme, selective

Cox-2 inhibitors have a significantly reduced incidence of both

minor and severe gastrointestinal side effects

Regulation of Reproductive Function

The eicosanoids, particularly prostaglandin family

mem-bers, are regulators of many aspects of the reproductive

process PGE2 stimulates LHRH secretion and may also

directly stimulate ovarian follicle maturation PGE2 also

promotes both ejaculation and implantation of the embryo in

the uterine wall PGE2 and PGF2afrom seminal fluid

pro-mote fertility by enhancing transport of sperm into the

fal-lopian tube Eicosanoids also regulate gestational length and

parturition Levels of PGE2, PGF2a, and LTB4 are elevated

in the maternal circulation prior to the onset of spontaneous

labor, and exogenous administration of PGE2 or PGF2a

induces softening of the cervix and uterine contractions in

both full-term and preterm labor Although their use in

preg-nancy is somewhat controversial, both PGE2 and the

syn-thetic prostaglandin misoprostol (PGE1) have been used

successfully for induction of labor

Role in Ischemia—Reperfusion Injury

Eicosanoids are important mediators of the harmful

con-sequences of tissue ischemia and reperfusion Although

decreased tissue perfusion causes compensatory increases in

PGI2levels, ischemia also stimulates thromboxane synthesis

and release Upon reestablishment of blood flow to an

ischemic organ, the ratio of TxA2 to PGI2 is increased,

producing a net vasoconstrictive effect Together with

lipoxygenase metabolites produced during ischemia, TxA2

activates neutrophils, which become sequestered in the

ischemic organ and the lung The products of neutrophil

activation include locally destructive proteases and reactive

oxygen species, as well as inflammatory cytokines that

con-tribute to increased capillary permeability and edema As a

result, tissue injury and decreased capacity for oxygenation

occur both in the ischemic organ and at the diffusional

sur-faces of the lung Leukotrienes produced as a result of

myocardial ischemia can be particularly damaging upon

restoration of coronary blood flow, as these agents may

have negative inotropic and arrhythmogenic effects There

is no one pharmacologic agent able to counteract the

harmful effects of ischemia—reperfusion injury Once

tissue perfusion has been reestablished, pharmacological

therapy focuses upon limiting leukocyte activation and the

resulting tissue damage For example, vasodilatation can be

promoted by nitrates, calcium channel blockers will limit

neutrophil superoxide formation and release, and

angiotensin-converting enzyme inhibitors can prevent

leukocyte adhesion

Eicosanoids and Tumorigenesis

In 1968, Williams recognized that tumors contained

increased levels of prostaglandins compared to adjacent

normal tissue Since that time, data from a wide array of studies suggest that prostaglandins stimulate tumorigenesis.By-products of eicosanoid production include a number

of potentially genotoxic substances, including organic freeradicals, peroxides, and activated oxygen species Thesesubstances are suspected to play a role in every stage of carcinogenesis, including activation of environmental car-cinogens, direct DNA damage, stimulation of proliferation,inhibition of apoptosis, suppression of antitumor immunity,and stimulation of metastasis

The cellular processes responsible for mediated tumorigenesis are incompletely understood Thereare numerous clinical associations and experimental linksbetween inflammation and epithelial cancers Inflammatorybowel disease, burn injuries, chronic ulcers, and long-standing cirrhosis examples of conditions that carry a cancerrisk proportional to their duration in an individual Initiation

eicosanoid-of the inflammatory response activates intracellular signalingcascades that govern cell proliferation and motility Whenthis condition becomes chronic, it provides a setting forselection of cells with other defects in growth control, even-tually producing a clone of cells with a malignant phenotype.Recently, it was recognized that abnormal cell proliferation

in a terminally differentiated epithelial cell population leads

to progressive telomere shortening, resulting eventually inanaphase bridging, chromosomal instability, “telomere crisis,” and the emergence of cells with unlimited prolifera-tive potential due to reactivation of telomerase

An interesting new observation in the field of eicosanoidbiology comes from study of the peroxisome proliferated-activated receptor (PPAR) transcription factors Thesereceptors were initially cloned as a family of orphan recep-tors, but are now known to interact with a wide variety ofligands, including hypolipidemic drugs and the eicosanoids

8-S-HETE, LTB4, and prostaglandins of the J series In thiscapacity, certain eicosanoids resemble steroid and thyroidhormones Cell culture data also suggests that PPARs may

be a target of NSAID activity, although in vivo data firming this have yet to be reported

con-The antitumor effects of NSAIDs have been examined inboth animal models and human clinical trials Many anti-tumor effects have been ascribed to NSAID-mediated inhi-bition of cyclooxygenase activity In particular, upregulation

of Cox-2 may be a key component of epithelial esis, and its suppression the main factor associated with theantitumor activity of NSAIDs Tissue-selective overexpres-sion of Cox-2 by promoter-specific targeting of murineepithelial cells induced tumorigenesis In an animal model

tumorigen-of FAP, intestinal tumor formation was dramaticallydecreased by either genetic deletion of Cox-2 or its inhibi-tion by a Cox-2 specific NSAID Recent studies in humantumor xenografts that constitutively expressed both Cox-1and Cox-2 showed that selective inhibition of Cox-2decreased intratumoral PGE2 and reduced tumor growth.This result was also achieved by specifically inhibitingPGE2with a neutralizing antibody, but not by selective inhi-bition of Cox-1 with a new NSAID, SC-560

In addition to enhanced expression in tumors, Cox-2 and

PGE2are also increased in fibroblasts and endothelial cells

associated with intestinal tumors Disruption of the PGE2

receptor, EP2, in Apc-mutant mice produces tumor

suppres-sion, an effect primarily due to a positive feedback

mecha-nism for Cox-2 expression by PGE2 in adenoma stromal

cells Cox-2 is highly expressed in tumor-associated

endothelial cells, and PGE2supports angiogenesis in human

tumors These observations led to the hypothesis that Cox-2

upregulation supports tumor angiogenesis, and that NSAIDs

are antiangiogenic because of their ability to suppress

cyclooxygenase activity This concept is supported by data

showing that selective Cox-2 inhibitors suppress

angiogen-esis in the FGF-rat corneal micropocket assay

Lipoxygenase metabolites may also play a role in tumor

formation Because they promote cell proliferation and

angiogenesis and suppress tumor cell apoptosis, the 5-, 8-,

and 12-LOX isoforms are characterized as “tumorigenic,”

whereas the opposite effects of 15-LOX suggest that this

enzyme may inhibit tumor formation In support of this

characterization, human epithelial tumors exhibit decreased

levels of 15-LOX compared to normal tissues, and in vitro

treatment of colorectal cancer cells with NSAIDs increased

levels of 15-LOX, augmented tumor apoptosis, and

decreased cell growth This effect appeared to be a direct

effect of NSAIDs on 15-LOX expression rather than a shift

of substrate from cyclooxygenase to lipoxygenase metabolic

pathways

Activity and Specificity of Inhibitors of

Arachidonic Acid Metabolism

NSAIDsBecause cyclooxygenases are the main target of NSAID

therapy, the role of cyclooxygenase-derived lipid mediators

has been widely studied Inhibition of cyclooxygenase leads

to a decrease in the production of all prostaglandins and

thromboxanes, and this accounts for the observed effects of

NSAIDs as anti-inflammatory, antipyretic, analgesic, and

antithrombotic agents It also explains their gastrointestinal

and renal side effects Enormous effort has been expended

to develop NSAIDs whose specificity of action will enhance

the benefits of eicosanoid inhibition yet minimize the

harm-ful effects on gastric mucosa and renal vasculature The

dis-covery of Cox-2 and its role in inflammation but not gastric

protection led not only to the development of specific

inhibitors of Cox-2, but also to studies examining the

differ-ential effects of existing NSAIDs upon the cyclooxygenase

isoforms

Aspirin is currently the only NSAID that covalently

modifies cyclooxygenase Aspirin has greater inhibitory

activity against Cox-1 than against Cox-2, and this explains

its antiplatelet and cardiovascular effects, as well as its

ten-dency to produce ulceration of gastric mucosa

Cyclooxyge-nase blockade by the other known NSAIDs occurs as a

result of reversible binding of the drug to the nase molecule The kinetics of NSAID—cyclooxygenaseinteractions are quite complex, with both competitive andtime-dependent elements This, together with the complex-ity of prostaglandin biology in vivo, makes it difficult tocompare the Cox-1/Cox-2 selectivity of different NSAIDs.Depending upon dosage, cell type, and assay conditions,every NSAID exhibits some degree of inhibition of bothCox-1 and Cox-2 In general, however, most NSAIDs, such

cyclooxyge-as cyclooxyge-aspirin, indomethacin, and piroxicam, are relatively specific A few, such as meloxicam, have some degree ofincreased specificity for Cox-2 A new class of NSAIDs, theselective Cox-2 inhibitors, include NS398, celecoxib, androfecoxib These agents are strong inhibitors of Cox-2 withminimal effect on Cox-1

non-Leukotriene ModifiersLeukotrienes, through their ability to modulate leuko-cyte—endothelial cell interactions, are thought to mediateNSAID-associated gastric mucosal damage Leukotrienesare also potent vasoconstrictors and inducers of bron-chospasm in susceptible individuals The enzyme responsi-ble for leukotriene synthesis, 5-LOX, is expressed only in alimited repertoire of cells, mostly leukocytes Leukotrienereceptors, however, are widely distributed among smoothmuscle cells of the vasculature and respiratory tract.Leukotriene modifiers, such as zileuton and montelukast,are 5-LOX inhibitors used clinically for asthma therapy Forunknown reasons, these agents are particularly useful forexercise-induced and aspirin-intolerant asthma

Combination Agents

A promising therapeutic approach to minimize the gastricside effects of aspirin while providing antithrombotic therapy is to concurrently suppress the activities of bothcyclooxygenase and 5-LOX enzymes Based upon the activ-ity profiles of cyclooxygenase and 5-LOX products, theseagents would be clinically useful in a wide variety of dis-eases, including inflammatory states, cancer prevention, andcardiovascular disorders Several of these dual inhibitors ofprostaglandin and leukotriene synthesis have been devel-oped A few of these, including the agent licofelone, areunder evaluation in Phase III clinical trials for the treatment

of osteoarthritis

Conclusion

Studies of eicosanoid biology have provided great insightinto normal physiology and the pathogenesis of disease Therapidly responsive, tissue-localized nature of eicosanoidactivities make them ideal targets for therapeutic interven-tion, and it is therefore easy to see why modulators ofeicosanoid synthesis, such as aspirin, are among the oldestknown therapeutics Because eicosanoids play central roles

C HAPTER 107 Eicosanoids 733

in a wide range of disease states, inhibitors of eicosanoid

synthesis can achieve a broad spectrum of activity This is

clearly demonstrated by the use of NSAIDs for conditions

as diverse as pain relief, prevention of cardiovascular

dis-ease, and inhibition of tumor formation In the future, the

development of specific agonists and antagonists for

eicosanoid receptors will yield further insight into the

rele-vance of various pathways to disease states and provide

new, more specific avenues for therapy

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Baker, R R (1990) The eicosanoids: A historical review Clin Biochem.

23, 455–458 A concise review of early studies of eicosanoid biology.

Bishop-Bailey, D., and Wray, J (2003) Peroxisome proliferator-activated

receptors: A critical review on endogenous pathways for ligand

gener-ation Prostaglandins Other Lipid Mediat 71, 1–22 A detailed

analy-sis of multiple signaling networks containing eicosanoids.

Funk, C D (2001) Prostaglandins, and leukotrienes: Advances in

eicosanoid biology Science 294, 1871–1875 Basic current review of

eicosanoid biology and potential for targeted therapies.

OTHERUSEFULREFERENCES Kudo, I., and Murakami, M (2003) Regulatory functions of prostaglandin

E2 synthases Adv Exp Med Biol 525, 103–106.

Nie, D., and Honn, K V (2002) Cyclooxygenase, lipoxygenase and tumor

angiogenesis Cell Mol Life Sci 59, 799–807.

Thun, M J., Henley, S J., and Patrono, C (2002) Nonsteroidal inflammatory drugs as anticancer agents: Mechanistic, pharmacologic,

anti-and clinical issues J Natl Cancer Inst 94, 4–20.

Capsule Biography

Monica M Bertagnolli is an Associate Professor of Surgery at Harvard Medical School and a member of the Division of Surgical Oncology at Brigham and Women’s Hospital and the Dana Farber Cancer Institute Her primary research interest is the impact of nonsteroidal anti-inflammatory drugs on early tumorigenesis.

C HAPTER 108

Sepsis and the Microvasculature

Alpha A Fowler III

Division of Pulmonary Disease and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University

Nitric Oxide Induces Microcirculatory Dysfunction

Inducible Nitric Oxide Synthase MediatesMicrovascular Dysfunction

A consensus has slowly emerged that organ failure andmortality from sepsis arise from injury and disordered cir-culatory homeostasis and hyperdynamic states Hypoxemiaand hypotension unresponsive to pharmacologic interven-tion are commonly present during sepsis Despite the presence of enhanced oxygen delivery associated withhyperdynamic states, defects in oxygen extraction and tissueoxygen utilization produce lactic acidosis, strongly suggest-ing that a microcirculatory dysfunction is present Cumula-tive research indicates that all anatomic compartments of the

microcirculation are dysregulated Cryer et al demonstrated

loss of vascular tone with significant dilatation of third- andfourth-order skeletal muscle arterioles (20 to 50mM) fol-

lowing onset of hyperdynamic Escherichia coli sepsis [2].

Subsequently, workers demonstrated that resistance oles are hyporeactive to the vasoconstrictive effects of nor-epinephrine in organ-specific resistance microvasculature(e.g., liver, lung) in sepsis Significant research indicatesthat the reactive nitrogen intermediate nitric oxide (NO) is akey factor producing disordered vasoregulation in sepsis.Under physiologic conditions, NO is continuously produced

arteri-at low levels by endothelium and vascular smooth musclecells through transcription of the constitutive NO synthasegene (NOSIII) However, abrupt increases of inducible NOsynthase (iNOS or NOSII) expression by endothelium, vas-cular smooth muscle cells, and monocyte/macrophagesoccur following onset of sepsis, producing remarkablesurges of detectable NO in the circulation

Sepsis Epidemiology

New technology and specialized medical practices have

evolved over the past five decades that permit support of

acute and chronic organ failure The current era in medicine

is remarkable for rapid progress in diverse fields such as

cancer therapy and transplantation of bone marrow and solid

organs Consequently, patients suffering from diseases that

were formerly fatal now more often than not move to

“postacute” or chronic phases of illness, necessitating

fre-quent or extended hospitalizations A striking increase in the

incidence of sepsis has accompanied medical advances

Recent studies suggest that more than 750,000 new cases of

sepsis occur in the United States annually Mortality rates

attributable to sepsis range from 25 percent to 30 percent

with higher mortality linked to increasing age Preexisting

or comorbid medical conditions as well as greater numbers

of organs systems failed are important factors that determine

outcomes Thus, expectations are that greater than 200,000

deaths will occur annually from sepsis with annual total

costs to the U.S economy alone exceeding 16 billion

dol-lars Given a host of biological factors combined with aging

populations and increased need for care of chronic illness,

conservative projections call for a 1.5 percent increase per

annum in the incidence of sepsis [1] Worldwide incidence

figures may vary, but sepsis exacts a huge toll in lost human

life and productivity Microvascular endothelial cells (ECs)

are integrally involved in regulating blood flow,

coagula-tion, leukocyte trafficking, edema formacoagula-tion, and

angiogen-esis Insights into the pathogenesis of sepsis are gained by

examining important concepts established through careful

study of microcirculatory biology

Copyright © 2006, Elsevier Science (USA).