NEUROVASCULAR MEDICINE - Pursuing Cellular Longevity for Healthy Aging Part 10 pptx

Serum levels of glucose-derived advanced glycation end products are associated with the severity of diabetic retinopathy in type 2 diabetic patients without renal dysfunction.. Telmisart

NOVEL CELLULAR PATHWAYS

528

The local RAS is activated under diabetes (Anderson 1997) We have recently found that angiotensin II (Ang II) stimulated intracellular ROS generation in retinal pericytes through an interac- tion with type 1 receptor Further, Ang II decreased DNA synthesis and simultaneously upregulated VEGF mRNA levels in pericytes, both of which were blocked by treatment with telmisartan, a com- mercially available Ang II type 1 receptor blocker,

or an antioxidant NAC (Yamagishi, Amano, Inagaki

et al 2003; Amano, Yamagishi, Inagaki et al 2003) These results suggest that Ang II-type 1 receptor interaction could induce pericyte loss and dysfunc- tion through intracellular ROS generation, thus being involved in diabetic retinopathy Since Ang II induces the VEGF receptor, KDR, expression in reti- nal microvascular ECs, the retinal RAS might aug- ment the permeability- and angiogenesis-inducing activity of VEGF, thus implicated in the progression of diabetic retinopathy as well (Otani, Takagi, Suzuma

et al 1998).

Blockade of the RAS by inhibitors of angiotensin converting enzyme or Ang II type 1 receptor antag- onists can reduce retinal overexpression of VEGF and hyperpermeability and neovascularization in experimental diabetes (Babaei-Jadidi, Karachalias, Ahmed et al 2003; Anderson 1997; Yamagishi, Amano, Inagaki et al 2003) Funatsu et al (Funatsu, Yamashita, Nakanishi et al 2002) recently found that the vitreous fl uid level of Ang II was signifi cantly correlated with that of VEGF, and both of them were signifi cantly higher in patients with active prolifer- ative diabetic retinopathy than in those with qui- escent proliferative diabetic retinopathy (Amano, Yamagishi, Inagaki et al 2003) These fi ndings fur- ther support the concept that Ang II contributes to development and progression of proliferative dia- betic retinopathy in combination with VEGF In the EUCLID Study, the angiotensin-converting enzyme inhibitor, lisinopril, reduced the risk of progression

of retinopathy by approximately 50% and also nifi cantly reduced the risk of progression to prolif- erative retinopathy although retinopathy was not a primary end point and the study was not suffi ciently powered for eye-related outcomes (Otani, Takagi, Suzuma et al 1998) The interaction of the RAS and AGE–RAGE system has also been proposed We have found that Ang II potentiates the deleterious effects

sig-of AGEs on pericytes by inducing RAGE protein expression (Yamagishi, Takeuchi, Matsui et al 2005)

In vivo, AGE injection stimulated RAGE expression

in the eye of spontaneously hypertensive rats, which was blocked by telmisartan In vitro, Ang II-type 1

receptor- mediated ROS generation elicited RAGE

gene expression in retinal pericytes through NF- κB

the characteristic changes of the early phase of

dia-betic retinopathy, in streptozotocin-induced diadia-betic

rats These observations suggest that SDH-mediated

conversion of sorbitol into fructose and the resultant

ROS generation may play a role in the pathogenesis of

diabetic retinopathy Since fructose is a stronger

gly-cating agent than glucose, intracellular AGEs

forma-tion via the SDH pathway might be involved in glucose

toxicity to retinal pericytes (Rosen, Nawroth, King

et al 2001).

There is a growing body of evidence that

gener-ation of ROS is increased in diabetes High glucose

concentrations, via various mechanisms such as

glucose autoxidation, increased the production

of AGEs, activation of PKC, and stimulation of the

polyol pathway, and it enhanced ROS generation

(Rosen, Nawroth, King et al 2001;

Bonnefont-Rousselot 2002) Increased ROS generation has been

found to regulate vascular infl ammation, altered

gene expression of growth factors and cytokines,

and platelet and macrophage activation, thus

play-ing a central role in the pathogenesis of diabetic

vas-cular complications (Yamagishi, Edelstein, Du et al

2001; Yamagishi, Edelstein, Du et al 2001; Yamagishi,

Okamoto, Amano et al 2002; Spitaler, Graier 2002;

Yamagishi, Inagaki, Amano et al 2002; Yamagishi

S, Amano S, Inagaki et al 2003) Further, we have

recently found that high glucose–induced

mito-chondrial overproduction of superoxide serves as a

causal link between elevated glucose and

hyperglyce-mic vascular damage in ECs (Nishikawa, Edelstein,

Du et al 2000; Brownlee 2001) Normalizing levels

of mitochondrial ROS prevent glucose-induced

for-mation of AGEs, activation of PKC, sorbitol

accu-mulation, and NF- κB activation These observations

suggest that the three main mechanisms implicated

in the pathogenesis of diabetic vascular

complica-tions might refl ect a single hyperglycemia-induced

process, thus providing a novel therapeutic

tar-get for diabetic angiopathies Recently, Hammes

et al (Hammes, Du, Edelstein et al 2003) have

dis-covered that the lipid-soluble thiamine derivative

benfotiamine can inhibit the three major

biochem-ical pathways as well as hyperglycemia-associated

NF- κB activation (Hammes, Du, Edelstein et al

2003) They showed that benfotiamine prevented

experimental diabetic retinopathy by activating

the pentose phosphate pathway enzyme,

transketo-lase, in the retinas, which converts

glyceraldehyde-3-phosphate and fructose-6-phosphate into

pentose-5-phosphates and other sugars (Hammes, Du,

Edelstein et al 2003) Thiamine and benfotiamine

therapy is reported to prevent streptozotocin-induced

incipient diabetic nephropathy as well (Babaei-Jadidi,

Karachalias, Ahmed et al 2003).

(Cooper, Bonnet, Oldfi eld et al 2001) Evidence has implicated the TGF- β system as a major etiologic agent in the pathogenesis of glomerulosclerosis and tubulointerstitial fi brosis in diabetic nephropathy (Sharma, Ziyadeh 1995; Aoyama, Shimokata, Niwa 2000; Wang, LaPage, Hirschberg 2000).

AGEs induce apoptotic cell death and VEGF expression in human-cultured mesangial cells, as the case in pericytes (Yamagishi, Inagaki, Okamoto

et al 2002) Mesangial cells occupy a central tomical position in the glomerulus, playing crucial roles in maintaining structure and function of glo- merular capillary tufts (Dworkin, Ichikawa, Brenner 1983) They actually provide structural support for capillary loops and modulate glomerular fi ltration

ana-by its smooth muscle activity (Dworkin, Ichikawa, Brenner 1983; Kreisberg, Venkatachalam, Troyer 1985; Schlondorff 1987) Therefore, it is conceivable that the AGE-induced mesangial apoptosis and dys- function may contribute in part to glomerular hyper-

fi ltration, an early renal dysfunction in diabetes Several experimental and clinical studies support the pathological role for VEGF in diabetic nephropathy Indeed, antibodies against VEGF have been found to improve hyperfi ltration and albuminuria in strepto- zotocin-induced diabetic rats (De Vriese, Tilton, Elger

et al 2001) Inhibition of VEGF also prevents ular hypertrophy in a model of obese type 2 diabetes, the Zucker diabetic fatty rat (Schrijvers, Flyvbjerg, Tilton et al 2006) Further, urinary VEGF levels are positively correlated with the urinary albumin to cre- atinine ratio and negatively correlated with creatinine clearance in type 2 diabetic patients (Kim, Oh, Seo

glomer-et al 2005) These observations suggest that urinary VEGF might be used as a sensitive marker of diabetic nephropathy VEGF overproduction elicited by AGEs may be involved in diabetic nephropathy.

Moreover, we have recently found that AGE–RAGE interaction stimulates MCP-1 expression in mesangial cells through ROS generation (Yamagishi, Inagaki, Okamoto et al 2002) Increased MCP-1 expression associated with monocyte infi ltration in mesangium has been observed in the early phase of diabetic nephropathy as well (Banba, Nakamura, Matsumura

et al 2000) Plasma MCP-1 was positively correlated with urinary albumin excretion rate in type 1 diabetic patients (Chiarelli, Cipollone, Mohn et al 2002) AGE accumulation in glomerulus could also be implicated

in the initiation of diabetic nephropathy by ing the secretion of MCP-1.

promot-AGE formation on extracellular matrix proteins alters both matrix–matrix and cell–matrix interac- tions, involved in the pathogenesis of diabetic glom- erulosclerosis For example, nonenzymatic glycations

of type IV collagen and laminin reduce their ability

activation Further, Ang II augmented AGE-induced

pericyte apoptosis, the earliest hallmark of diabetic

retinopathy Further, we have recently found that

telmisartan blocks the Ang II-induced RAGE

expres-sion in ECs as well (Nakamura, Yamagishi, Nakamura

et al 2005) Telmisartan could decrease endothelial

RAGE levels in patients with essential hypertension

Taken together, these observations provide the

func-tional interaction between the AGE–RAGE system and

the RAS in the pathogenesis of diabetic retinopathy,

thus suggesting a novel benefi cial aspect of

telmisar-tan on the devastating disorder We posit a table that

presents the etiologies of diabetic retinopathy and its

possible therapeutic agents (Table 21.2).

ROLE OF AGES IN DIABETIC

NEPHROPATHY

Diabetic nephropathy is a leading cause of ESRD and

accounts for disabilities and the high mortality rate in

patients with diabetes (Krolewski, Warram, Valsania

et al 1991) Development of diabetic nephropathy

is characterized by glomerular hyperfi ltration and

thickening of glomerular basement membranes,

fol-lowed by an expansion of extracellular matrix in

mesangial areas and increased urinary albumin

excre-tion rate (UAER) Diabetic nephropathy ultimately

progresses to glomerular sclerosis associated with

renal dysfunction (Sharma, Ziyadeh 1995) Further, it

has recently been recognized that changes within

tub-ulointerstitium, including proximal tubular cell

atro-phy and tubulointerstitial fi brosis, are also important

in terms of renal prognosis in diabetic nephropathy

(Ziyadeh, Goldfarb 1991; Lane, Steffes, Fioretto et al

1993; Taft, Nolan, Yeung et al 1994; Jones, Saunders,

Qi et al 1999; Gilbert, Cooper 1999) Such tubular

changes have been reported to be the dominant lesion

in about one-third of patients with type 2 diabetes

(Fiorreto, Mauer, Brocco et al 1996) It appears that

both metabolic and hemodynamic factors interact to

stimulate the expression of cytokines and growth

fac-tors in glomeruli and tubules from the diabetic kidney

Table 21.2 Diabetic Retinopathy

Etiology Cellular Pathway Treatment Regimen

BenfotiamineTelmisartan

NOVEL CELLULAR PATHWAYS

530

malondialdehyde-lysine accumulate in the expanded mesangial matrix and thickened glomerular base- ment membranes of early diabetic nephropathy, and

in nodular lesions of advanced disease, further gesting the active role of AGEs for diabetic nephropa- thy (Suzuki, Miyata, Saotome et al 1999).

sug-A number of studies have demonstrated that oguanidine decreased AGE accumulation and plasma protein trapping in the glomerular basement mem- brane (Matsumura, Yamagishi, Brownlee 2000) In streptozocin-induced diabetic rats, aminoguanidine treatment for 32 weeks dramatically reduced the level

amin-of albumin excretion and prevented the development

of mesangial expansion (Soulis-Liparota Cooper, Papazoglou et al 1991) Furthermore, aminoguani- dine treatment was found to prevent albuminuria

in diabetic hypertensive rats without affecting blood pressure (Edelstein, Brownlee 1992) Whether inhi- bition by aminoguanidine of inducible nitric oxide synthase (iNOS) could contribute to these renopro- tective effects remains to be elucidated However, methylguanidine, which inhibits iNOS but not AGE formation, was reported not to retard the develop- ment of albuminuria in diabetic rats (Soulis, Cooper, Sastra et al 1997) These observations suggest that the benefi cial effects of aminoguanidine could be medi- ated predominantly by decreased AGE formation rather than by iNOS inhibition A recent randomized, double-masked, placebo-controlled study (ACTION I trial) revealed that pimagedineR (aminoguanidine) reduced the decrease in glomerular fi ltration rate and 24-hour total proteinuria in type 1 diabetic patients (Bolton, Cattran, Williams et al 2004) Although the time for doubling of serum creatinine, a primary end point of this study, was not signifi cantly improved by pimagedineR treatment (P = 0.099), the trial provided the fi rst clinical proof of the concept that blockade of AGE formation could result in a signifi cant attenua- tion of diabetic nephropathy.

We have found that OPB-9195, a synthetic dine derivative and novel inhibitor of AGEs, prevented the progression of diabetic nephropathy by lowing serum concentrations of AGEs and their deposition

thiazoli-of glomeruli in Otsuka–Long–Evans–Tokushima– Fatty rats, a type 2 diabetes mellitus model animal (Tsuchida, Makita, Yamagishi et al 1999) OPB-9195 was also found to retard the progression of diabetic nephropathy by blocking type IV collagen produc- tion and suppressing overproduction of two growth factors, TGF- β and VEGF.

Recently, Degenhardt and Baynes et al hardt, Alderson, Arrington et al 2002) reported that pyridoxamine inhibited the progression of renal dis- ease and decreases hyperlipidemia and apparent redox imbalances in diabetic rats Pyridoxamine and aminoguanidine had similar effects on parameters

(Degen-to interact with negatively charged proteoglycans,

increasing vascular permeability to albumin (Silbiger,

Crowley, Shan et al 1993) Furthermore, AGE

forma-tion on various types of matrix proteins impairs their

degradation by matrix metalloproteinases,

contribut-ing to basement membrane thickencontribut-ing and

mesan-gial expansion, hallmarks of diabetic nephropathy

(Brownlee 1993; Mott, Khalifah, Nagase et al 1997)

AGEs formed on the matrix components can trap and

covalently cross-link with the extravasated plasma

proteins such as lipoproteins, thereby exacerbating

diabetic glomerulosclerosis (Brownlee 1993).

AGEs stimulate insulin-like growth factor-I, -II,

PDGF and TGF- β in mesangial cells, which in turn

mediate production of type IV collagen, laminin, and

fi bronectin (Matsumura, Yamagishi, Brownlee 2000;

Yamagishi, Takeuchi, Makita 2001) AGEs induce

TGF- β overexpression in both podocytes and

proxi-mal tubular cells as well (Wendt TM, Tanji N, Guo J,

et al 2003; Yamagishi, Inagaki, Okamoto et al 2003)

Recently, Ziyadeh et al (2000) reported that

long-term treatment of type 2 diabetic model mice with

blocking antibodies against TGF- β suppressed excess

matrix gene expression, glomerulosclerosis, and

pre-vented the development of renal insuffi ciency These

observations suggest that AGE-induced TGF- β

expres-sion plays an important role in the pathogenesis of

glomerulosclerosis and tubulointerstitial fi brosis in

diabetic nephropathy (Raj, Choudhury, Welbourne

et al 2000; Yamagishi, Koga, Inagaki et al 2002).

In vivo, the administration of AGE-albumin to

normal healthy mice for 4 weeks has been found to

induce glomerular hypertrophy with overexpression

of type IV collagen, laminin B1, and TGF- β genes

(Yang, Vlassara, Peten et al 1994) Furthermore,

chronic infusion of AGE-albumin to otherwise

healthy rats leads to focal glomerulosclerosis,

mesan-gial expansion, and albuminuria (Vlassara H,

Striker LJ, Teichberg et al 1994) Recently,

RAGE-overexpressing diabetic mice have been found to show

progressive glomerulosclerosis with renal

dysfunc-tion, compared with diabetic littermates lacking the

RAGE transgene (Yamamoto, Kato, Doi et al 2001)

Further, diabetic homozygous RAGE null mice failed

to develop signifi cantly increased mesangial matrix

expansion or thickening of the glomerular basement

membrane (Wendt, Tanji, Guo et al 2003) Taken

together, these fi ndings suggest that the activation of

AGE–RAGE axis contributes to expression of VEGF

and enhanced attraction/activation of infl ammatory

cells in the diabetic glomerulus, thereby setting the

stage for mesangial activation and TGF- β production;

processes that converge to cause albuminuria and

glomerulosclerosis.

AGEs including glycoxidation or lipoxidation

pro-ducts such as Nε-(carboxymethyl)lysine, pentosidine,

is responsible for about 70% of all causes of death

in patients with type 2 diabetes (Laakso 1999) In Framingham study, the incidence of CVD was 2 to

4 times greater in diabetic patients than in general polulation (Haffner, Lehto, Ronnemaa et al 1998) Conventional risk factors, including hyperlipidemia, hypertension, smoking, obesity, lack of exercise, and a positive family history, contribute similarly

to macrovascular complications in type 2 diabetic patients and nondiabetic subjects (Laakso 1999) The levels of these factors in diabetic patients were certainly increased, but not enough to explain the exaggerated risk for macrovascular complications in diabetic population (Standl, Balletshofer, Dahl et al 1996) Therefore, specifi c diabetes-related risk fac- tors should be involved in the excess risk in diabetic patients.

A variety of molecular mechanisms underlying the actions of AGEs and their contribution to diabetic macrovascular complications have been proposed (Stitt, Bucala, Vlassara 1997; Bierhaus, Hofmann, Ziegler et al 1998; Schmidt, Stern 2000; Vlassara, Palace 2002; Wendt, Bucciarelli, Qu et al 2002) AGEs formed on the extracellular matrix results in decreased elasticity of vasculatures, and quench nitric oxide, which could mediate defective endothelium- dependent vasodilatation in diabetes (Bucala, Tracey, Cerami 1991) AGE modifi cation of low-density lipo- protein (LDL) exhibits impaired plasma clearance and contributes signifi cantly to increased LDL in vivo, thus being involved in atherosclerosis (Bucala, Mitchell, Arnold et al 1995) Binding of AGEs to RAGE results in generation of intracellular ROS generation and subsequent activation of the redox- sensitive transcription factor NF- κB in vascular wall cells, which promotes the expression of a variety of atherosclerosis-related genes, including ICAM-1, vas- cular cell adhesion molecule-1, MCP-1, PAI-1, tissue factor, VEGF, and RAGE (Stitt, Bucala, Vlassara 1997; Bierhaus, Hofmann, Ziegler et al 1998; Schmidt, Stern 2000; Tanaka, Yonekura, Yamagishi et al 2000; Vlassara, Palace 2002; Wendt, Bucciarelli, Qu et al 2002) AGEs have the ability to induce osteoblas- tic differentiation of microvascular pericytes, which would contribute to the development of vascular cal- cifi cation in accelerated atherosclerosis in diabetes as well (Yamagishi, Fujimori, Yonekura et al 1999) The interaction of the RAS and AGEs in the development

of diabetic macrovascular complications has also been proposed AGE–RAGE interaction augments

measured, supporting a mechanism of action

involv-ing AGE inhibition (Degenhardt, Alderson, Arrinvolv-ington

et al 2002) Although the results of AGE inhibitors

in animal models of diabetic nephropathy are

prom-ising, effectiveness of these AGE inhibitors must be

confi rmed by multicenter, randomized, double-blind

clinical studies.

Cross Talk between the AGE–RAGE Axis

and the RAS in Diabetic Nephropathy

Recent experiments have focused on the interaction

of the AGE–RAGE axis and the RAS thought to be

critical to the development of diabetic nephropathy

Indeed, angiotensin converting enzyme inhibition

reduces the accumulation of renal and serum AGEs,

probably via effects on oxidative pathways (Forbes,

Cooper, Thallas et al 2002) Long-term treatment

with Ang II receptor 1 antagonist may exert

salu-tary effects on AGEs levels in the rat remnant

kid-ney model, probably due to improved renal function

(Sebekova, Schinzel, Munch et al 1999) Ramipril

administration has been recently shown to result in a

mild decline of fl uorescent

non-carboxymethyllysine-AGEs and malondialdehyde concentrations in

nondi-abetic nephropathy patients (Sebekova, Gazdikova,

Syrova et al 2003) Further, we have recently found

that the AGE–RAGE-mediated ROS generation

activates TGF- β-Smad signaling and subsequently

induces mesangial cell hypertrophy and fi bronectin

synthesis by autocrine production of Ang II (Fukami,

Ueda, Yamagishi et al 2004) In addition, AGEs

induce mitogenesis and collagen production in renal

interstitial fi broblasts as well via Ang II-connective

tissue growth factor pathway (Lee, Guh, Chen et al

2005) Moreover, olmesartan medoxomil, an Ang II

type 1 receptor blocker, protects against

glomeru-losclerosis and renal tubular injury in AGE-injected

rats, thus further supporting the concept that AGEs

could induce renal damage in diabetes partly via the

activation of RAS (Yamagishi, Takeuchi, Inoue et al

2005) We posit a table that presents the etiologies

of diabetic nephropathy and its possible therapeutic

agents (Table 21.3).

Table 21.3 Diabetic Nephropathy

Etiology Cellular Pathway Treatment Regimen

Hyperfi ltration

NOVEL CELLULAR PATHWAYS

532

fi vefold lower AGE content signifi cantly decreased serum levels of AGEs, soluble form of VCAM-1 and C-reactive protein (CRP), compared to equivalent regular diets (Vlassara, Cai, Crandall et al 2002) AGE-poor diets also reduced peripheral mononu- clear cell tumor necrosis factor- α (TNF-α) expres- sion at both mRNA and protein levels (Vlassara, Cai, Crandall et al 2002) Further, LDL pooled from dia- betic patients on a standard diet for 6 weeks (high AGE-LDL) was more glycated and oxidized than that from diabetic patients on an AGE-poor diet (low AGE-LDL) (Cai, He, Zhu et al 2004) High AGE-LDL signifi cantly induced soluble form of VCAM-1 expres- sion in human umbilical vein ECs via redox-sensitive MAPK activation, compared to native LDL or low AGE-LDL (Cai, He, Zhu et al 2004) In addition, AGE pronyl-glycine, a food-derived AGE, was reported to elicit infl ammatory response to cellular proliferation

in an intestinal cell line, Caco-2, through the mediated MAPK activation (Zill, Bek, Hofmann et al 2003) These observations suggest the causal link between dietary intake of AGEs and proinfl amma- tion and vascular injury, thus providing the clinical relevance of dietary AGE restriction in the prevention

RAGE-of accelerated atherosclerosis in diabetes We have very recently found that PAI-1 and fi brinogen levels are positively associated with serum AGE levels in nondiabetic general population Food-derived AGEs may also be associated with thrombogenic tendency

in nondiabetic subjects (Enomoto, Adachi, Yamagishi

et al 2006).

CONCLUSION

In the DCCT-EDIC, the reduction in the risk of progressive diabetic micro- and macroangipathies resulting from intensive therapy in patients with type 1 diabetes persisted for at least several years, despite increasing hyperglycemia (DCCT-EDIC Research Group 2000; Writing Team for DCCT-EDIC Research Group 2003; Nathan, Lachin, Cleary et al 2003; Nathan, Cleary, Backlund et al 2005) These

clinical studies strongly suggest that so-called glycemic memory is involved in the pathogenesis of dia-

hyper-betic vascular complications, AGE hypothesis seems

to be most compatible with this theory Moreover, large clinical investigations will be needed to clar- ify whether the inhibition of AGE formation or the blockade of their downstream signaling could pre- vent the development and progression of vascular complications in diabetes Until the specifi c remedy that targets diabetic vascular complications are devel- oped, multifactorial intensifi ed intervention will be a promising therapeutic strategy for the prevention of these devastating disorders.

Ang II-induced smooth muscle cell proliferation and

activation, thus being involved in accelerated

ath-erosclerosis in diabetes (Shaw, Schmidt, Banes et al

2003) AGEs have been actually detected within

ath-erosclerotic lesions in both extra- and

intracellu-lar locations (Nakamura, Horii, Nishino et al 1993;

Niwa, Katsuzaki, Miyazaki et al 1997; Sima, Popov,

Starodub et al 1997).

In animal models, Park et al (1998) has

demon-strated that diabetic apolipoprotein E (apoE) null

animals receiving soluble RAGE (sRAGE) display a

dose-dependent suppression of accelerated

athero-sclerosis in these mice Lesions that formed in

ani-mals receiving sRAGE appeared largely arrested at

the fatty streak stage; the number of complex

ath-erosclerotic lesions was strikingly reduced in diabetic

apoE null mice The tissue and plasma AGE burden

was suppressed in diabetic apoE null mice receiving

sRAGE, suggesting that the AGE–RAGE-induced

oxi-dative stress generation might participate in AGEs

formation themselves Treatment with sRAGE did

not affect the levels of established risk factors in these

mice These observations suggest the active

involve-ment of AGE–RAGE interaction in the pathogenesis

in accelerated atherosclerosis in diabetes The same

group has recently reported that the AGE–RAGE

sys-tem contributes to the atherosclerotic lesion

progres-sion as well, and RAGE blockade stabilizes the leprogres-sions

in these mice (Bucciarelli, Wendt, Qu et al 2002)

Another study shows a correlation between AGE

lev-els and the degree of atheroma in cholesterol-fed

rabbits, and aminoguanidine has an antiatherogenic

effect in these rabbits by inhibiting AGEs formation

(Panagiotopoulos, O’Brien, Bucala et al 1998) In

humans, RAGE overexpression is associated with

enhanced infl ammatory reaction and

cyclooxyge-nase-2 and prostaglandin E synthase-1 expression in

diabetic plaque macrophages, and this effect may

con-tribute to plague destabilization by inducing culprit

metalloproteinase expression (Cipollone, Iezzi, Fazia

et al 2003).

Recently, food-derived AGEs are reported to

induce oxidative stress and promote infl ammatory

signals (Cai, Gao, Zhu et al 2002) Dietary glycotoxins

promote diabetic atherosclerosis in apoE-defi cient

mice (Lin, Reis, Dore et al 2002; Lin, Choudhury, Cai

et al 2003) Further, an AGE-poor diet that contained

four- to fi vefold lower AGE contents for 2 months

also decreased serum levels of AGEs and markedly

reduced tissue AGE and RAGE expression, numbers

of infl ammatory cells, tissue factor, VCAM-1, and

MCP-1 levels in diabetic apolipoprotein E-defi cient

mice (Lin, Choudhury, Cai et al 2003).

Diet is a major environmental source of

pro-infl ammatory AGEs in humans as well (Vlassara, Cai,

Crandall et al 2002) In diabetic patients, diets with

Boeri D, Maiello M, Lorenzi M 2001 Increased prevalence

of microthromboses in retinal capillaries of diabetic

individuals Diabetes 50:1432–1439.

Bolton WK, Cattran DC, Williams ME et al 2004 ACTION

I Investigator Group Randomized trial of an tor of formation of advanced glycation end products in

inhibi-diabetic nephropathy Am J Nephrol 24:32–40.

Bonnefont-Rousselot D 2002 Glucose and reactive oxygen

species Curr Opin Clin Nutr Metab Care 5:561–568.

Brownlee M, Vlassara H, Kooney A, Ulrich P, Cerami A 1986 Aminoguanidine prevents diabetes-induced arterial

wall protein cross-linking Science 232:1629–1632.

Brownlee M, Cerami A, Vlassara H 1988 Advanced cosylation end products in tissue and the biochemi-

gly-cal basis of diabetic complications N Engl J Med

318:1315–1321

Brownlee M 1994 Lilly Lecture 1993 Glycation and

diabetic complications Diabetes 43:836–841.

Brownlee M 1995 Advanced protein glycosylation in

diabetes and aging Ann Rev Med 46:223–234.

Brownlee M 2001 Biochemistry and molecular cell biology

of diabetic complications Nature 414:813–820.

Bucala R, Tracey KJ, Cerami A 1991 Advanced sylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in

glyco-experimental diabetes J Clin Invest 87:432–438.

Bucala, Cerami A 1992 Advanced glycosylation: chemistry,

biology, and implications for diabetes and aging Adv

Pharmacol 23:1–34.

Bucala R, Mitchell R, Arnold K, Innerarity T, Vlassara H, Cerami, A 1995 Identifi cation of the major site of apo-lipoprotein B modifi cation by advanced glycosylation end products blocking uptake by the low density lipo-

protein receptor J Biol Chem 270:10828–10832.

Bucciarelli LG, Wendt T, Qu W et al 2002 RAGE blockade stabilizes established atherosclerosis in diabetic apoli-

poprotein E-null mice Circulation 106:2827–2835.

Cai W, Gao QD, Zhu L, Peppa M, He C, Vlassara H 2002 Oxidative stress-inducing carbonyl compounds from common foods: novel mediators of cellular dysfunc-

tion Mol Med 8:337–346.

Cai W, He JC, Zhu L et al 2004 High levels of dietary advanced glycation end products transform low- density lipoprotein into a potent redox-sensitive mitogen- activated protein kinase stimulant in diabetic patients

Cipollone F, Iezzi A, Fazia M et al 2003 The receptor RAGE

as a progression factor amplifying dependent infl ammatory and proteolytic response in human atherosclerotic plaques: role of glycemic con-

arachidonate-trol Circulation 108:1070–1077.

Cogan DG, Toussaint D, Kuwabara T 1961 Retinal

vascu-lar patterns IV Diabetic retinopathy Arch Ophthalmo

66:366–378

Cooper ME, Bonnet F, Oldfi eld M, Jandeleit-Dahm K 2001

Mechanisms of diabetic vasculopathy: an overview Am

J Hypertens 14:475–486.

REFERENCES

Abe R, Shimizu T, Sugawara H et al 2004 Regulation of

human melanoma growth and metastasis by AGE-AGE

receptor interactions J Invest Dermatol 122:461–467.

Adamis AP, Miller JW, Bernal MT et al 1994 Increased

vascular endothelial growth factor levels in the

vitre-ous of eyes with proliferative diabetic retinopathy Am J

Ophthalmol 118:445–450.

Aiello LP, Avery RL, Arrigg PG et al 1994 Vascular

endo-thelial growth factor in ocular fl uid of patients with

dia-betic retinopathy and other retinal disorders N Engl J

Med 331:1480–1487.

Amano S, Yamagishi S, Kato N et al 2002 Sorbitol

dehydro-genase overexpression potentiates glucose toxicity to

cultured retinal pericytes Biochem Biophys Res Commun

299:183–188

Amano S, Yamagishi S, Inagaki Y, Okamoto T 2003

Angiotensin II stimulates platelet-derived growth

factor-B gene expression in cultured retinal pericytes

through intracellular reactive oxygen species

genera-tion Int J Tissue React 25:51–55.

Amano S, Yamagishi S, Koda Y et al 2003

Polymorph-isms of sorbitol dehydrogenase (SDH) gene and

susceptibility to diabetic retinopathy Med Hypotheses

60:550–551

Anderson S 1997 Role of local and systemic angiotensin in

diabetic renal disease Kidney Int Suppl 63:S107–S110.

Antonelli-Orlidge A, Saunders KB, Smith SR, D’Amore PA

1989 An active form of transforming growth factor-β

was produced by co-cultures of ECs and pericytes Proc

Natl Acad Sci U S A 86:4544–4548.

Aoyama I, Shimokata K, Niwa T 2000 Oral adsorbent

AST-120 ameliorates interstitial fi brosis and transforming

growth factor-beta(1) expression in spontaneously

dia-betic (OLETF) rats Am J Nephrol 20:232–241.

Babaei-Jadidi R, Karachalias N, Ahmed N, Battah S,

Thornalley PJ 2003 Prevention of incipient diabetic

nephropathy by high-dose thiamine and benfotiamine

Diabetes 52:2110–2120.

Banba N, Nakamura T, Matsumura M, Kuroda H, Hattori

Y, Kasai K 2000 Possible relationship of monocyte

chemoattractant protein-1 with diabetic nephropathy

Kidney Int 58:684–690.

Barile GR, Pachydaki SI, Tari SR et al 2005 The RAGE axis

in early diabetic retinopathy Invest Ophthalmol Vis Sci

46:2916–2924

Benjamin LE, Hemo I, Keshet E 1998 A plasticity

win-dow for blood vessel remodelling is defi ned by

peri-cyte coverage of the preformed endothelial network

and is regulated by PDGF-B and VEGF Development

125:1591–1598

Bierhaus A, Hofmann MA, Ziegler R, Nawroth PP 1998

AGEs and their interaction with AGE-receptors in

vascular disease and diabetes mellitus I The AGE

concept Cardiovasc Res 37:586–600.

Boehm BO, Lang G, Volpert O et al 2003 Low content of

the natural ocular anti-angiogenic agent pigment

epi-thelium-derived factor (PEDF) in aqueous humor

pre-dicts progression of diabetic retinopathy Diabetologia

46:394–400

NOVEL CELLULAR PATHWAYS

534

and without prior myocardial infarction N Engl J Med

339:229–234

Hammes HP, Martin S, Federlin K, Geisen K, Brownlee M

1991 Aminoguanidine treatment inhibits the

develop-ment of experidevelop-mental diabetic retinopathy Proc Natl

Acad Sci U S A 88:11555–11558.

Hammes HP, Lin J, Renner O et al 2002 Pericytes and

the pathogenesis of diabetic retinopathy Diabetes

tissue sequestration Diabetes 48:1308–1315.

Herman IM, D’Amore PA 1985 Microvascular pericytes

contain muscle and nonmuscle actins J Cell Biol

101:43–52

Inagaki Y, Yamagishi S, Okamoto T, Takeuchi M, Amano

S 2003 Pigment epithelium-derived factor prevents advanced glycation end products-induced monocyte chemoattractant protein-1 production in microvascular endothelial cells by suppressing intracellular reactive

oxygen species generation Diabetologia 46:284–287.

Ishida S, Usui T, Yamashiro K et al 2003 VEGF164 is proinfl ammatory in the diabetic retina Invest

Ophthalmol Vis Sci 44:2155–2162.

Jones SC, Saunders HJ, Qi W, Pollock CA 1999 Intermittent high glucose enhances cell growth and collagen synthesis in cultured human tubulointerstitial cells

Diabetologia 42:1113–1119.

Jonnes N, Iljin K, Dumont DJ, Alitalo K 2001 Tie receptors: new modulators of angiogenic and lymphangiogenic

responses Nat Rev Mol Cell Biol 2:257–267.

Joussen AM, Poulaki V, Qin W et al 2002 Retinal vascular endothelial growth factor induces intercellular adhe-sion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte

adhesion in vivo Am J Pathol 160:501–509.

Joyce NC, Haire MF, Palade GE 1985 Contractile proteins

in pericytes II Immunocytochemical evidence for the presence of two isomyosins in graded concentrations

J Cell Biol 100:1387–1395.

Kaji Y, Usui T, Ishida S et al 2007 Inhibition of diabetic leukostasis and blood-retinal barrier breakdown with a soluble form of a receptor for advanced glycation end

products Invest Ophthalmol Vis Sci 48:858–865.

Khalifah RG, Baynes JW, Hudson BG 1999 Amadorins: novel post-Amadori inhibitors of advanced glycation

reactions Biochem Biophys Res Commun 257:251–258.

Kim NH, Oh JH, Seo JA et al 2005 Vascular thelial growth factor (VEGF) and soluble VEGF

endo-receptor FLT-1 in diabetic nephropathy Kidney Int

67:167–177

Koga K, Yamagishi S, Okamoto T et al 2002 Serum levels

of glucose-derived advanced glycation end products are associated with the severity of diabetic retinopathy

in type 2 diabetic patients without renal dysfunction

Int J Clin Pharmacol Res 22:13–17.

De Vriese AS, Tilton RG, Elger M, Stephan CC, Kriz W,

Lameire 2001 Antibodies against vascular

endothe-lial growth factor improve early renal dysfunction in

experimental diabetes J Am Soc Nephrol 12:993–1000.

Degenhardt TP, Alderson NL, Arrington DD et al 2002

Pyridoxamine inhibits early renal disease and

dyslipi-demia in the streptozotocin-diabetic rat Kidney Int

6:939–950

Dworkin LD, Ichikawa I, Brenner BM 1983 Hormonal

modulation of glomerular function Am J Physiol

244:F95–F104

Dyer DG, Blackledge JA, Thorpe SR, Baynes JW 1991

Formation of pentosidine during nonenzymatic

brown-ing of proteins by glucose Identifi cation of glucose and

other carbohydrates as possible precursors of

pentosi-dine in vivo J Biol Chem 266:11654–11660.

Edelstein D, Brownlee M 1992 Aminoguanidine

ame-liorates albuminuria in diabetic hypertensive rats

Diabetologia 35:96–97.

Enomoto M, Adachi H, Yamagishi S et al 2006 Positive

association of serum levels of advanced glycation end

products with thrombogenic markers in humans

Metabolism 55:912–917.

Fiorreto P, Mauer M, Brocco E et al 1996 Patterns of renal

injury in NIDDM patients with microalbuminuria

Diabetologia 39:1569–1576.

Folkman J, D’Amore PA 1996 Blood vessel formation: what

is its molecular basis? Cell 87:1153–1155.

Forbes JM, Cooper ME, Thallas V et al 2002 Reduction of

the accumulation of advanced glycation end products

by ACE inhibition in experimental diabetic

nephropa-thy Diabetes 51:3274–3282.

Frank RN 1991 On the pathogenesis of diabetic

retinopa-thy A 1990 update Ophthalmology 98:586–593.

Fukami K, Ueda S, Yamagishi S et al 2004 AGEs activate

mesangial TGF-beta-Smad signaling via an

angio-tensin II type I receptor interaction Kidney Int

66:2137–2147

Funatsu H, Yamashita H, Nakanishi Y, Hori S 2002

Angiotensin II and vascular endothelial growth factor

in the vitreous fl uid of patients with proliferative

diabetic retinopathy Br J Ophthalmol 86: 311–315.

Gilbert RE, Cooper ME 1999 The tubulointerstitium in

progressive diabetic kidney disease: more than an

after-math of glomerular injury? Kidney Int 56:1627–1637.

Gitlin JD, D’Amore PA 1983 Culture of retinal

capil-lary cells using selective growth media Microvasc Res

26:1455–1462

Glomb MA, Monnier VM 1995 Mechanism of protein

modi-fi cation by glyoxal and glycolaldehyde, reactive

intermedi-ates of the Maillard reaction J Biol Chem 70:10017–10026.

Goldberg T, Cai W, Peppa M et al 2004. Advanced

glycoxi-dation end products in commonly consumed foods

J Am Diet Assoc 104:1287–1291.

Grandhee SK, Monnier VM 1991 Mechanism of

forma-tion of the Maillard protein cross-link pentosidine

Glucose, fructose, and ascorbate as pentosidine

pre-cursors J Biol Chem 266:11649–11653.

Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M

1998 Mortality from coronary heart disease in subjects

with type 2 diabetes and in nondiabetic subjects with

Mott JD, Khalifah RG, Nagase H, Shield CF III, Hudson JK, Hudson BG 1997 Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility

Kidney Int 52:1302–1312.

Murata T, Ishibashi T, Khalil A et al 1995 Vascular thelial growth factor plays a role in hyperpermeability

endo-of diabetic retinal vessels Ophthalmic Res 27:48–52.

Nakamura Y, Horii Y, Nishino T et al 1993 tochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in

Immunohis-diabetes mellitus Am J Pathol 143:1649–1656.

Nakamura K, Yamagishi S, Nakamura Y, et al 2005 Telmisartan inhibits expression of a receptor for advanced glycation end products (RAGE) in angio-tensin-II-exposed endothelial cells and decreases serum levels of soluble RAGE in patients with essential

hypertension Microvasc Res 70:137–141.

Nathan DM, Lachin J, Cleary P et al 2003 Intensive tes therapy and carotid intima-media thickness in type

diabe-1 diabetes mellitus N Engl J Med 348:2294–2303.

Nathan DM, Cleary PA, Backlund JY et al 2005 Intensive betes treatment and cardiovascular disease in patients

dia-with type 1 diabetes N Engl J Med 353:2643–2653.

Nishikawa T, Edelstein D, Du XL et al 2000 Normalizing mitochondrial superoxide production blocks three path-

ways of hyperglycaemic damage Nature 404:787–790.

Niwa T, Katsuzaki T, Miyazaki S et al 1997 tochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of

Immunohis-diabetic patients J Clin Invest 99:1272–1280.

Nomura M, Yamagishi S, Harada S et al 1995 Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced prolif-

eration of endothelial cells and pericytes J Biol Chem

270:28316–28324

Okamoto T, Yamagishi S, Inagaki Y et al 2002 Angiogenesis induced by advanced glycation end products and its

prevention by cerivastatin FASEB J 16:1928–1930.

Okamoto T, Yamagishi S, Inagaki Y et al 2002 Incadronate disodium inhibits advanced glycation end products-

induced angiogenesis in vitro Biochem Biophys Res

Commun 297:419–424.

Otani A, Takagi H, Suzuma K, Honda Y 1998 Angiotensin

II potentiates vascular endothelial growth induced angiogenic activity in retinal microcapillary

factor-endothelial cells Circ Res 82:619–628.

Ottonello L, Morone MP, Dapino P, Dallegri F 1995 Tumor necrosis factor-alpha-induced oxidative burst

in neutrophils adherent to fi bronectin: effects of cyclic

AMP-elevating agents Br J Haematol 91:566–570.

Panagiotopoulos S, O’Brien RC, Bucala R, Cooper ME, Jerums G 1998 Aminoguanidine has an anti-athero-

genic effect in the cholesterol-fed rabbit Atherosclerosis

perspective Am J Kidney Dis 35:365–380.

Koschinsky T, He CJ, Mitsuhashi T et al 1997 Orally

absorbed reactive glycation products (glycotoxins):

an environmental risk factor in diabetic nephropathy

Proc Natl Acad Sci U S A 94:6474–6479.

Kreisberg JI, Venkatachalam M, Troyer D 1985 Contractile

properties of cultured glomerular mesangial cells

Am J Physiol 249:F457–F463.

Krolewski AS, Warram JH, Valsania P et al 1991 Evolving

natural history of coronary artery disease in diabetes

mellitus Am J Med 90:56S–61S.

L’Esperance FA, James WA, Judson PH 1990 The eye and

diabetes mellitus In Lifkin H, Porte D, ed Ellenberg

and Rifkin’s Diabetes Mellitus, Theory and Practice

New York: Elsevier 661–683

Laakso M 1999 Hyperglycemia and cardiovascular disease

in type 2 diabetes Diabetes 48:937–942.

Lane PH, Steffes MW, Fioretto P, Mauer SM 1993 Renal

interstitial expansion in insulin-dependent diabetes

mellitus Kidney Int 43:661–667.

Lee CI, Guh JY, Chen HC,Hung WC, Yang YL, Chuang LY

2005 Advanced glycation end-product-induced

mito-genesis and collagen production are dependent on

angiotensin II and connective tissue growth factor in

NRK-49F cells J Cell Biochem 95:281–292.

Lin RY, Reis ED, Dore AT et al 2002 Lowering of

dietary advanced glycation endproducts (AGE)

reduces neointimal formation after arterial injury in

genetically hypercholesterolemic mice Atherosclerosis

163:303–311

Lin RY, Choudhury RP, Cai W et al 2003 Dietary

glycotox-ins promote diabetic atherosclerosis in apolipoprotein

E-defi cient mice Atherosclerosis 68:213–220.

Lu M, Kuroki M, Amano S et al 1998 Advanced glycation

end products increase retinal vascular endothelial

growth factor expression J Clin Invest 101:1219–1224.

Lu M, Perez VL, Ma N et al 1999 VEGF increases retinal

vascular ICAM-1 expression in vivo Invest Ophthalmol

Vis Sci 40:1808–1812.

Maisonpierre PC, Suri C, Jones PF et al 1997 Angiopoietin-2,

a natural antagonist for Tie2 that disrupts in vivo

angiogenesis Science 277:55–60.

Mandarino LJ 1992 Current hypotheses for the

bioche-mical basis of diabetic retinopathy Diabetes Care

15:1892–1901

Matsumura T, Yamagishi S, Brownlee M 2000 Advanced

glycation end products and the pathogenesis of

dia-betic complications In Leroith D, Taylor SI, Olefsky

JM, ed Diabetes Mellitus: a Fundamental and Clinical Text

New York: Lippincott-Raven Publishers 983–991

Mitamura Y, Takeuchi S, Matsuda A, Tagawa, Y Mizue, J

Nishihira 2000 Monocyte chemotactic protein-1 in

the vitreous of patients with proliferative diabetic

retinopathy Ophthalmologica 215:415–418.

Miura J, Yamagishi S, Uchigata Y et al 2003 Serum levels of

non-carboxymethyllysine advanced glycation

endprod-ucts are correlated to severity of microvascular

com-plications in patients with Type 1 diabetes J Diabetes

Complications 17:16–21.

Moore TC, Moore JE, Kaji Y et al 2003 The role of advanced

glycation end products in retinal microvascular

leuko-stasis Invest Ophthalmol Vis Sci 44:4457–4464.

NOVEL CELLULAR PATHWAYS

536

Sima A, Popov D, Starodub O et al 1997 Pathobiology of the heart in experimental diabetes: immunolocaliza-tion of lipoproteins, immunoglobulin G, and advanced glycation endproducts proteins in diabetic and/or

hyperlipidemic hamster Lab Invest 77:3–18.

Sims DE 1991 Recent advances in pericyte

biology–implica-tions for health and disease Can J Cardiol 7:431–443.

Soulis T, Cooper ME, Sastra S et al 1997 Relative tions of advanced glycation and nitric oxide synthase inhibition to aminoguanidine-mediated renoprotec-

contribu-tion in diabetic rats Diabetologia 40:1141–1151.

Soulis-Liparota T, Cooper M, Papazoglou D, Clarke B, Jerums G 1991 Retardation by aminoguanidine of development of albuminuria, mesangial expansion, and tissue fl uorescence in streptozocin-induced

diabetic rat Diabetes 40:1328–1334.

Spitaler MM, Graier WF 2002 Vascular targets of redox

sig-nalling in diabetes mellitus Diabetologia 45:476–494.

Spranger J, Osterhoff M, Reimann M et al 2002 Loss of the antiangiogenic pigment epithelium-derived fac-

tor in patients with angiogenic eye disease Diabetes

50:2641–2645

Standl E, Balletshofer B, Dahl B et al 1996 Predictors of 10-year macrovascular and overall mortality in patients with NIDDM: the Munich General Practitioner Project

Diabetologia 39:1540–1545.

Stitt A, Gardiner TA, Alderson NL et al 2002 The AGE inhibitor pyridoxamine inhibits development of retin-

opathy in experimental diabetes Diabetes 51:2826–2832.

Stitt AW, Bucala R, Vlassara H 1997 Atherogenesis and advanced glycation: promotion, progression, and

prevention Ann N Y Acad Sci 811:115–127.

Stitt AW, Li YM, Gardiner TA, et al 1997 Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and

AGE-infused rats Am J Pathol 150:523–531.

Stitt AW, Bhaduri T, McMullen CB, Gardiner TA, Archer

DB 2000 Advanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic

rats Mol Cell Biol Res Commun 3:380–388.

Suzuki D, Miyata T, Saotome N et al 1999 tochemical evidence for an increased oxidative stress and carbonyl modifi cation of proteins in diabetic

Immunohis-glomerular lesions J Am Soc Nephrol 10:822–832 Taft J, Nolan CJ, Yeung SP, Hewitson TD, Martin FI 1994

Clinical and histological correlations of decline in renal function in diabetic patients with proteinuria

Diabetes 43:1046–1051.

Takenaka K, Yamagishi S, Matsui T, Nakamura K, Imaizumi

T 2006 Role of advanced glycation end products (AGEs) in thrombogenic abnormalities in diabetes

Curr Neurovasc Res 3:73–77.

Takeuchi M, Yamagishi S 2004 Alternative routes for the formation of glyceraldehyde-derived AGEs (TAGE) in

vivo Med Hypotheses 63:453–455.

Takeuchi M, Yamagishi S 2004 TAGE (toxic AGEs)

hypothesis in various chronic diseases Med Hypotheses

63:449–452

Tanaka N, Yonekura H, Yamagishi S et al 2000 The tor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis

recep-Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ,

Packer L 2001 The role of oxidative stress in the

onset and progression of diabetes and its

complica-tions: a summary of a Congress Series sponsored by

UNESCO-MCBN, the American Diabetes Association

and the German Diabetes Society Diabetes Metab Res

Rev 17:189–212.

Sato T, Iwaki M, Shimogaito N, X Wu, S Yamagishi, M

Takeuchi 2006 TAGE (toxic AGEs) theory in diabetic

complications Curr Mol Med 6:351–358.

Sato T, Shimogaito N, Wu X, Kikuchi, S.-i Yamagishi, M

Takeuchi 2006 Toxic advanced glycation end products

(TAGE) theory in Alzheimer’s disease Am J Alzheimers

Dis Other Demen 21:197–208.

Schlondorff D 1987 The glomerular mesangial cell: an

expanding role for a specialized pericyte FASEB J

1:272–281

Schmidt AM, Stern D 2000 Atherosclerosis and diabetes:

the RAGE connection Curr Atheroscler Rep 2:430–436.

Schrijvers BF, Flyvbjerg A, Tilton RG, Lameire NH, De

Vriese AS 2006 A neutralizing VEGF antibody

pre-vents glomerular hypertrophy in a model of obese type

2 diabetes, the Zucker diabetic fatty rat Nephrol Dial

Transplant 21:324–329.

Schroder S, Palinski W, Schmid-Schonbein GW 1991

Activated monocytes and granulocytes, capillary

non-perfusion, and neovascularization in diabetic

retinop-athy Am J Pathol 139:81–100.

Sebekova K, Schinzel R, Munch G, Krivosikova Z, Dzurik R,

Heidland A 1999 Advanced glycation end-product

lev-els in subtotally nephrectomized rats: benefi cial effects

of angiotensin II receptor 1 antagonist losartan Miner

Electrolyte Metab 25:380–383.

Sebekova K, Gazdikova K, Syrova D et al 2003 Effects

of ramipril in nondiabetic nephropathy: improved

parameters of oxidatives stress and potential

mod-ulation of advanced glycation end products J Hum

Hypertens 17:265–270.

Segawa Y, Shirao Y, Yamagishi S et al 1998 Upregulation

of retinal vascular endothelial growth factor mRNAs

in spontaneously diabetic rats without

ophthal-moscopic retinopathy A possible participation of

advanced glycation end products in the development

of the early phase of diabetic retinopathy Ophthalmic

Res 30:333–339.

Sharma K, Ziyadeh FN 1995 Hyperglycemia and diabetic

kidney disease The case for transforming growth

factor-beta as a key mediator Diabetes 44:1139–1146.

Sharma NK, Gardiner TA, Archer DB 1985 A morphologic

and autoradiographic study of cell death and

regenera-tion in the retinal microvasculature of normal and

dia-betic rats Am J Ophthalmol 100:51–60.

Shaw SS, Schmidt AM, Banes AK, Wang X, Stern DM,

Marrero MB 2003 S100B-RAGE-mediated

augmen-tation of angiotensin II-induced activation of JAK2 in

vascular smooth muscle cells is dependent on PLD2

Diabetes 52:2381–2388.

Silbiger S, Crowley S, Shan Z, Brownlee M, Satriano J,

Schlondorff D 1993 Nonenzymatic elevated glucose

reduces collagen synthesis and proteoglycan charge

Kidney Int 43:853–864.

chemical modifi cation of proteins by scavenging carbonyl intermediates of carbohydrate and lipid

degradation J Biol Chem 277:3397–3403.

Wang SN, LaPage J, Hirschberg R 2000 Role of ular ultrafi ltration of growth factors in progressive

glomer-interstitial fi brosis in diabetic nephropathy Kidney Int

57:1002–1014

Wendt T, Bucciarelli L, Qu W, Lu Y, Yan SF et al 2002 Receptor for advanced glycation endproducts (RAGE) and vascular infl ammation: insights into the pathogen-

esis of macrovascular complications in diabetes Curr

Atheroscler Rep 4:228–237.

Wendt TM, Tanji N, Guo J et al 2003 RAGE drives the opment of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy

devel-Am J Pathol 162:1123–1137.

Williamson JR, Chang K, Frangos M et al 1993 glycemic pseudohypoxia and diabetic complications

Hyper-Diabetes 42:801–813.

Winocour PH 2003 In Fisher M, ed Heart Disease and

Diabetes London: Martin Dunitz Ltd 121–170.

Writing Team For The Diabetes Control And Complications Trial/Epidemiology Of Diabetes Interventions And Complications Research Group 2003 Sustained effect

of intensive treatment of type 1 diabetes mellitus on development and progression of diabetic nephropa-thy: the Epidemiology of Diabetes Interventions and

Complications (EDIC) study JAMA 290:2159–2167.

Yabe-Nishimura C 1998 Aldose reductase in glucose icity: a potential target for the prevention of diabetic

tox-complications Pharmacol Rev 50:21–33.

Yamagishi S, Hsu CC, Kobayashi K, Yamamoto H 1993 Endothelin 1 mediates endothelial cell-dependent

proliferation of vascular pericytes Biochem Biophys Res

Commun 191:840–846.

Yamagishi S, Kobayashi K, Yamamoto H 1993 Vascular pericytes not only regulate growth, but also preserve prostacyclin-producing ability and protect against lipid peroxide-induced injury of co-cultured endothelial

cells Biochem Biophys Res Commun 190:418–425.

Yamagishi S, Hsu CC, Taniguchi M et al 1995 mediated toxicity to pericytes of advanced glycosyla-tion end products: a possible mechanism of pericyte

Receptor-loss in diabetic microangiopathy Biochem Biophys Res

Commun 213:681–687.

Yamagishi S, Yamamoto Y, Harada S, Hsu CC, Yamamoto

H 1996 Advanced glycosylation end products late the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with

stimu-their receptors FEBS Lett 384:103–106.

Yamagishi S, Yonekura H, Yamamoto Y et al 1997 Advanced glycation end products-driven angiogenesis

in vitro Induction of the growth and tube formation of human microvascular endothelial cells through auto-

crine vascular endothelial growth factor J Biol Chem

272:8723–8730

Yamagishi S, Fujimori H, Yonekura H, Yamamoto Y, Yamamoto H 1998 Advanced glycation endproducts inhibit prostacyclin production and induce plasmi-nogen activator inhibitor-1 in human microvascular

endothelial cells Diabetologia 41:1435–1441.

factor-alpha through nuclear factor-kappa B, and by

17beta-estradiol through Sp-1 in human vascular

endo-thelial cells J Biol Chem 275:25781–25790.

The Diabetes Control and Complications Trial Research

Group 1993 The effect of intensive treatment of

diabetes on the development and progression of

long-term complications in insulin-dependent diabetes

mellitus N Engl J Med 329:977–986.

The Diabetes Control and Complications Trial/Epidemiology

of Diabetes Interventions and Complications Research

Group 2000 Retinopathy and nephropathy in patients

with type 1 diabetes four years after a trial of intensive

therapy N Engl J Med 342:381–389.

Thornalley PJ 2003 Use of aminoguanidine (Pimagedine)

to prevent the formation of advanced glycation

end-products Arch Biochem Biophys 419:31–40.

Tombran-Tink J, Chader CG, Johnson LV 1991 PEDF:

Pigment epithelium-derived factor with potent

neuro-nal differentiative activity Exp Eye Res 53:411–414.

Tsuchida K, Makita Z, Yamagishi S et al 1999 Suppression

of transforming growth factor beta and vascular

endo-thelial growth factor in diabetic nephropathy in rats

by a novel advanced glycation end product inhibitor,

OPB-9195 Diabetologia 4:579–588.

UK Prospective Diabetes Study (UKPDS) Group 1998

Intensive blood-glucose control with sulphonylureas or

insulin compared with conventional treatment and risk

complications in patients with type 2 diabetes (UKPS

33) Lancet 352:837–853.

Uribarri J, Cai W, Sandu O, Peppa M, Goldberg T, Vlassarah

H 2005 Diet-derived advanced glycation end products

are major contributors to the body’s AGE pool and

induce infl ammation in healthy subjects Ann N Y Acad

Sci 1043:461–466.

Van den Enden MK, Nyengaard JR, Ostrow E, Burgan JH,

Williamson JR 1995 Elevated glucose levels increase

retinal glycolysis and sorbitol pathway metabolism

Implications for diabetic retinopathy Invest Ophthalmol

Vis Sci 36:1675–1685.

Vasan S, Foiles PG, Founds HW 2001 Therapeutic

poten-tial of AGE inhibitors and breakers of AGE protein

cross-links Expert Opin Investig Drugs 10:1977–1987.

Vlassara H, Bucala R, Striker L 1994 Pathogenic effects

of advanced glycosylation: biochemical, biologic, and

clinical implications for diabetes and aging Lab Invest

70:138–151

Vlassara H, Striker LJ, Teichberg S et al 1994 Advanced

glycation end products induce glomerular

sclero-sis and albuminuria in normal rats Proc Natl Acad Sci

U S A 91:11704–11708.

Vlassara H, Cai W, Crandall J et al 2002 Infl ammatory

mediators are induced by dietary glycotoxins, a major

risk factor for diabetic angiopathy Proc Natl Acad Sci

U S A 99:15596–15601.

Vlassara H, Palace MR 2002 Diabetes and advanced

glyca-tion endproducts J Intern Med 251:87–101.

Vlassara H 2005 Advanced glycation in health and

dis-ease: role of the modern environment Ann N Y Acad

Sci 1043:452–460.

Voziyan PA, Metz TO, Baynes JW, Hudson BG 2002 A

post-Amadori inhibitor pyridoxamine also inhibits

NOVEL CELLULAR PATHWAYS

538

Yamagishi S, Amano S, Inagaki Y, Okamoto T, Takeuchi M, Inoue H 2003 Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppress-ing vascular endothelial growth factor gene expres-

sion through anti-oxidative properties Microvasc Res

65:186–190

Yamagishi S, Amano S, Inagaki Y et al 2003 Angiotensin II-type 1 receptor interaction upregulates vascular endothelial growth factor messenger RNA levels

in retinal pericytes through intracellular reactive

oxygen species generation Drugs Exp Clin Res

29:75–80

Yamagishi S, Inagaki Y, Okamoto T et al 2003 Advanced glycation end products inhibit de novo protein synthe-sis and induce TGF-beta overexpression in proximal

tubular cells Kidney Int 63:464–473.

Yamagishi S, Takeuchi M, Inagaki Y, Nakamura K, Imaizumi

T 2003 Role of advanced glycation end products (AGEs) and their receptor (RAGE) in the pathogen-

esis of diabetic microangiopathy Int J Clin Pharmacol

Res 23:129–134.

Yamagishi S, Nakamura K, Takeuchi M, Imaizumi T 2004 Molecular mechanism for accelerated atherosclerosis

in diabetes and its potential therapeutic intervention

Int J Clin Pharmacol Res 24:129–134.

Yamagishi S, Imaizumi T 2005 Diabetic vascular tions: pathophysiology, biochemical basis and potential

complica-therapeutic strategy Curr Pharm Des 11:2279–2299.

Yamagishi S, Nakamura K, Inoue H 2005 Possible ticipation of advanced glycation end products in the

par-pathogenesis of osteoporosis in diabetic patients Med

Hypotheses 65:1013–1015.

Yamagishi S, Nakamura K, Inoue H, Kikuchi S, Takeuchi M

2005 Serum or cerebrospinal fl uid levels of hyde-derived advanced glycation end products (AGEs) may be a promising biomarker for early detection of

glyceralde-Alzheimer’s disease Med Hypotheses 64:1205–1207.

Yamagishi S, Takeuchi M, Matsui T et al 2005 Angiotensin

II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression

FEBS Lett 579:4265–4270.

Yamagishi S, Takeuchi M, Inoue H 2005 Olmesartan medoxomil, a newly developed angiotensin II type 1 receptor antagonist, protects against renal damage in advanced glycation end product (age)-injected rats

Drugs Exp Clin Res 31:45–51.

Yamagishi S, Matsui T, Nakamura K, et al 2006 Pigment epithelium-derived factor (PEDF) prevents diabetes-

or advanced glycation end products (AGE)-elicited

ret-inal leukostasis Microvasc Res 72:86–90.

Yamagishi S, Nakamura K, Matsui T 2006 Advanced tion end products (AGEs) and their receptor (RAGE)

glyca-system in diabetic retinopathy Curr Drug Discov Technol

3:83–88

Yamagishi S, Nakamura K, Matsui T et al 2006 Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeabil-ity by blocking reactive oxygen species-mediated vas-

cular endothelial growth factor expression J Biol Chem

281:20213–20220

Yamagishi S, Fujimori H, Yonekura H, Tanaka N, Yamamoto

H 1999 Advanced glycation endproducts accelerate

calcifi cation in microvascular pericytes Biochem Biophys

Res Commun 258:353–357.

Yamagishi S, Kawakami T, Fujimori H et al 1999 Insulin

stimulates the growth and tube formation of human

microvascular endothelial cells through autocrine

vascular endothelial growth factor Microvasc Res

57:329–339

Yamagishi S, Yonekura H, Yamamoto Y et al 1999 Vascular

endothelial growth factor acts as a pericyte mitogen

under hypoxic conditions Lab Invest 79:501–509.

Yamagishi S, Edelstein D, Du XL, Brownlee M 2001

Hyperglycemia potentiates collagen-induced platelet

activation through mitochondrial superoxide

overpro-duction Diabetes 50:1491–1494.

Yamagishi S, Edelstein D, Du XL, Kaneda Y, Guzman M,

Brownlee M 2001 Leptin induces mitochondrial

superoxide production and monocyte

chemoattrac-tant protein-1 expression in aortic endothelial cells by

increasing fatty acid oxidation via protein kinase A

J Biol Chem 276:25096–25100.

Yamagishi S, Takeuchi M, Makita Z 2001 Advanced

gly-cation end products and the pathogenesis of diabetic

nephropathy In Tomino Y, ed Type-2 Diabetic Nephropathy

in Japan, from Bench to Bedside Basel: Karger 30–35.

Yamagishi S, Amano S, Inagaki Y et al 2002 Advanced

glycation end products-induced apoptosis and

over-expression of vascular endothelial growth factor in

bovine retinal pericytes Biochem Biophys Res Commun

290:973–978

Yamagishi S, Amano S, Inagaki Y, Okamoto T, Takeuchi M,

Makita Z 2002 Beraprost sodium, a prostaglandin

I2 analogue, protects against advanced glycation end

products-induced injury in cultured retinal pericytes

Mol Med 8:546–550.

Yamagishi S, Inagaki Y, Amano S, Okamoto T, Takeuchi M

2002 Up-regulation of vascular endothelial growth

factor and down-regulation of pigment

epithelium-derived factor messenger ribonucleic acid levels in

leptin-exposed cultured retinal pericytes Int J Tissue

React 24:137–142.

Yamagishi S, Inagaki Y, Amano S, Okamoto T, Takeuchi

M, Makita Z 2002 Pigment epithelium-derived

fac-tor protects cultured retinal pericytes from advanced

glycation end product-induced injury through its

antioxidative properties Biochem Biophys Res Commun

296:877–882

Yamagishi S, Inagaki Y, Okamoto T et al 2002 Advanced

glycation end product-induced apoptosis and

overex-pression of vascular endothelial growth factor and

mono-cyte chemoattractant protein-1 in human- cultured

mesangial cells J Biol Chem 277:20309–20315.

Yamagishi S, Koga K, Inagaki Y et al 2002 Dilazep

hydro-chloride, an antiplatelet drug, prevents

progres-sion of diabetic nephropathy in Otsuka Long-Evans

Tokushima fatty rats Drugs Exp Clin Res 28:221–227.

Yamagishi S, Okamoto T, Amano S et al 2002

Palmitate-induced apoptosis of microvascular endothelial cells

and pericytes Mol Med 8:178–183.

Yamagishi S, Matsui T, Nakamura K et al 2007

Pigment-epithelium-derived factor suppresses expression of

receptor for advanced glycation end products in the

eye of diabetic rats Ophthalmic Res 39:92–97.

Yamagishi SI, Nakamura K, Matsui T, et al 2007 Pigment

epithelium-derived factor (PEDF) blocks advanced

glycation end product (AGE)-induced angiogenesis in

vitro Horm Metab Res 39:233–235.

Yamamoto Y, Kato I, Doi T et al 2001 Development and

prevention of advanced diabetic nephropathy in

RAGE-overexpressing mice J Clin Invest 108:261–268.

Yang CW, Vlassara H, Peten EP et al 1994 Advanced

glyca-tion end products up-regulate gene expression found

in diabetic glomerular disease Proc Natl Acad Sci U S A

91:9436–9440

Yokoi M, Yamagishi S, Takeuchi M et al 2005 Elevations

of AGE and vascular endothelial growth factor with

decreased total antioxidant status in the vitreous fl uid

of diabetic patients with retinopathy Br J Ophthalmol

89:673–675

Yoshida Y, Yamagishi S, Matsui T et al 2007 Positive

cor-relation of pigment epithelium-derived factor (PEDF)

and total anti-oxidant capacity in aqueous humor

of patients with uveitis and proliferative diabetic

reti-nopathy Br J Ophthalmol 91:1133–1134.

Yokoi M, Yamagishi S, Sato A et al 2007 Positive association

of pigment epithelium-derived factor (PEDF) with total anti-oxidant capacity in the vitreous fl uid of patients

with proliferative diabetic retinopathy Br J Ophthalmol

91:885–887

Yokoi M, Yamagishi S, Takeuchi M et al 2007 Positive relation between vitreous levels of advanced glycation end products and vascular endothelial growth factor in patients with diabetic retinopathy suffi ciently treated

cor-with photocoagulation Br J Ophthalmol 91:397–398.

Zill H, Bek S, Hofmann T et al 2003 RAGE-mediated MAPK activation by food-derived AGE and non-AGE

products Biochem Biophys Res Commun 300:311–315.

Ziyadeh FN, Goldfarb S 1991 The renal tubulointerstitium

in diabetes mellitus Kidney Int 39:464–475.

Ziyadeh FN, Hoffman BB, Han DC et al 2000 Long-term prevention of renal insuffi ciency, excess matrix gene expression, and glomerular mesangial matrix expan-sion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice

Proc Natl Acad Sci U S A 97:8015–8020.

DURING DIABETES MELLITUS

Kenneth Maiese, Zhao Zhong Chong, and Faqi Li

ABSTRACT

Our book Neurovascular Medicine: Pursuing Cellular

Longevity for Healthy Aging provides a unique

perspec-tive from a diverse group of internationally

recog-nized investigators with a broad range of experience

in neuronal, vascular, and immune-mediated disease

processes to translate basic cellular mechanisms into

viable therapeutic measures Yet, as with any form

of published literature, the work presented is not

all encompassing and intends to not only highlight

and explore new avenues to extend cell longevity for

healthy aging but also outline the potential concerns

and limitations of novel treatment approaches for

patients With this in mind, this concluding chapter

of the book serves to exemplify the raves and risks of

novel therapeutic strategies that are translational in

nature by focusing upon the complications of

oxida-tive stress and diabetes mellitus in the neuronal and

vascular systems.

Both type 1 and type 2 diabetes mellitus (DM)

can lead to signifi cant disability in the nervous and

cardiovascular systems, such as cognitive loss and

cardiac insuffi ciency Intimately connected to these

disorders in the nervous and vascular systems are the pathways of oxidative stress Furthermore, oxidative stress is a principal pathway for the destruction

of cells in several disease entities including tes mellitus As a result, innovative strategies that directly target oxidative stress to preserve neuronal and vascular longevity could offer viable therapeu- tic options to diabetic patients in addition to the more conventional treatments that are designed to control serum glucose levels Here we discuss the novel applications of nicotinamide, Wnt signaling, and erythropoietin (EPO) that modulate cellular oxi- dative stress and offer signifi cant promise for the prevention of diabetic complications in the nervous and vascular systems Essential to this process is the precise focus upon the cellular pathways governed

diabe-by nicotinamide, Wnt signaling, and EPO to avoid detrimental clinical complications and offer the development of effective and safe future therapy for patients.

Keywords: endothelial, neurodegeneration,

oxi-dative stress, erythropoietin, Wnt, FoxO, forkhead, nicotinamide, diabetes, cardiovascular.

THE CLINICAL RELEVANCE OF DM

IN THE NEUROVASCULAR SYSTEMS

D M is a signifi cant health concern in the

clinical population (Maiese, Chong, Shang

2007a) The disease is present in at least

16 million individuals in the United States

and in more than 165 million individuals

world-wide (Quinn 2001) Furthermore, by the year 2030,

it is predicted that more than 360 million

individu-als will be affected by DM (Wild, Roglic, Green et al

2004) At least 80% of all diabetic patients have type

2 DM, which is increasing in incidence as a result of

changes in human behavior relating to diet and daily

exercise (Laakso 2001) Although type 1

(insulin-dependent) DM accounts for only 5% to 10% of all

diabetic patients (Maiese, Morhan, Chong 2007c),

its incidence is increasing in adolescent minority

groups (Dabelea, Bell, D’Agostino et al 2007) Of

potentially greater concern is the incidence of

undi-agnosed diabetes that consists of impaired glucose

tolerance and fl uctuations in serum glucose levels

that can increase the risk for the development of DM

(Jacobson, Musen, Ryan et al 2007) Individuals with

impaired glucose tolerance have a more than two

times the risk for the development of diabetic

com-plications than individuals with normal glucose

toler-ance (Harris, Eastman 2000).

Both acute and long-term occurrence of type 1 and

type 2 DM can result in complications of the neuronal

and vascular systems For example, DM can impair

vascular integrity and alter cardiac output (Donahoe,

Stewart, McCabe et al 2007), which eventually

dimin-ish the capacity of sensitive cognitive regions of the

brain, leading to functional impairment and

demen-tia (Schnaider Beeri, Goldbourt, Silverman et al

2004; Chong, Li, Maiese 2005b; Li, Chong, Maiese

2006a) Disease of the nervous system can become

the most debilitating complication for DM and affect

the sensitive cognitive regions of the brain, such as

the hippocampus that modulates memory function,

resulting in signifi cant functional impairment and

dementia (Awad, Gagnon, Messier 2004) DM has

also been found to increase the risk for

vascu-lar dementia in elderly subjects (Schnaider Beeri,

Goldbourt, Silverman et al 2004; Xu, Qiu, Wahlin

et al 2004), as well as potentially alter the course of

Alzheimer’s disease Although some studies have

found that diabetic patients may have less neuritic

plaques and neurofi brillary tangles than nondiabetic

patients (Beeri, Silverman, Davis et al 2005),

con-trasting work suggests the modest adjusted relative

risk of Alzheimer’s disease in patients with diabetes

as compared with those without diabetes to be 1.3

(Luchsinger, Tang, Stern et al 2001) Furthermore,

costs to care for cognitive impairments resulting from diabetes that can mimic Alzheimer’s disease can approach $100 billion a year (McCormick, Hardy, Kukull et al 2001; Mendiondo, Kryscio, Schmitt 2001; Maiese, Chong 2004).

OXIDATIVE PATHWAYS AND DM

Closely tied to the development of insulin resistance and the complications of DM in the nervous and vascular systems is the presence of cellular oxida- tive stress and the release of reactive oxygen species (Maiese, Morhan, Chong 2007c) Oxidative stress occurs as a result of the development of reactive oxy- gen species that consist of oxygen free radicals and other chemical entities Oxygen consumption in organisms, or at least the rate of oxygen consumption

in organisms, has intrigued a host of investigators and may have had some of its origins in the work of Pearl Pearl proposed that increased exposure to oxygen through an increased metabolic rate could lead to

a shortened lifespan (Pearl, 1928) Subsequent work

by multiple investigators has furthered this esis by demonstrating that increased metabolic rates could be detrimental to animals in an environment

hypoth-of elevated oxygen (Muller, Lustgarten, Jang et al 2007) When one moves to more current work, oxygen free radicals and mitochondrial DNA mutations have become associated with oxidative stress injury, aging mechanisms, and accumulated toxicity in an organ- ism (Yui, Matsuura 2006).

Oxidative stress represents a signifi cant nism for the destruction of cells that can involve apo- ptotic neuronal and vascular cell injury (Lin, Maiese 2001; Chong, Li, Maiese 2006b; De Felice, Velasco, Lambert et al 2007) In fact, it has recently been shown that genes involved in the apoptotic process are replicated early during processes that involve cell replication and transcription, suggesting a much broader role for these genes than originally antici- pated (Cohen, Cordeiro-Stone, Kaufman 2007) Apoptotic-induced oxidative stress in conjunction with processes of mitochondrial dysfunction can con- tribute to a variety of disease states such as diabetes, ischemia, general cognitive loss, Alzheimer’s disease, and trauma (Chong, Li, Maiese 2005b, 2005d; Harris, Fox, Wright et al 2007; Leuner, Hauptmann, Abdel- Kader et al 2007; Okouchi, Ekshyyan, Maracine

mecha-et al 2007) Oxidative stress can lead to apoptosis in

a variety of cell types including neurons, endothelial cells (ECs), cardiomyocytes, and smooth muscle cells through multiple cellular pathways (Kang, Chong, Maiese 2003b; Chong, Kang, Maiese 2004a; Harris, Fox, Wright et al 2007; Karunakaran, Diwakar, Saeed

NOVEL CELLULAR PATHWAYS

542

with oxidative stress (Maiese, Chong 2004; Chong,

Li, Maiese 2005b) Blockade of the electron transfer chain at the fl avin mononucleotide group of complex

I or at the ubiquinone site of complex III results in the active generation of free radicals that can impair mitochondrial electron transport and enhance free radical production (Li, Chong, Maiese 2006a; Chong, Maiese 2007b) Furthermore, mutations in the mito- chondrial genome have been associated with the potential development of a host of disorders, such as hypertension, hypercholesterolemia, and hypomag- nesemia (Wilson, Hariri, Farhi et al 2004; Li, Chong, Maiese 2004b) Reactive oxygen species may also lead

to the induction of acidosis-induced cellular toxicity and subsequent mitochondrial failure (Chong, Li, Maiese 2005d) Disorders, such as hypoxia (Roberts, Chih 1997), diabetes (Cardella 2005; Kratzsch, Knerr, Galler et al 2006), and excessive free radical pro- duction (Ito, Bartunek, Spitzer et al 1997; Vincent, TenBroeke, Maiese 1999a, 1999b), can result in the disturbance of intracellular pH.

In disorders such as DM, elevated levels of ceruloplasmin have been suggested to represent increased concentration of reactive oxygen species (Memisogullari, Bakan 2004) and acute glucose fl uc- tuations have been described as a potential source

of oxidative stress (Monnier, Mas, Ginet et al 2006) Elevated serum glucose levels have also been shown

to lead to increased production of reactive oxygen species in ECs, but prolonged duration of hyperglyce- mia is not necessary to lead to oxidative stress injury, since even short periods of hyperglycemia can gener- ate reactive oxygen species in vascular cells (Yano, Hasegawa, Ishii et al 2004) Recent clinical correlates support these experimental studies to show that acute glucose swings in addition to chronic hyperglycemia can trigger oxidative stress mechanisms during type 2

DM, illustrating the importance of therapeutic ventions during acute and sustained hyperglycemic episodes (Monnier, Mas, Ginet et al 2006).

inter-The maintenance of cellular energy reserves and mitochondrial integrity also becomes a signifi cant factor in DM (Newsholme, Haber, Hirabara et al 2007) During DM, fatty acid accumulation leads to both the generation of reactive oxygen species and mitochondrial DNA damage (Rachek, Thornley, Grishko et al 2006) A decrease in the levels of mito- chondrial proteins and mitochondrial DNA in adi- pocytes has been correlated with the development of type 2 DM (Choo, Kim, Kwon et al 2006) In addi- tion, insulin resistance in the elderly has been linked

to fat accumulation and reduction in mitochondrial oxidative and phosphorylation activity (Petersen, Befroy, Dufour et al 2003; Pospisilik, Knauf, Joza

et al 2007).

et al 2007; Verdaguer, Susana Gde, Clemens et al

2007; Chong Li, Maiese et al 2007c).

Membrane phosphatidylserine (PS)

externaliza-tion is an early event during cell apoptosis (Maiese,

Vincent, Lin et al 2000; Mari, Karabiyikoglu, Goris

et al 2004) and can signal the phagocytosis of cells

(Lin, Maiese 2001; Chong, Kang, Li et al 2005e;

Li, Chong, Maiese 2006c) The loss of membrane

phospholipid asymmetry leads to the externalization

of membrane PS residues and assists microglia to

tar-get cells for phagocytosis (Maiese, Chong 2003; Kang,

Chong, Maiese 2003a, 2003b; Chong, Kang, Maiese

2003c; Mallat, Marin-Teva, Cheret 2005) This process

occurs with the expression of the phosphatidylserine

receptor (PSR) on microglia during oxidative stress

(Li, Chong, Maiese 2006a, 2006b), since blockade

of PSR function in microglia prevents the activation

of microglia (Kang, Chong, Maiese 2003a; Chong,

Kang, Maiese 2003b) As an example, externalization

of membrane PS residues occurs in neurons during

anoxia (Maiese, Boccone 1995; Vincent, Maiese 1999b;

Maiese 2001), during nitric oxide exposure (Maiese,

TenBroeke, Kue 1997; Chong, Lin, Kang et al 2003e),

and during the administration of agents that induce

the production of reactive oxygen species, such as

6-hydroxydopamine (Salinas, Diaz, Abraham et al

2003) Membrane PS externalization on platelets has

also been associated with clot formation in the

vascu-lar system (Leytin, Allen, Mykhaylov et al 2006).

The cleavage of genomic DNA into fragments

(Maiese, Ahmad, TenBroeke et al 1999; Maiese,

Vincent 2000a, 2000b) is considered to be a later

event during apoptotic injury (Chong, Kang Maiese

2004c) Several enzymes responsible for DNA

deg-radation have been differentiated and include the

acidic, cation-independent endonuclease (DNase II),

cyclophilins, and the 97-kDa magnesium-dependent

endonuclease (Chong, Li, Maiese 2005b; Chong,

Maiese 2007b) Three separate endonuclease

activi-ties are present in neurons that include a constitutive

acidic cation-independent endonuclease, a

constitu-tive calcium-/magnesium-dependent endonuclease,

and an inducible magnesium-dependent

endonu-clease (Vincent, Maiese 1999a; Vincent, TenBroeke,

Maiese 1999a).

During oxidative stress, the mitochondrial

mem-brane transition pore permeability is also increased

(Lin, Vincent, Shaw 2000; Di Lisa, Menabo, Canton

et al 2001; Chong, Kang, Maiese 2003a; Kang, Chong,

Maiese 2003b), a signifi cant loss of mitochondrial

nicotinamide adenine dinucleotide (NAD+) stores

occurs, and further generation of superoxide radicals

leads to cell injury (Maiese, Chong 2003; Chong, Lin,

Li et al 2005f) In addition, mitochondria are a

signif-icant source of superoxide radicals that are associated

organs of EPO production and secretion are the kidney, liver, brain, and uterus EPO production and secretion occurs foremost in the kidney (Fliser, Haller 2007) The kidney peritubular interstitial cells are responsible for the production and secretion of EPO (Fisher 2003) With the use of cDNA probes derived

from the EPO gene, peritubular ECs, tubular epithelial

cells, and nephron segments in the kidney have also been demonstrated to be vital cells for the production and secretion of EPO (Lacombe, Da Silva, Bruneval

et al 1991; Mujais, Beru, Pullman et al 1999) During periods of acute renal failure, EPO may provide assistance for the protection of nephrons (Sharples, Thiemermann, Yaqoob 2005; Sharples, Yaqoob 2006) Secondary sites of EPO production and secretion are the liver and the uterus (Chong, Kang, Maiese 2002c) Hepatocytes, hepatoma cells, and Kupffer cells of the liver can produce EPO (Fisher 2003), and in turn, EPO may provide a protective environment for these cells (Schmeding, Neumann, Boas-Knoop et al 2007) In regards to the uterine production of EPO,

it is believed that neonatal anemia that can occur in the early weeks after birth may partly result from the loss of EPO production and secretion by the placenta (Davis, Widness, Brace 2003) In the nervous system, the major sites of EPO production and secretion are in the hippocampus, internal capsule, cortex, midbrain, cerebral ECs, and astrocytes (Chong, Kang, Maiese 2002c; Li, Chong, Maiese 2004a) Further work has revealed several other organs as secretory tissues for EPO that include peripheral ECs (Anagnostou, Liu, Steiner et al 1994), myoblasts (Ogilvie, Yu, Nicolas- Metral et al 2000), insulin-producing cells (Fenjves, Ochoa, Cabrera et al 2003), and cardiac tissue (Maiese, Li, Chong 2005b; Fliser, Haller 2007).

As a strong cytoprotectant against oxidative stress, EPO can enhance the survival of a number of cells in the nervous system (Maiese, Li, Chong 2004, 2005b; Lykissas, Korompilias, Vekris et al 2007) (Table 22.1)

In cells of the brain or the retina, EPO can prevent injury from hypoxic ischemia (Chong, Kang, Maiese 2002b, 2003b; Yu, Xu, Zhang et al 2005; Liu, Suzuki, Guo et al 2006; Meloni, Tilbrook, Boulos et al 2006), excitotoxicity (Yamasaki, Mishima, Yamashita et al 2005; Montero, Poulsen, Noraberg et al 2007), infec- tion (Kaiser, Texier, Ferrandiz et al 2006), free radi- cal exposure (Chong, Kang, Maiese 2003a; Chong, Lin, Kang et al 2003d; Yamasaki, Mishima, Yamashita

et al 2005), amyloid exposure (Chong, Li, Maiese 2005c), staurosporine (Pregi, Vittori, Perez et al 2006), and dopaminergic cell injury (McLeod, Hong, Mukhida et al 2006) In addition, administration of EPO also represents a viable option for the prevention

of retinal cell injury during glutamate toxicity (Zhong, Yao, Deng et al 2007) and glaucoma (Tsai, Song, Wu

INNOVATIVE DIRECTIONS FOR

NEUROVASCULAR PROTECTION

DURING DM

Possible pathways that may decrease neuronal and

vascular longevity during DM are broad in scope and

involve multiple precipitating factors Yet, oxidative

stress-induced cellular signaling is believed to be a

signifi cant factor responsible for cell injury that is

initially set in motion following hyperglycemia For

example, studies have shown that administration of

insulin or insulin growth factor at concentrations

that were insuffi cient to reverse hyperglycemia could

nevertheless reduce oxidative stress injury to cells

and maintain mitochondrial inner membrane

poten-tial (Maiese, Chong, Shang 2007a; Maiese, Morhan,

Chong 2007c) As a result, innovative strategies that

directly target the reduction of oxidative stress

toxic-ity to neuronal and vascular cells could offer viable

therapeutic options to patients with DM in addition

to the more conventional treatments that are targeted

to control serum glucose levels.

A Growth Factor and Cytokine

EPO is a 30.4-kDa glycoprotein with approximately

50% of its molecular weight derived from

carbohy-drates (Maiese, Li, Chong 2005b) As a growth

fac-tor and cytokine, EPO is considered to be ubiquitous

in the body (Maiese, Chong, Shang 2007a; Maiese,

Morhan, Chong 2007c), since it can be detected in the

breath of healthy individuals (Schumann, Triantafi lou,

Krueger et al 2006) EPO may also provide

develop-mental cognitive support in humans, with the recent

observations that elevated EPO concentrations

dur-ing infant maturation have been correlated with

increased Mental Development Index scores (Bierer,

Peceny, Hartenberger et al 2006) Although EPO is

currently approved for the treatment of anemia, the

role of EPO has become far more reaching beyond

the need for erythropoiesis in other organs and

tis-sues, such as the brain, heart, and vascular system

(Chong, Kang, Maiese 2002b, 2003b; Moon, Krawczyk,

Paik et al 2006; Mikati, Hokayem, Sabban 2007; Um,

Gross, Lodish 2007; Chong, Maiese 2007a).

It is the discovery of EPO and the EPO receptor

(EPOR) in the nervous and vascular systems that has

resulted in a heightened level of interest and

enthu-siasm in the potential clinical applications of EPO,

such as in Alzheimer’s disease, cardiac insuffi ciency

(Palazzuoli, Silverberg, Iovine et al 2006; Assaraf,

Diaz, Liberman et al 2007), and cardiac

transplan-tation (Gleissner, Klingenberg, Staritz et al 2006;

Mocini, Leone, Tubaro et al 2007) The primary

NOVEL CELLULAR PATHWAYS

544

vasoconstriction, and enhance neuronal survival and functional recovery following subarachnoid hemor- rhage (Olsen 2003).

EPO also plays a signifi cant role in the cular system (Maiese, Li, Chong 2004, 2005b) and

cardiovas-in the renal system (Sharples, Yaqoob 2006) to limit injury from oxidative stress that can ultimately affect the function of the nervous system (Table 22.1) For example, in patients with anemia, EPO administra- tion can increase left ventricular ejection fraction and stroke volume (Goldberg, Lundin, Delano et al 1992) More recent studies have shown that patients with acute myocardial infarction have increased plasma EPO levels within 7 days of a cardiac insult, suggesting a possible protective response from the body (Ferrario, Massa, Rosti et al 2007) In addition, EPO administration in patients with anemia and con- gestive heart failure can improve exercise tolerance, renal function, and left ventricular systolic function (Palazzuoli, Silverberg, Iovine et al 2006, 2007) Tightly integrated with cardiac performance, pulmo- nary function is also believed to be enhanced during EPO administration, especially in the setting of isch- emic reperfusion injury of the lung (Wu, Ren, Zhu

et al 2006) Serum levels of EPO may also function

et al 2007) Systemic application of EPO also can

improve functional outcome and reduce cell loss

dur-ing spinal cord injury (Kdur-ing, Averill, Hewazy et al

2007; Okutan, Solaroglu, Beskonakli et al 2007),

traumatic cerebral edema (Verdonck, Lahrech,

Francony et al 2007), cortical trauma (Cherian,

Goodman, Robertson 2007), and epileptic activity

(Mikati, Hokayem, Sabban 2007; Nadam, Navarro,

Sanchez et al 2007) In direct relation to the

poten-tial cerebroprotective effects of EPO, enhanced

sur-vival by EPO also extends to afford protection to the

neurovascular unit during cerebral vascular disease

(Maiese, Chong 2004; Keogh, Yu, Wei 2007) In

addi-tion, EPO can protect sensitive hippocampal neurons

from both focal and global ischemic brain injury

(Yu, Xu, Zhang et al 2005; Zhang, Signore, Zhou

et al 2006) Systemic administration of EPO also

rep-resents a viable option for several other disorders

EPO administration for retinal cell injury can

pro-tect retinal ganglion cells from apoptosis (Grimm,

Wenzel, Groszer et al 2002); EPO can also improve

functional outcome and reduce lipid peroxidation

during spinal cord injury (Kaptanoglu, Solaroglu,

Okutan et al 2004), and can maintain

autoregula-tion of cerebral blood fl ow, reverse basilar artery

Table 22.1 Therapeutic Potential and Adverse Effects of Erythropoietin

Diabetes mellitus Cytoprotection

Cardiac function improvement

Silverberg et al 2006; Chong et al 2007bAlzheimer’s disease Neuroprotection Chong et al 2005c; Assaraf et al 2007

Epilepsy Decrease epileptic activity Mikati et al 2007; Nadam et al 2007

Parkinson’s disease Reduce functional diability McLeod et al 2006

Cardiac transplantation Resolution of anemia Gleissner et al 2006

Congestive heart failure or anemia Functional tolerance is increased,

improvement in left ventricular function and renal function

Maiese et al., 2005b; Palazzuoli et al

2006, 2007Chronic heart failure Functional capacity is increased Goldberg et al 1992; Mancini et al 2003

Acute renal failure Nephron protection Sharples et al 2005; Sharples, Yaqoob 2006Cerebral ischemia Neuroprotection Yu et al 2005; Zhang et al 2006

Subarachnoid hemorrhage Autoregulation of cerebral blood

fl ow, basilar artery dilation, and neuroprotection

Olsen 2003

Neurotrauma Neuroprotection and functional

improvement

King et al 2007; Okutan et al 2007;

Verdonck et al 2007; Cherian et al 2007

Adverse effects

Vascular intima hyperplasia Excessive neointima formation Reddy et al 2007

Cardiac dysfunction Potential impaired prognosis with

elevated erythropoietin levels

van der Meer et al 2007Cancer progression Tumor cell growth is increased,

progression of metastases, survival of cancer patients is decreased

Leyland-Jones et al 2005; Hardee et al

2006; Lai, Grandis 2006

offer an attractive alternative therapy to maintain proper cellular metabolism and mitochondrial mem- brane potential (∆ Ψm ) during DM (Fig 22.1) In clini- cal studies with DM, plasma EPO level is often low in diabetic patients with anemia (Mojiminiyi, Abdella, Zaki et al 2006) or without anemia (Symeonidis, Kouraklis-Symeonidis, Psiroyiannis et al 2006) Furthermore, the failure of these individuals to pro- duce EPO in response to a declining hemoglobin level suggests an impaired EPO response in diabetic patients (Thomas, Cooper, Tsalamandris et al 2005) Yet, increased EPO secretion during diabetic preg- nancies may represent the body’s attempt at endog- enous protection against the complications of DM (Teramo, Kari, Eronen et al 2004) Similar to the

as a biomarker of cardiovascular injury (Fu, Van Eyk

2006) Work from experimental studies illustrates that

EPO plays a critical role in the vascular and renal

sys-tems by maintaining erythrocyte (Foller, Kasinathan,

Koka et al 2007) and podocyte (Eto Wada, Inagi

et al 2007) integrity , regulating the survival of ECs

(Chong, Kang, Maiese 2002b, 2003a), and acting as

a powerful endogenous protectant during cardiac

injury (Asaumi, Kagaya, Takeda et al 2007).

In light of the fact that during elevated glucose

concentrations antioxidants can block free radical

production and prevent the production of

advan-ced glycation end-products known to produce reac

-tive oxy gen species and oxidative stress during

DM (Giardino, Edelstein, Brownlee 1996), EPO may

DNAfragmentation

Apoptosis

Cytochrome candcaspase releaseIAPs

14-3-3p-FOXO3aFOXO3a

Akt

Pl 3-K

Nicotinamide

LRP5/6Frizzled

β-CateninLef/Tcf

Genetranscription

PS exposure

Activation ofmicroglia

Phagocytosis

p-β-CateninMito∆Ψm↓

IKK

IκBNF-κB

p

p

p

Bcl-xL

Figure 22.1 Erythropoietin (EPO), nicotinamide, and Wnt use diverse as well as common pathways to foster cellular longevity EPO and

the EPO receptor (EPOR) can increase cellular longevity through protein kinase B (Akt), the forkhead transcription factor family member FOXO3a, glycogen synthase kinase-3β (GSK-3β), nuclear factor-κB (NF-κB), and Bcl-xL Similar to EPO, nicotinamide modulates the activ-ity of FOXO3a through phosphorylation (p) along with 14–3-3 protein and can maintain cellular integrity and prevent infl ammatory activa-tion of microglia that ultimately can lead to apoptosis through the maintenance of mitochondrial membrane potential (∆Ψm), the release

of cytochrome c, and the prevention of caspase activation Wnt signaling begins with Frizzled receptors resulting in the activation of Dishevelled followed by the inhibition of glycogen synthase kinase (GSK-3β) through phosphorylation (p) The suppressed GSK-3β along with other Wnt signaling complexes prevents phosphorylation (p) of β-catenin and leads to the accumulation of β-catenin β-catenin enters into cellular nucleus and contributes to the formation of lymphocyte enhancer factor/T cell factor (Lef/Tcf) and the β-catenin com-plex that leads to gene transcription, resulting in cellular proliferation, differentiation, survival, and apoptosis Interconnected pathways with EPO, nicotinamide, and Wnt involve IκB kinase (IKK), IκB, inhibitors of apoptotic protein (IAPs), GSK-3β, NF-κB, mitochondrial mem-brane potential (∆Ψm), and cytochrome c Ultimately, these pathways converge upon early apoptotic injury with phosphatidylserine (PS) exposure and later apoptotic DNA degradation that can impact the activation of microglia PI3-K, phosphatidylinositol-3-kinase

NOVEL CELLULAR PATHWAYS

546

2004a), heat-acclimation protection (Shein, Tsenter, Alexandrovich et al 2007), metabotropic receptor signaling (Maiese, Chong, Li 2005a; Chong, Kang Li

et al 2005e; Chong, Li, Maiese 2006a), cell metabolic pathways (Maiese, Chong 2003; Chong, Lin, Li et al 2005f), and oxidative stress (Kang, Chong, Maiese 2003a, 2003b; Chong, Kang, Maiese 2004a), increases cell survival and protects against these toxic insults Cytoprotection through Akt also can involve control

of infl ammatory cell activation (Chong, Kang, Maiese 2003a; Kang, Chong, Maiese 2003a, 2003b), tran- scription factor regulation (Chong, Maiese 2007a), maintenance of mitochondrial membrane potential (∆ Ψm), prevention of cytochrome c release (Chong, Kang, Maiese 2003a, 2003b; Chong, Lin, Kang et al 2003d), and blockade of caspase activity (Chong, Kang, Maiese 2002b, 2003a, 2003b), each of which is relevant to the protection offered by EPO (Maiese, Chong, Kang 2003) (Table 22.2).

Other studies suggest that EPO interfaces with the mammalian forkhead transcription factor family that oversees processes that can involve cell metabo- lism, hormone modulation, and apoptosis (Cuesta, Zaret, Santisteban 2007; Maiese, Chong, Shang 2007a; Maiese, Chong, Shang 2007b) The fi rst mem-

ber of this family was the Drosophila melanogaster gene Forkhead Since this time, more than 100 forkhead

genes and 19 human subgroups extending from FOXA

to FOXS have been discovered (Maiese, Chong, Shang

2007b) The forkhead box (FOX) family of genes is

characterized by a conserved forkhead domain monly noted as a “forkhead box” or a “winged helix”

com-as a result of the butterfl y-like appearance on X-ray crystallography (Clark, Halay, Lai et al 1993) and nuclear magnetic resonance (Jin, Marsden, Chen

et al 1998) All Fox proteins contain the 100-amino acid winged helix domain, but it should be noted that not all winged helix domains are Fox proteins (Larson, Eilers, Menon et al 2007).

potential protective role of insulin (Duarte, Proenca,

Oliveira et al 2006), EPO administration has been

shown both in diabetic and nondiabetic patients

with severe, resistant congestive heart failure to

decrease fatigue, increase left ventricular ejection

fraction, and signifi cantly decrease the number of

hospitalization days (Silverberg, Wexler, Iaina et al

2006) In studies that examine the toxic effects of

elevated glucose levels upon vascular cells, EPO

was found to be protective and prevent early

apop-totic membrane PS exposure and late DNA

degrada-tion at concentradegrada-tions that were clinically relevant

(Chong, Kang, Maiese 2002b) to cellular protection

in patients with cardiac or renal disease

(Mason-Garcia, Beckman, Brookins et al 1990; Namiuchi,

Kagaya, Ohta et al 2005).

Also relevant to cellular metabolism and DM

man-agement, cellular protection by EPO is closely tied

to protein kinase B (Akt) to prevent cell injury and

the subsequent induction of the apoptotic cascades

(Chong, Kang, Maiese 2002b; Mikati, Hokayem,

Sabban 2007; Chong, Maiese 2007a) (Fig 22.1)

Phosphorylation of Akt leads to its activation and

protects cells against genomic DNA degradation and

membrane PS exposure (Chong, Kang, Maiese 2003a,

2003b; Chong, Lin, Kang et al 2003d) Upregulation

of Akt activity during multiple injury paradigms,

such as vascular and cardiomyocyte ischemia (Parsa,

Matsumoto, Kim et al 2003; Miki, Miura, Yano et al

2006), free radical exposure (Matsuzaki, Tamatani,

Mitsuda et al 1999; Chong, Kang, Maiese 2003b),

N-methyl-d-aspartate toxicity (Dzietko,

Felderhoff-Mueser, Sifringer et al 2004), hypoxia (Chong, Kang,

Maiese 2002b; Zhang, Park, Gidday et al 2007),

β-amyloid toxicity (Nakagami, Nishimura, Murasugi

et al 2002; Du, Ohmichi, Takahashi et al 2004;

Chong, Li, Maiese 2005c), DNA damage (Henry,

Lynch, Eapen et al 2001; Chong, Kang, Maiese 2002b;

Kang, Chong, Maiese 2003a; Chong, Kang, Maiese

Table 22.2 Cellular Pathways Modulated by Erythropoietin

Cellular Mechanisms Possible Biological and Clinical Effects Selected References

Akt activation and maintenance of

mitochondrial potential

Inhibition of cytochrome c release and apoptosis; increase in cell survivalInhibition of infl ammatory cell activationBlockade of caspase activation

Chong et al 2002b; Parsa et al 2003; Miki

et al 2006; Chong, Maiese 2007; Mikati et al 2007

Chong et al 2003dChong et al 2002b, 2003a, 2003b, 2003dFOXO3a inactivation Inhibition of FOXO3a activation,

maintenance of FOXO3a in the cytoplasm

Chong, Maiese 2007a

Nuclear factor (NF)-κB activation Inhibition of apoptosis against oxidative

stress

Bittorf et al 2001; Chong et al 2005c;

Spandou et al 2006; Li et al 2006cWnt signaling Increase of Wnt expression, cytoprotection

of vascular cells during elevated glucose

Chong et al 2007b

GSK-3β inactivation Inhibition of cell injury, potential benefi ts

with exercise against diabetes mellitus

Howlett et al 2006; Li et al 2006c;

Wu et al 2007; Chong et al 2007b

can affect Akt signaling and prevent FoxO3a tion and nuclear translocation (Anitha, Gondha, Sutliff et al 2006) Interestingly, the ability of Akt to also inhibit pyruvate dehydrogenase kinase 4 expres- sion, a protein that conserves gluconeogenic sub- strates during DM, requires the inhibition of FoxO3a activity (Kwon, Huang, Unterman et al 2004).

activa-As a result, FoxO3a has emerged as an important target for DM Akt can phosphorylate FoxO3a and inhibit its activity to sequester FoxO3a in the cytoplasm

by association with 14–3–3 proteins (Brunet, Kanai, Stehn et al 2002; Kino, De Martino, Charmandari

et al 2005; Dong, Kang, Gu et al 2007; Munoz-Fontela, Marcos-Villar, Gallego et al 2007; Chong, Maiese 2007a) (Fig 22.1) In the absence of inhibitory Akt1 phosphorylation, FoxO3a is active, can translocate to the nucleus, and controls a variety of functions that involve cell cycle progression, cell longevity, and apop- tosis (Lehtinen, Yuan, Boag et al 2006; Li, Chong, Maiese 2006a; Maiese, Chong, Shang 2007a) Control

of FoxO3a is considered to be a viable therapeutic target for agents such as metabotropic glutamate receptors (Chong, Li, Maiese 2006a), neurotrophins (Zheng, Kar, Quirion 2002), and cytokines such as EPO (Chong, Maiese 2007a) to increase cell survival (Table 22.2) EPO controls the phosphorylation and degradation of FoxO3a to retain it in the cytoplasm through binding to 14–3–3 protein and to foster vas- cular cell protection during oxidative stress (Chong, Maiese 2007a) (Fig 22.2).

Cytoprotection by EPO also is mediated through the activation of nuclear factor- κB (NF-κB) tied to Akt (Fig 22.1) NF- κB proteins are composed of several homo- and heterodimer proteins that can bind to common DNA elements It is the phospho- rylation of I κB proteins by the IκB kinase (IKK) and their subsequent degradation that lead to the release of NF- κB for its translocation to the nucleus

to initiate gene transcription (Hayden, Ghosh 2004) Dependent upon Akt-controlled pathways, the trans- activation domain of the p65 subunit of NF- κB is activated by IKK and the IKK α catalytic subunit to lead to the induction of protective antiapoptotic path- ways (Chong, Li, Maiese 2005a) Increased expres- sion of NF- κB during injury can occur in cells, such

as infl ammatory microglial cells (Chong, Li, Maiese 2005c; Guo, Bhat 2006; Chong, Li, Maiese 2007c) and neurons (Sanz, Acarin, Gonzalez et al 2002) NF- κB represents a critical pathway that is responsible for the activation of inhibitors of apoptotic proteins (IAPs), the maintenance of Bcl-xL expression (Chen, Edelstein, Gelinas 2000; Chong, Li, Maiese 2005d), and protection against cell injury during oxidative stress (Chong, Li, Maiese 2005c) EPO employs NF- κB

to prevent apoptosis through the enhanced sion and translocation of NF- κB to the nucleus to

expres-Of the forkhead transcription factors, FOXO3a is

one member that exemplifi es the ability to function

as a versatile component during normal

physiologi-cal conditions as well as during disorders such as DM

(Maiese, Chong, Shang 2007b) The nomenclature

for human Fox proteins places all letters in

case, otherwise only the initial letter is listed as

upper-case for the mouse, and for all other chordates the

initial and subclass letters are in uppercase FOXO3a

appears to be involved in several pathways

responsi-ble for cell metabolism, DM onset, and diabetic

com-plications (Maiese, Li, Chong 2004; Maiese, Chong,

Li 2005a; Maiese, Li, Chong 2005b; Chong, Maiese

2007b) A clinical study of 734 individuals that

exam-ined all exons of the FOXO genes—FOXO1a, FOXO3a,

and FOXO4—found one promoter single nucleotide

polymorphism in the 5’ fl anking region of FOXO3a

that displayed a signifi cant association with body mass

index such that the highest body mass index was

pres-ent in individuals who were homozygous for this allele

(Kim, Jung, Bae et al 2006) Although other

stud-ies have reported that haplotype analyses of FOXO1a

rather than FOXO3a in individuals is associated with

higher HbA1c levels to suggest evidence of at least

an association with disorders of glucose intolerance,

FOXO3a haplotypes also have been associated with an

increased risk for stroke (Kuningas, Magi, Westendorp

et al 2007) In addition, the human immunodefi

-ciency virus (HIV) 1 accessory protein Vpr has been

reported to contribute to insulin resistance in HIV

patients by interfering with FoxO3a signaling with

protein 14–3–3 (Kino, De Martino, Charmandari

et al 2005).

Experimental work on DM has indicated that

administration of a high-fat diet in animals that lead

to hyperinsulinemic insulin-resistant obesity was

associated with an increased expression of FoxO3a

(Relling, Esberg, Fang et al 2006) Some studies

have suggested that FoxO3a may be benefi cial

dur-ing elevated glucose exposure and DM For example,

interferon γ–driven expression of tryptophan

catab-olism by cytotoxic T-lymphocyte antigen 4 may

acti-vate FoxO3a to protect dendritic cells from injury in

nonobese diabetic mice (Fallarino, Bianchi, Orabona

et al 2004) Yet, the role of forkhead transcription

fac-tors can vary among different cell types and tissues

Mice overexpressing FoxO1 in skeletal muscle suffer

from reduced skeletal muscle mass and poor glycemic

control (Kamei, Miura, Suzuki et al 2004) Additional

investigations have linked diabetic nephropathy to

FoxO3a by demonstrating that phosphorylation of

FoxO3a increases in rat and mouse renal cortical

tis-sues 2 weeks after the induction of diabetes by

strep-tozotocin (Kato, Yuan, Xu et al 2006) Furthermore,

enteric neurons can be protected from hyperglycemia

by glial cell line–derived neurotrophic factors that

NOVEL CELLULAR PATHWAYS

548

relevant to diabetic patients with renal failure, tinamide has been shown to also reduce intestinal absorption of phosphate and prevent the development

nico-of hyperphosphatemia and progressive renal tion (Eto, Miyata, Ohno et al 2005) In animal and cell culture studies, nicotinamide can also maintain normal fasting blood glucose in animals with strep- tozotocin-induced diabetes (Reddy, Bibby, Wu et al 1995b; Hu, Wang, Wang et al 1996), reduce periph- eral nerve injury during elevated glucose (Stevens, Li, Drel et al 2007), lead to the remission of type 1 DM

dysfunc-in mice with acetyl-1-carnitdysfunc-ine (Cresto, Fabiano de Bruno, Cao et al 2006), and inhibit oxidative stress pathways that lead to apoptosis (Chong, Lin, Maiese 2002a; Chong, Lin, Li et al 2005f; Ieraci, Herrera 2006; Chlopicki, Swies, Mogielnicki et al 2007; Hara, Yamada, Shibata et al 2007) (Table 22.3).

Nicotinamide can exert protean endocrine effects in the body (Aoyagi, Archer 2008) and derive its protective capacity through a number of cellular pathways In addition to the neuroprotec- tive attributes of nicotinamide (Chong, Lin, Maiese 2004b; Anderson, Bradbury, Schneider 2006; Feng, Paul, LeBlanc 2006), one potential pathway to con- sider for the protective capacity of nicotinamide in

DM involves the maintenance of vascular integrity (Maiese, Chong 2003; Li, Chong, Maiese 2004b, 2006a) For example, nicotinamide can protect the function of the blood–brain barrier (Hoane, Kaplan,

elicit antiapoptotic gene activation (Bittorf, Buchse,

Sasse et al 2001; Chong, Li, Maiese 2005c; Spandou,

Tsouchnikas, Karkavelas et al 2006; Li, Chong, Maiese

2006c) (Table 22.2).

A Precursor for the Coenzyme 𝛃-Nicotinamide

Adenine Dinucleotide

As the amide form of niacin or vitamin B3,

nicotin-amide plays a critical role in cellular metabolism

and can offer signifi cant neuronal and vascular cell

protection during a wide range of disorders that

include DM Nicotinamide is the precursor for the

coenzyme β-NAD+ and is essential for the

synthe-sis of nicotinamide adenine dinucleotide phosphate

(NADP+) (Maiese, Chong 2003; Li, Chong, Maiese

2004b) Nicotinamide and nicotinic acid can be

obtained either through synthesis in the body, such as

in the liver, or through a dietary source that is

rap-idly absorbed through the gastrointestinal epithelium

Once nicotinamide is available to the body, it is

uti-lized to synthesize NAD+ (Li, Chong, Maiese 2006a).

In clinical studies for DM, oral nicotinamide

pro-tects β-cell function, prevents clinical disease in islet

cell–antibody-positive fi rst-degree relatives of type 1

DM (Olmos, Hodgson, Maiz et al 2005), and in

com-bination therapy with insulin reduces HbA1c levels

(Crino, Schiaffi ni, Ciampalini et al 2005) Potentially

Figure 22.2 Erythropoietin (EPO) maintains FOXO3a in the cytoplasm during oxygen-glucose deprivation (OGD) Administration of

EPO (10 ng/ml) with an 8 hour period of OGD, OGD alone, or untreated cells (Control) was followed at 6 hours with immunofl uorescent staining for FOXO3a (Texas-red) in endothelial cells (ECs) The nuclei of ECs were counterstained with DAPI In merged images, control cells or cells with combined EPO and OGD show EC nuclei with minimal FOXO3a staining (control, blue/white, EPO/OGD, green/white) and show EC cytoplasm with signifi cant FOXO3a staining (red) This is in contrast to cells with OGD alone with signifi cant FOXO3a staining

in both the cytoplasm and the nuclei of ECs, demonstrating the ability of EPO to maintain FOXO3a in the cytoplasm

Shaw 2000; Chong, Lin, Maiese 2002a) (Table 22.3) Interestingly, nicotinamide appears to act directly at the level of mitochondrial membrane pore formation

to prevent cytochrome c release (Lin, Vincent, Shaw 2000; Chong, Lin, Maiese 2002a).

Nicotinamide can also prevent infl ammatory cell demise through the maintenance of membrane asym- metry, the activation of Akt, and the inhibition of cytokine release (Maiese, Chong 2003; Li, Chong, Maiese 2004b, 2006a) Nicotinamide blocks mem- brane PS externalization during a variety of insults that involve anoxia, free radical exposure, and oxy- gen-glucose deprivation (Lin, Vincent, Shaw 2000; Lin, Chong, Maiese 2001; Chong, Lin, Maiese 2002a) Nicotinamide regulates membrane PS exposure and microglial activation through activation of Akt, a cen- tral pathway for cytoprotection (Chong, Lin, Maiese 2004b) (Fig 22.1).

In addition to targeting the activity of brane PS exposure and microglial activation, nicoti- namide inhibits several proinfl ammatory cytokines, such as interleukin-1 β, interleukin-6, interleukin-8, tissue factor, and tumor necrosis factor α (TNF-α) (Reddy, Young, Ginn 2001; Chen, Wang, Hwang

mem-et al 2001a; Moberg, Olsson, Berne mem-et al 2003; Ungerstedt, Blomback, Soderstrom 2003) Nicotin- amide also can alter major histocompatibility complexes (Fukuzawa, Satoh, Muto et al 1997), inhibit intracellular adhesion molecule expression

Ellis et al 2006a; Hoane, Gilbert, Holland et al

2006b), infl uence arteriolar dilatation and blood fl ow

(Giulumian, Meszaros, Fuchs 2000), potentially lead

to decreased atherosclerotic plaque through

inhibi-tion of poly(ADP-ribose) polymerase

(Oumouna-Benachour, Hans, Suzuki et al 2007), and promote

platelet production through megakaryocyte

matura-tion (Giammona, Fuhrken, Papoutsakis et al 2006)

(Table 22.3) Nicotinamide can also maintain EC

viability during reactive oxygen species exposure

(Autor, Bonham, Thies 1984; Lin, Chong, Maiese

2001; Chong, Lin, Maiese 2002a; Maiese, Chong

2003) Nicotinamide is believed to be responsible for

the preservation of cerebral (Sadanaga-Akiyoshi, Yao,

Tanuma et al 2003) and endocardial (Bowes, Piper,

Thiemermann 1998; Cox, Sood, Hunt et al 2002)

ECs during models of oxidative stress Interestingly,

during periods of ischemia and oxidative stress,

aci-dosis-induced cellular toxicity may ensue (Chong, Li,

Maiese 2005d) and lead to subsequent mitochondrial

failure (Sensi, Jeng 2004) Yet, nicotinamide cannot

prevent cellular injury during intracellular acidifi

ca-tion paradigms (Lin, Vincent, Shaw 2000).

An alternative mechanism for nicotinamide may

require the maintenance of the mitochondrial

mem-brane potential (∆ Ψm) to protect cells from injury

(Fig 22.1) Nicotinamide can preserve mitochondrial

NAD-linked respiration and block the

depolariza-tion of the mitochondrial membrane (Lin, Vincent,

Table 22.3 Therapeutic Potential of Nicotinamide

Diabetes mellitus May prevent clinical disease

Reduces HbA1C levelsMaintains normal fasting blood glucose levels

in animal models with streptozotocinReduces peripheral nerve injury during elevated glucose

Olmos et al 2005Crino et al 2005Reddy et al 1995b; Hu et al 1996Stevens et al 2007

Traumatic brain injury Maintains the integrity of the BBB

Reduces cortical neuronal death and edema

Hoane et al 2006aHoane et al 2006bAtherosclerotic diseases Increases arteriolar dilation and blood fl ow

Decreases atherosclerotic plaquePromotes platelet production

Giulumian et al 2000Oumouna-Benachour et al 2007Giammona et al 2006

Oxidative stress Maintains EC viability

Inhibits PARP and protects human cardiac blasts and endocardial ECs

Maintains mitochondrial membrane potential

Autor et al 1984; Lin et al 2001; Maiese, Chong 2003

Bowes et al 1998; Cox et al 2002Lin et al 2000; Chong et al 2002aInfl ammation Inhibits microglial activation

Inhibits the release of interleukin-1β, -6, and -8, and TNF

Lin et al 2001; Chong et al 2004bReddy et al 2001; Chen et al 2001a;

Maiese, Chong 2003; Moberg et al

2003; Ungerstedt et al 2003Cytokine modulation Alters major histocompatibility complexes

Inhibits the expression of intracellular adhesion molecules

Modulates the production of TNFReduces demyelination

Fukuzawa et al 1997Hiromatsu et al 1992Fukuzawa et al 1997; Kaneko et al 2006BBB, blood–brain barrier; EC, endothelial cell; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor

NOVEL CELLULAR PATHWAYS

550

Patapoutian, Reichardt 2000; Li, Chong, Maiese 2005) The Wnt-FZD transduction pathway plays a signifi cant role in the control of the pattern of the body axis as well as in the development and matura- tion of the central nervous system (Augustine, Liu, Sadler 1993; Ikeya, Lee, Johnson et al 1997), cardio- vascular system (Marvin, Di Rocco, Gardiner et al 2001; Naito, Shiojima, Akazawa et al 2006; Palpant, Yasuda, MacDougald et al 2007; Singh, Li, Hamazaki

et al 2007), and the limbs (Kengaku, Twombly, Tabin 1997) (Table 22.4) During embryological develop- ment, alternations of the Wnt-FZD pathway can lead

to abnormal morphogenesis in animal models (Stark, Vainio, Vassileva et al 1994; Ikeya, Lee, Johnson

et al 1997; Liu, Wakamiya, Shea et al 1999) and congenital defects in humans (Jordan, Mohammed, Ching et al 2001; Rodova, Islam, Maser et al 2002; Niemann, Zhao, Pascu et al 2004) In mature tissues, the Wnt-FZD pathway is involved in the self-renewal of pluripotent embryonic stem cells (Bakre, Hoi, Mong

et al 2007) and bone formation (Canalis, Giustina, Bilezikian 2007), and may be responsible for the maintenance of many normal tissues (Ross, Hemati, Longo et al 2000; Reya, Duncan, Ailles et al 2003; Willert, Brown, Danenberg et al 2003; He, Zhang, Tong et al 2004) as well as cellular senescence (Liu, Fergusson, Castilho et al 2007) (Table 22.4) Other studies have revealed that dysfunction of the Wnt- FZD pathway can lead to neurodegenerative disor- ders, such as Alzheimer’s disease (Soriano, Kang, Fu

et al 2001; Marambaud, Shioi, Serban et al 2002; Morin, Medina, Semenov et al 2004; Balaraman, Limaye, Levey et al 2006; Chong, Li, Maiese 2007a) and heart failure (Barandon, Couffi nhal, Ezan

et al 2003; Barandon, Dufourcq, Costet et al 2005;

Li, Chong, Maiese 2006b; van de Schans, van den Borne, Strzelecka et al 2007).

(Hiromatsu, Sato, Yamada et al 1992), and modulate

the production of TNF in vascular cells (Fukuzawa,

Satoh, Muto et al 1997) that may be responsible for

the ability of nicotinamide to reduce

demyelina-tion in models of multiple sclerosis (Kaneko, Wang,

Kaneko et al 2006) However, translation of these

experimental studies to clinical effi cacy appears to

require further work, since some studies show that

oral nicotinamide administration following

endo-toxin challenge in healthy volunteers did not

dem-onstrate a signifi cant effect upon serum cytokine

levels (Soop, Albert, Weitzberg et al 2004).

Similar to EPO, nicotinamide may also require

other substrates of the Akt pathway, such as the

fork-head transcription factorFoxO3a, to prevent cell

injury (Fig 22.1) FoxO3a interfaces with several

path-ways that regulate cellular lifespan (Lehtinen, Yuan,

Boag et al 2006) and function to control neoplastic

growth (Li, Wang, Kong et al 2007) Given the

poten-tial treatment advantages of nicotinamide in DM, it

should be of interest that nicotinamide may be

cyto-protective through two separate mechanisms of

post-translational modifi cation of FoxO3a Nicotinamide

can not only maintain phosphorylation of FoxO3a

and inhibit its activity but also preserve the integrity

of the FoxO3a protein (Chong, Lin, Maiese 2004b)

to block FoxO3a proteolysis that can yield

poten-tially proapoptotic amino-terminal (Nt) fragments

(Charvet, Alberti, Luciano et al 2003).

Cysteine-Rich Glycosylated Wnt Proteins

Wnt proteins are secreted cysteine-rich glycosylated

proteins that can be dependent upon Akt signaling

and oversee embryonic cell proliferation,

differentia-tion, survival, and death (Li, Chong, Maiese 2006b;

Speese, Budnik 2007; Chong, Li, Maiese 2007a;

Chong, Shang, Maiese 2007b) More than 80 target

genes of Wnt signaling pathways have been

demon-strated in humans, mouse, Drosophila, Xenopus, and

zebrafi sh This representation encompasses several

cellular populations, such as neurons,

cardiomyo-cytes, endothelial cells, cancer cells, and

preadipo-cytes (Chong, Maiese 2004; Li, Chong, Maiese 2005)

In addition, at least 19 of 24 Wnt genes that express

Wnt proteins have been identifi ed in humans.

In general, all Wnt signaling pathways are

initi-ated by interaction of Wnt proteins with Frizzled

(FZD) receptors and by the binding of the Wnt

pro-tein to the FZD transmembrane receptor in the

pres-ence of the co-receptor LRP-5/6 (Mao, Wang, Liu

et al 2001) (Fig 22.1) Once Wnt protein binds to

the FZD transmembrane receptor and the

co-recep-tor LRP-5/6, Dishevelled, a cytoplasmic

multifunc-tional phosphoprotein, is recruited (Salinas 1999;

Table 22.4 Cellular Expression of Wnt Protein and the

Biological Response

Cellular Expression of Wnt Biological Response

Neurons Brain development and resistance

to injuryAstrocytes Brain development and protectionEndothelial cells Angiogenesis

Vascular smooth muscle cells

Angiogenesis, vascular remodeling, and cytoprotection

Progenitor cardiac stem cells

CardiomyogenesisEndocardial cells Endocardial cushion formationCardiomyocytes Cardiac remodeling and

cytoprotectionAdipocytes, bone cells Adipogenesis, metabolism, bone

formationCancer cells Cell growth

(Chong, Li, Maiese 2007a) Inhibition of the phatidylinositol-3-kinase (PI 3-K) pathway or gene silencing of Akt expression prevents Wnt from block- ing apoptotic injury and microglial activation (Chong,

phos-Li, Maiese 2007a).

Abnormalities in the Wnt signaling pathways,

such as with transcription factor 7-like 2 gene, may lead

to increased risk for type 2 DM in some populations (Grant, Thorleifsson, Reynisdottir et al 2006; Scott, Bonnycastle, Willer et al 2006; Lehman, Hunt, Leach

et al 2007), as well as have increased association with obesity (Guo, Xiong, Shen et al 2006) (Table 22.5) Additional work has described the expression of Wnt5b in adipose tissue, the pancreas, and the liver

in diabetic patients, suggesting a potential regulation

of adipose cell function (Kanazawa, Tsukada, Sekine

et al 2004) Clinical observations in patients with coronary artery disease and the combined metabolic syndrome with hypertension, hyperlipidemia, and

DM have indicated impaired Wnt signaling through a missense mutation in LRP-6 (Mani, Radhakrishnan, Wang et al 2007) Experimental studies in mice that develop hyperglycemia through a high-fat diet also demonstrate increased expression of some Wnt fam- ily members, such as Wnt3a and Wnt7a (Al-Aly, Shao, Lai et al 2007) Yet, intact Wnt family members may

Wnt signaling can prevent cell injury through

β-catenin/Tcf transcription-mediated pathways (Chen,

Guttridge, You et al 2001b) and against c-myc-induced

apoptosis through cyclooxygenase-2- and Wnt-induced

secreted protein (You, Saims, Chen et al 2002)

However, more recent work has linked Wnt

cytopro-tection in neuronal and vascular cells with more

unconventional pathways of Wnt that involve Akt

(Fig 22.1) For example, neuronal cell differentiation

that is dependent upon Wnt signaling and trophic

factor induction is blocked during the repression of

Akt activity (Fukumoto, Hsieh, Maemura et al 2001)

and Wnt differentiation of cardiomyocytes does not

proceed without Akt activation (Naito, Akazawa,

Takano et al 2005) Soluble secreted FZD-related

pro-teins, which can modulate Wnt signaling, also employ

Akt for cardiac tissue repair (Mirotsou, Zhang, Deb

et al 2007) (Table 22.5) Reduction in tissue injury

through Wnt signaling during pressure overload

car-diac hypertrophy is linked to Akt activation (van de

Schans, van den Borne, Strzelecka et al 2007), and

the benefi ts of cardiac ischemic preconditioning

appear to rely upon Akt (Barandon, Dufourcq, Costet

et al 2005) In the neuronal system, Wnt

overexpres-sion can independently increase the phosphorylation

and activation of Akt to promote neuronal protection

Table 22.5 Wnt Signaling Pathways in Disease

Physiological and

Pathological Entities

Wnt Signaling Components

Development and

maturation

Wnt-Frizzled activation Control of body pattern; normal

morphogenesis; self-renewal of pluripotent embryonic stem cells;

bone formation

Maintenance of normal tissuesCellular senescence

Augustine et al 1993; Kengaku

et al 1997; Ikeya et al 1997; Marvin et al 2001;

Natio et al 2006; Palpant

et al 2007; Singh et al 2007; Canalis et al 2007

Ross et al 2000; Reya et al 2003; Willert et al 2003; He et al 2004Liu et al 2007

Alzheimer’s disease Wnt-Frizzled dysfunction;

increased production of Aβ

Decrease in amyloid production and toxicity; increase in β-catenin degradation; increase in GSK-3β activity and decrease in β-catenin activity; increase in microglial activation

Soriano et al 2001; Marambaud

et al 2002; Morin et al 2004; Li

et al 2005; Balaraman et al 2006; Chong et al 2007a

Diabetes mellitus Increased expression of Wnt5b,

Wnt3a, Wnt7aAbnormalities of transcription factor 7-like 2 gene

Wnt expression

Association with obesityIncreased risk for type 2 diabetes mellitus

Decreased obesityHigh glucose-induced injury in ECs reduced with inhibition of GSK-3β;

mesangial cells protected

Guo et al 2006; Al-Aly et al 2007Grant et al 2006; Scott et al 2006; Lehman et al 2007;

Wright et al 2007;

Lin et al 2006; Chong et al 2007b

Myocardial infarction Over-expression

of Frizzled AWnt-Frizzled signaling modulation

Reduced cardiac infarction; enhanced ischemic preconditioning; infl uencedAkt activation; reduction in pressure overload–induced cardiac hypertrophy

Barandon et al 2003; Barandon

et al 2005;

Li et al 2006b; Van de Schans

et al 2007Cardiac repair Release of SFRP

modulates Wnt signaling

Akt activation with cardiac repair Mirotsou et al 2007

Aβ, beta-amyloid; EC, endothelial cell; GSK-3β, glycogen synthase kinase-3β; SFRP, secreted Frizzled-related protein

NOVEL CELLULAR PATHWAYS

552

prevents toxicity from high concentrations of glucose and increases rat β-cell replication, suggesting a pos- sible target of GSK-3 β for pancreatic β-cell regenera- tion (Mussmann, Geese, Harder et al 2007) Clinical applications for Wnt that involve GSK-3 β are attrac- tive (Rowe, Wiest, Chuang 2007), especially in con- cert with EPO (Table 22.2) For example, both the potential benefi ts of EPO to improve cardiovascular function in diabetic patients (Silverberg, Wexler, Sheps et al 2001; Silverberg, Wexler, Iaina et al 2006) and the positive effects of exercise to improve glycemic control during DM (Maiorana, O’Driscoll, Goodman et al 2002) appear to rely upon the inhibi- tion of GSK-3 β activity EPO blocks GSK-3β activity (Li, Chong, Maiese 2006c; Wu, Shang, Sun et al 2007; ChongShang, Maiese 2007b), and when combined with exercise, it may offer synergistic benefi ts, since physical exercise has also been shown to phosphory- late and inhibit GSK-3 β activity (Howlett, Sakamoto,

con-in the United States for EPO reported to approach

9 billion dollars (Donohue, Cevasco, Rosenthal 2007), adverse effects or lack of effi cacy during treat- ment with EPO is also becoming increasingly evident (Table 22.1) Some cardiac injury experimental mod- els do not consistently demonstrate a benefi t with EPO (Olea, Vera Janavel, De Lorenzi et al 2006), and elevated plasma levels of EPO independent of hemo- globin concentration can be associated with increased severity of disease in individuals with congestive heart failure (van der Meer, Voors, Lipsic et al 2004) or can contribute to vascular stenosis with intima hyperpla- sia (Reddy, Vasir, Hegde et al 2007) Other adverse conditions associated with EPO include increased incidence of thrombotic vascular effects, elevation

in mean arterial pressure, and increased metabolic rate and blood viscosity (Maiese, Li, Chong 2005b; Corwin, Gettinger, Fabian et al 2007) The potential progression of cancer has been another signifi cant concern raised with EPO administration (Maiese,

Li, Chong 2005c; Kokhaei, Abdalla, Hansson et al 2007) Not only has both EPO and its receptor been

offer glucose tolerance and increased insulin

sensi-tivity (Wright, Longo, Dolinsky et al 2007), as well

as protect glomerular mesangial cells from elevated

glucose–induced apoptosis (Lin, Wang Huang 2006)

(Table 22.5) Animals that overexpressed Wnt10b and

were placed on a high-fat diet had a reduction in body

weight, hyperinsulinemia, and triglyceride plasma

levels, and improved glucose homeostasis (Aslanidi,

Kroutov, Philipsberg et al 2007).

These clinical and experimental investigations

for the Wnt pathway suggest a potentially protective

cellular mechanism for Wnt during DM Recent in

vitro studies demonstrate that the Wnt1 protein is

necessary and suffi cient to provide cellular protection

during elevated glucose exposure (Chong, Shang,

Maiese 2007b) (Table 22.2) Administration of

exog-enous Wnt1 protein can signifi cantly prevent

apop-totic EC injury during elevated glucose exposure

Interestingly, this protection by Wnt1 can be regulated

by the growth factor and cytokine EPO (Maiese, Li,

Chong 2004, 2005b; Nangaku, Fliser 2007) Through

the Wnt pathway, EPO may offer an attractive therapy

to maintain proper cellular metabolism and

mitochon-drial membrane potential (∆ Ψm ) during conditions of

oxidative stress and DM In cell culture and animal

studies, EPO is cytoprotective during elevated glucose

levels (Chong, Shang, Maiese 2007b), and it has the

capacity to prevent the depolarization of the

mito-chondrial membrane, which also affects the release of

cytochrome c (Chong, Kang, Maiese 2002b; Chong,

Lin, Kang et al 2003d; Miki, Miura, Yano et al 2006)

With the Wnt pathway, EPO maintains the expression

of Wnt1 during elevated glucose exposure and

pre-vents the loss of Wnt1 expression that would normally

occur in the absence of EPO during elevated glucose

levels In addition, blockade of Wnt1 with a Wnt1

anti-body can neutralize the protective capacity of EPO,

illustrating that Wnt1 is a critical component in the

cytoprotection of EPO during elevated glucose

expo-sure (Chong, Shang, Maiese 2007b) (Table 22.5).

Interestingly, Wnt also can protect cells during

oxidative stress (Chong, Maiese 2004) and other toxic

injuries such as β-amyloid toxicity (Chong, Maiese

2004) through the modulation of glycogen synthase

kinase-3 β (GSK-3β) and β-catenin (Chong, Li, Maiese

2007a) (Fig 22.1) Inhibition of GSK-3 β activity can

increase cell survival during oxidative stress, and as a

result, GSK-3 β is considered to be a therapeutic

tar-get for some neurodegenerative disorders (Chong, Li,

Maiese 2005b; Balaraman, Limaye, Levey et al 2006;

Nurmi, Goldsteins, Narvainen et al 2006; Qin, Peng,

Ksiezak-Reding et al 2006) GSK-3 β also may infl

u-ence infl ammatory cell survival (Chong, Li, Maiese

2007c) and activation (Tanuma, Sakuma, Sasaki et al

2006) In metabolic disease, inactivation of GSK-3 β

by small molecule inhibitors or RNA interference

In the Wnt pathway, Wnt signaling can either facilitate or prevent apoptosis depending upon the environmental stimuli For example, Wnt proteins can enhance apoptosis within rhombomeres 3 and 5

in the developing hindbrain and in limb buds ing vertebrate limb development to control growth of the hindbrain and limbs (Ellies, Church, Francis-West

dur-et al 2000; Grotewold, Ruther 2002a, 2002b) Wnt signaling has also been closely linked to tumorigen- esis for a number of years (Li, Chong, Maiese 2006b; Emami, Corey 2007) Furthermore, in studies that involve DM, neuronal disorders, or vascular disease, it

is not consistently clear whether mutations in genes of the Wnt pathway or alterations in protein expression

of the Wnt pathway components during these ders confer protective or detrimental effects.

disor-For innovative strategies to effectively and safely work against a variety of disorders, future investiga- tions that utilize data from basic and clinical research must translate and integrate this knowledge to effec- tively balance the potential for high impact clinical success with the avoidance of treatment complications Paramount to achieving these goals is the targeted focus upon intricate and often common cellular pathways governed by potential strategies, such as EPO, nicotinamide, and Wnt signaling, to overcome the present challenges and controversies of existing

or developing therapies With such an approach, the fruitful development of new therapeutic agents

to preserve neuronal and vascular longevity during debilitating conditions such as DM will continue

to grow at an exponential pace to yield substantial benefi ts for clinical care.

Acknowledgment This work was supported by the following grants (KM): American Diabetes Association, American Heart Association (National), Bugher Foundation Award, Janssen Neuroscience Award, LEARN Foundation Award, MI Life Sciences Challenge Award, Nelson Foundation Award, NIH NIEHS (P30 ES06639), and NIH NINDS/NIA

REFERENCES

Al-Aly Z, Shao JS, Lai CF et al 2007 Aortic Msx2-Wnt

calci-fi cation cascade is regulated by TNF-alpha-dependent signals in diabetic Ldlr–/–mice Arterioscler Thromb Vasc

Biol 27:2589–2596.

Anagnostou A, Liu Z, Steiner M et al 1994 Erythropoietin receptor mRNA expression in human endothelial cells

Proc Natl Acad Sci U S A 91:3974–3978.

Anderson DW, Bradbury K A, Schneider JS 2006 roprotection in Parkinson models varies with toxin

Neu-administration protocol Eur J Neurosci 24:3174–3182.

Anitha M, Gondha C, Sutliff R et al 2006 GDNF rescues hyperglycemia-induced diabetic enteric neuropathy

demonstrated in tumor specimens, but under some

conditions EPO expression has also been suggested

to block tumor cell apoptosis through Akt (Hardee,

Rabbani, Arcasoy et al 2006), enhance tumor

pro-gression, increase metastatic disease (Lai, Grandis

2006), decrease survival in cancer patients

(Leyland-Jones, Semiglazov, Pawlicki et al 2005), and negate the

effects of radiotherapy by assisting with tumor

angio-genesis (Ceelen, Boterberg, Smeets et al 2007) When

evaluating the possible tumor-promoting ability of

EPO (Rades, Golke, Schild et al 2007), a number of

competing factors must be considered including the

possible benefi ts of EPO administration in patients

with cancer that involve the synergistic effects of

EPO with chemotherapeutic modalities (Sigounas,

Sallah, Sigounas 2004; Ning, Hartley, Molineux et al

2005), potential protection against chemotherapy

tis-sue injury (Joyeux-Faure 2007), and the treatment of

cancer-related anemia.

Nicotinamide also has been reported to have

diverse biological roles that include cellular lifespan

reduction Prolonged exposure to nicotinamide in

some studies can lead to impaired β-cell function and

reduction in cell growth (Reddy, Salari-Lak, Sandler

1995a; Liu, Green, Flatt et al 2004) Nicotinamide may

also inhibit P450 and hepatic metabolism (Gaudineau,

Auclair 2004) and play a role in the progression of

Parkinson’s disease if cellular compartmentation is

abruptly changed (Williams, Cartwright, Ramsden

2005) Under other conditions, nicotinamide has

been described as an agent that limits cell growth and

promotes cell injury Nicotinamide in the presence

of transforming growth factor β-1 can block hepatic

cell proliferation and lead to apoptosis with caspase

3 activation (Traister, Breitman, Bar-Lev et al 2005)

During moderate temperature hyperthermia or

car-bogen breathing, nicotinamide can also result in

enhanced solid tumor radiosensitivity and assist with

tumor load reduction (Griffi n, Ogawa, Williams et al

2005) In addition, nicotinamide offers cellular

pro-tection in millimole concentrations against oxidative

stress, but in relation to cell longevity, lower

concen-trations of nicotinamide can function as an inhibitor

of sirtuins, which are necessary for the promotion of

increased lifespan in yeast and metazoans (Porcu,

Chiarugi 2005; Li, Chong, Maiese 2006a; Saunders,

Verdin 2007) Interestingly, it has been postulated

that sirtuins may prevent nicotinamide from

assist-ing with DNA repair by alterassist-ing the accessibility of

DNA-damaged sites for repair enzymes (Kruszewski,

Szumiel 2005) Given the intimate and inverse

rela-tionship of sirtuins with nicotinamide and the latter’s

ability to alter cell longevity, alternative approaches for

the protection of neuronal and vascular cells during

DM may be required that may involve the tight

modu-lation of intracellular nicotinamide accumumodu-lation.