Role of h2s in acute and chronic inflammation

... expression and reduced H2S biosynthesis in the models of high fat fed and endotoxin tolerant mice These results give us an insight into the roles of H2S during the early stages of atherosclerosis and. .. from the H2S concentration at the site of inflammation, another factor which may play a part in determining the involvement of this gas in inflammation, is the duration and perhaps also the intensity... platelets and triggering of the clotting cascade Depending on the location of the clot, the thrombotic event may be serious or life-threatening H2S performs several roles during inflammation These include

ROLE OF HYDROGEN SULFIDE IN ACUTE AND CHRONIC

INFLAMMATION

DAVID NG SHEN WEN

(B.Sc (Hons.), NATIONAL UNIVERSITY OF SINGAPORE)

A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE

2014

ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude to my supervisor, Prof Philip Keith Moore for giving me the opportunity to work on this research project and his guidance I would also like

to thank my co-supervisor, A/Prof Low Chian Ming for his advice

I would like to thank Dr Mohamed Shirhan Bin Mohamed Atan, Dr Li Ling, Dr Tsai Chin-Yi, Ms Peh Meng Teng, Mr Huang Weihao Caleb and Ms Ng Li Theng, for their help over the course of the project

Finally, I would also like to thank my family for their support throughout my studies

TABLE OF CONTENTS

DECLARATION………I ACKNOWLEDGEMENTS……….II TABLE OF CONTENTS………III SUMMARY………VII LIST OF FIGURES……….VIII ABBREVIATIONS……….IX

CHAPTER 1: Introduction and Overview……… 1

1 Introduction……….2

1.1 Hydrogen Sulfide – overview and historical perspective………2

1.2 Physical and chemical properties of H2S……….2

1.3 Biosynthesis of H2S……….3

1.4 Biological effects and physiological functions of H2S………4

1.5 Research Objectives……….8

CHAPTER 2: Effects of administration of a high fat diet on H 2 S metabolism in mice…… 9

2 Introduction………10

2.1 Materials and methods……… 13

2.1.2 Measurement of tissue H2S synthesizing enzyme activity………13

2.1.3 Measurement of plasma H2S concentration……… 14

2.1.4 Western blotting for H2S synthesizing enzymes………15

2.1.5 Measurement of plasma cytokine and chemokine levels……… 16

2.1.6 Measurement of plasma SAA and CRP levels……… 16

2.1.7 Immunohistochemistry staining for CSE, CBS and 3-MST……… 16

2.1.8 Oil red O staining……… 17

2.1.9 Statistics……….17

2.2 Results………18

2.2.1 Effect of a high fat diet on plasma H2S concentration……… 18

2.2.2 Effect of a high fat diet on CSE, CBS and 3-MST expression………… 19

2.2.3 Effect of a high fat diet on tissue H2S synthesising activity……… 21

2.2.4 Effect of a high fat diet on vascular H2S synthesizing enzyme localisation……….23

2.2.5 Effect of a high fat diet on plasma cytokines and chemokines………… 25

2.2.6 Effect of a high fat diet on plasma SAA and CRP levels……… 28

2.2.7 Effect of a high fat diet on lipid deposition in aorta……… 29

2.3 Discussion……… 31

CHAPTER 3: Effects of endotoxin tolerance on H 2 S metabolism in mice……….35

3 Introduction………36

3.1 Materials and methods……… 39

3.1.1 Animals and treatment groups……… 39

3.1.2 Measurement of plasma IL-1β, IL-6 and TNF-α levels……….40

3.1.3 Measurement of myeloperoxidase activity in tissues………40

3.1.4 Haematoxylin and eosin staining……… 41

3.1.5 Measurement of plasma H2S concentration……… 41

3.1.6 Western blotting for H2S synthesizing enzymes………41

3.1.7 Measurement of tissue H2S synthesizing enzyme activity………41

3.1.8 Statistics……….42

3.2 Results………43

3.2.1 Effect of endotoxin tolerance on inflammatory cytokines in plasma……43

3.2.2 Effect of endotoxin tolerance on myeloperoxidase activity in tissues… 45

3.2.3 Effect of endotoxin tolerance on cell infiltration and tissue remodelling 46

3.2.4 Effect of endotoxin tolerance on plasma H2S concentration……….48

3.2.5 Effect of endotoxin tolerance on CBS, CSE and 3-MST expression……48

3.2.6 Effect of endotoxin tolerance on tissue H2S synthesising activity………51

3.3 Discussion……… 53

CHAPTER 4: Conclusion……… 55

4 Conclusion……….56

BIBLIOGRAPHY………58

H2S system as a therapeutic target in early atherosclerosis and endotoxin tolerance

LIST OF FIGURES

Figure 1 The biosynthesis of H2S within mammalian tissues……….4

Figure 2 Therapeutic targets for H2S……… 8

Figure 3 Plasma H2S concentration……… 18

Figure 4 Expression of CBS, CSE and 3-MST……….20

Figure 5 Biosynthesis of H2S………22

Figure 6 Representative photographs of IHC staining in aorta……….24

Figure 7 Plasma concentration of cytokines and chemokines……… 26

Figure 8 Plasma concentrations of biochemical markers of atherosclerosis………28

Figure 9 Representative photographs showing Oil red O staining for lipid in aorta………30

Figure 10 Plasma concentration of inflammatory cytokines………44

Figure 11 Myeloperoxidase activity……….46

Figure 12 Representative photographs of histological changes within liver and lung………….47

Figure 13 Plasma H2S concentration………48

Figure 14 Expression of CBS, CSE and 3-MST……… 50

Figure 15 Biosynthesis of H2S……… 52

Figure 16 Experimental flowchart…… ……… 57

ABBREVIATIONS Symbols Full name

3MP 3-mercaptopyruvate

3-MST 3-mercaptopyruvate sulfurtransferase

AP-1 Activator protein 1

ApoE-/- Apolipoprotein E-deficient

ATB-346 2-(6-methoxy-napthalen-2-yl)-propionic acid

4-thiocarbamoyl-phenyl ester BSA Bovine serum albumin

C19H42BrN Hexadecyltrimethylammonium bromide

cAMP Cyclic adenosine monophosphate

CARS Compensatory anti-inflammatory response syndrome

CAT Cysteine aminotransferase

CRP C-reactive protein

CSE Cystathionine γ lyase

DTT Dithiothreitol

ERK Extracellular signal-regulated kinases

FeCl3 Iron (III) chloride

G-CSF Granulocyte-colony stimulating factor

GMP Guanosine monophosphate

GYY4137 Morpholin-4-ium 4 methoxyphenyl (morpholino)

phosphinodithioate H2S Hydrogen sulfide

HCl Hydrochloric acid

HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A

HPLC High-performance liquid chromatography IACUC Institutional Advisory Care and Use Committee ICAM-1 Intracellular adhesion molecule 1

IFN-γ Interferon gamma

IHC Immunohistochemistry

IKK IκB kinase

IL Interleukin

KATP ATP-sensitive potassium

MCP-1 Monocyte chemotactic protein 1

MIP Macrophage inflammatory protein

MPO Myeloperoxidase

MyD88 Myeloid differentiation primary response gene 88

NaHS Sodium hydrosulfide

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NMDA N-methyl-D-aspartic acid

NO Nitric oxide

NUS National University of Singapore

OxLDL Oxidised low density lipoprotein

P5P Pyridoxal 5’-phosphate

PAMPs Pathogen-associated molecular patterns

PBS Phosphate buffered saline

PRR Pattern recognition receptors

RANTES Regulated on activation, normal T expressed and secreted ROS Reactive oxygen species

SAA Plasma serum amyloid A

SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of mean

TNF-α Tumor necrosis factor alpha

VCAM-1 Vascular cell adhesion molecule 1

Chapter 1

Introduction and Overview

1 Introduction

1.1 Hydrogen Sulfide – overview and historical perspective

Hydrogen sulfide (H2S) is a colourless gas with the distinctive odor of rotten eggs [1] Until recently, H2S was widely regarded as a noxious gas.H2S is naturally occurring within volcanoes, sulfur springs and swamps.H2S is also produced by the petrochemical industrial activities and by tanneries H2S has been known for many years to inhibit cytochrome c oxidase, an essential enzyme for mitochondrial respiration [2] Even with the characteristic odour of H2S, individuals may be unaware of its presence in the air as the sense of smell is severely compromised by H2S concentrations above 150 ppm [3] Exposure to H2S concentrations of above 500 ppm leads to respiratory paralysis, unconsciousness and may consequently result in death [4]

In the past two decades, the view that H2S is purely an environmental pollutant has been increasingly challenged and is no longer tenable Indeed, H2S is now seen as an endogenous biologically active molecule with a range of significant functions inside the body In this regard,

H2S influences numerous signaling processes within cells H2S is now classified alongside nitric oxide (NO) and carbon monoxide (CO) as gasotransmitters As the newest member of the gasotransmitter family there has been considerable interest in the biology of H2S

1.2 Physical and chemical properties of H 2 S

H2S is able to diffuse readily through plasma membranes without the help of membrane transporters H2S dissolves freely in water to form a weak acid which dissociates as follows: H2S

↔ HS- + H+ Dissolved H2S under physiological conditions of pH 7.4 at 37oC, will exist predominantly as the hydrosulfide anion (HS-, 81.5%) compared to 18.5% as H2S [5] H2S is a powerful reducing agent as it is easily oxidized

1.3 Biosynthesis of H 2 S

H2S production within mammalian tissues occurs via both enzymatic and non-enzymatic means [6; 7; 8; 9] Generation of H2S via non-enzymatic means occurs through the reduction of thiols Using L-cysteine as a starting material, H2S can be synthesized by at least four distinct routes enzymatically [6] These four routes are: (i) cystathionine γ lyase (CSE) produces thiocysteine from cystine, which then reorganizes to produce H2S; (ii) cystathionine β synthase (CBS) works

on L-cysteine to create H2S and L-serine; (iii) cysteine aminotransferase (CAT) working in concert with 3-mercaptopyruvate sulfurtransferase (3-MST) to produce H2S with 3-mercaptopyruvate as the intermediary product; (iv) cysteine lyase (CL) catalyzes L-cysteine and sulfite to H2S and L-cysteate These enzymes occur in mammalian tissues and are differentially expressed CBS is abundantly expressed in brain whereas CSE is the predominant enzyme in liver, kidney and smooth muscle cells CAT and 3-MST are primarily localized within mitochondria [10] CSE, CBS, CAT and CL need pyridoxal 5’-phosphate (P5P) as a cofactor 3-MST requires zinc as cofactor H2S can also be synthesized from homocysteine in a reaction catalyzed by CBS The four enzymatic pathways that catalyze H2S production are shown in Figure 1

Figure 1 The biosynthesis of H 2 S within mammalian tissues

Figure taken from Li et al., (2011) [6]

1.4 Biological effects and physiological functions of H 2 S

H2S, which has been long viewed purely as an environmental pollutant, has in recent years increasingly come to be recognized as an influential biological molecule with important physiological roles Recently, there has been an abundance of reports illustrating the role of H2S

in diverse fields such as cardiovascular, reproductive, neurobiology and inflammation [5; 6; 11; 12; 13; 14] These studies have not only considerably increased our understanding of H2S but have additionally illuminated the complexity of this seemingly chemically simple gas

Within the cardiovascular system, H2S acts as a physiological vasodilator, modulator of blood flow within the microcirculation and regulator of blood pressure through the opening of ATP-sensitive potassium (KATP) channels within vascular smooth muscle cells [15; 16; 17] In contrast, lack of endogenous H2S may contribute to the development of hypertension [18] H2S promotes angiogenesis and is anti-atherosclerotic in nature [19; 20] During atherosclerosis, there

is oxidative stress due to increased formation of reactive oxygen species (ROS) Oxidative stress promotes proliferation of vascular smooth muscle cells and increased foam cell formation which are key steps in atherosclerosis H2S can directly quench ROS with its strong reducing propertiesand inhibit ROS production [21; 22] Within atherosclerotic lesions, there is massive proliferation of vascular smooth muscle cells H2S inhibits vascular smooth muscle cells proliferation and also induces apoptosis of vascular smooth muscle cells [23] Foam cells formation from macrophages by OxLDL is critical for the initiation and progression of atherosclerotic lesions H2S was shown to inhibit formation of foam cells [24] In CSE knockout mice, a deficiency of endogenous H2S has been associated with accelerated atherosclerosis [25]

In reproductive biology, H2S relaxes both the corpus cavernosum and vaginal smooth muscles again through the modulation of KATP channels These data suggest that sexual function may, at least partly, be controlled by H2S [11; 12]

In neurobiology, H2S has been reported to alter activity of potassium channels and curb synaptic potentials in dorsal raphe serotonergic neurons [26] In neurons, H2S enhances N-methyl-D-aspartic acid (NMDA) receptor-mediated responses and modifies the induction of hippocampal long-term potentiation [27] Additionally, sensitizing of NMDA receptor by H2S been reported to

have a role in pain sensation [28] H2S also been shown to confer protection on neurons from oxidative stress and prevent neurodegeneration [29; 30]

In inflammation research, H2S exhibits both pro-inflammatory and anti-inflammatory properties

H2S been reported to be an endogenous potentiator of T cell activation [14] T cells interact with many cell types and recognize a large number of pathogens T cell activation leads to either production of antibodies, activation of phagocytic cells or direct cell killing which are essential components of host defense However, uncontrolled T cell activation might result in autoimmune destruction of cells and tissue [14] During inflammation, H2S increases leukocyte adhesion via

activation of the β2 integrin Mac-1 (CD11b/CD18) [31]

Remarkably, this gas also has opposing roles on the effect of administration of lipopolysaccharide (LPS) In a pro-inflammatory role, elevated H2S level and myeloperoxidase (MPO) activity, which serves as a marker for neutrophil infiltration, were observed after either administration of LPS or treatment with the H2S donor sodium hydrosulfide (NaHS) [32] On the other hand, H2S donors (NaHS and S-diclofenac) demonstrated anti-inflammatory effects through decreased activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and reduced production of inflammatory cytokines [33; 34] In a burn injury model,

H2S caused markedly increased inflammatory damage [35] Another study reported a negative association between endogenous H2S levels and the severity of chronic obstructive pulmonary disease (COPD) [36] It has been proposed that the pro-inflammatory properties of H2S might be mediated through increased release of the neuropeptide, substance P, leading to the resultant activation of extracellular signal-regulated kinases (ERK) and NF-κB to generate inflammatory

mediators such as interleukin (IL)-1β, tumor necrosis factor α (TNF-α) [37] H2S has been shown to ameliorate emphysema in a murine smoke exposure model [38] H2S donors have also been reported to possess anti-inflammatory properties in murine models of ischemia-reperfusion injury of small intestine and colitis [39; 40] The anti-inflammatory properties of H2S may be partly due to inhibition of phosphodiesterase activity resulting in an increased cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP) levels within aortic cells [41] Recently, H2S was reported to function as an endogenous mediator of the resolution phase of inflammation [42] H2S donors that release H2S rapidly over a short period (e.g NaHS) are typically pro-inflammatory [32; 43] On the other hand, H2S donors releasing H2S slowly over a long period (e.g morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) and 2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester (ATB-346)) are usually anti-inflammatory [44; 45] As such, the overall effect of H2S in inflammation most likely depends on the actual experimental conditions and the concentration of

H2S at the site of inflammation [6] Apart from the H2S concentration at the site of inflammation, another factor which may play a part in determining the involvement of this gas in inflammation,

is the duration and perhaps also the intensity of the inflammatory insult [6] In addition to the topics discussed above, H2S been additionally implicated in other disease states including cancer, organ transplant and metabolic syndromes [46; 47; 48] The above-mentioned examples serve to illustrate the complexity and wide ranging roles of H2S across different physiological and pathophysiological states Consequently, H2S, and its donor drugs, are increasingly being considered as potential therapeutic agents for a number of diseases [49; 50; 51] An illustration

of the various potential therapeutic targets for H2S is provided in Figure 2

Figure 2 Therapeutic targets for H 2 S

Figure taken from Predmore et al., (2012) [51]

shock following injection of mice with E coli LPS

Chapter 2

Effects of administration of a high fat

2 Introduction

Cardiovascular disease is currently one of the chief causes of mortality within developed countries and is a leading health concern worldwide [52] High cholesterol levels increases the risk of cardiovascular diseases [53] One third of heart disease cases worldwide can be attributed

to high blood cholesterol levels which is especially obvious within the developed world [52] One of the biggest influences on high blood cholesterol levels is diet high in saturated fats [54] Atherosclerosis is the principal cause of cardiovascular disease Atherosclerosis is characterized

by accumulation of lipids, fibrous elements, smooth muscle cells, endothelial cells, leukocytes and foam cells in large arteries [55] Atherosclerosis is increasingly being recognized as an inflammatory disease Over the past decades, the involvement of immune cells and inflammatory mechanisms in the development of atherosclerosis has become clear [56; 57] During atherogenesis, a process in which atherosclerotic plaques are formed, monocyte recruitment to sites of lesion in large arteries occurs as an early event [56] Circulating oxidised low density lipoprotein (OxLDL) leads to activation of endothelial cells The attachment of monocytes to the activated endothelial cells is facilitated by various adhesion molecules The two main molecules are vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1) The recruited monocytes undergo maturation into macrophages and releases several mediators to further drive atherogenesis [57] The macrophages migrate into the intima of vasculature and increase expression of surface scavenger receptors such as scavenger receptor class A (SRA) and cluster of differentiation 36 (CD36) These receptors bind and internalize lipoprotein with the cells The lipids accumulate in the macrophages to form foam cells Foam cells release pro-inflammatory cytokines and ROS which augments the inflammation within the lesion Foam cells also augment smooth muscle cell migration into the intima which drives the

progression of the atherosclerosis [58] These processes result in the formation of fatty steaks Over time, these fatty streaks evolve into atherosclerotic plaques The initiation and progression

of atherosclerotic plaques generally take place over many years without any symptoms In the event of a plaque rupture, acute thrombosis occurs by activation of platelets and triggering of the clotting cascade Depending on the location of the clot, the thrombotic event may be serious or life-threatening

H2S performs several roles during inflammation These include dilation of blood vessels and regulation of leukocyte adhesion [15; 31] The role of H2S in atherosclerosis has been well documented [59] It has been reported that apolipoprotein E-deficient (ApoE-/-) mice have lowered plasma H2S levels with increased plasma ICAM-1 concentrations [20] This is interesting since ApoE-/- mice are known to develop atherosclerosis spontaneously even when fed a normal diet Proliferation of vascular smooth muscle cells is one of the key features of atherosclerosis H2S been described to induce apoptosis and suppress the proliferation of vascular smooth muscle cells [23] These suggest that H2S may be protective against atherosclerosis [60] During the progression of atherosclerosis, the increased ROS formation within the lesion results in oxidative stress which plays an important function in driving the atherosclerotic process H2S can directly quench ROS with its strong reducing properties [21] Additionally, H2S also been shown to inhibit ROS production [22] The H2S donor, NaHS inhibited the formation of foam cells from macrophages by OxLDL [24] H2S also reduced the extent of aortic lesions in ApoE-/- mice [20; 61] CSE knockout mice, which suffer from deficiency of endogenous H2S have accelerated development of atherosclerosis [25] Taken together, a strong case can be made that deficiency of H2S is linked with the development of

atherosclerosis Whether there are any changes to the metabolism of H2S in mice before the development of atherosclerotic lesions in animals or in man is not clear.Moreover, whether such changes, if they occur, would be good predictors of future development of atherosclerosis is also unclear In this work, I examine whether administration of a high fat diet to mice affects tissue

H2S biosynthesis and synthesising enzymes as well as plasma markers of both inflammation and atherosclerosis

2.1 Materials and methods

2.1.1 Animals and diet

Male C57/Bl6 mice (23–25 g) were maintained in Comparative Medicine at National University

of Singapore (NUS) in an environment with regulated temperature (21-24oC) and lighting (12:12

h light-darkness cycle) Drinking water was provided ad libitum A period of at least three days

was allowed for animals to acclimatize before any experimental manipulations were undertaken Thereafter, mice were fed either a high fat (16% fat, 12.5% cholesterol and 5% sodium cholic acid) or normal diet (Research Diets Inc., NJ, USA) for up to 16 weeks At the end of 8, 12 or 16 weeks, groups of animals were anaesthetised with a mixture of ketamine (75 mg/kg, i.p.) and medetomidine (1 mg/kg, i.p.) and blood obtained by cardiac puncture, anticoagulated with heparin (100 U/ml) Blood was then centrifuged at 10000g for 3 min at 4oC to prepare plasma which was then stored at -80oC Livers, kidneys and lungs were rapidly excised and immediately snap frozen in liquid nitrogen prior to biochemical analyses Aortae were also removed and placed in 4% v/v paraformaldehyde in phosphate buffer for 24 hours and then embedded in paraffin for subsequent histology All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of NUS

2.1.2 Measurement of tissue H 2 S synthesizing enzyme activity

Tissue H2S synthesizing enzyme activity was determined as previously described [32] Lung, kidney and liver were homogenized in ice-cold potassium phosphate buffer (100 mM, pH 7.4)

The homogenates were centrifuged at 900g for 10 min at 4oC and the resulting supernatant were collected The assay mixture (500 µl) contained tissue homogenate (430 µl), L-cysteine (10 mM;

20 µl), P5P (2 mM; 20 µl) (Sigma–Aldrich Ltd.,MO, USA), and saline (30 µl) For the detection

of H2S synthesizing activity by 3-MST, the protocol was modified from a previous paper [62] All reagents in the assay mixture remained the same, except that L-cysteine was replaced with 3-mercaptopyruvate (3MP) (0.1 mM, 40 µl), P5P was omitted and dithiothreitol (DTT) (1 mM) (Sigma–Aldrich Ltd., MO, USA) was added The assay mixtures were incubated for 30 min at

37oC in parafilm-sealed microcentrifuge tubes Thereafter, zinc acetate (1% w/v, 250 µl) was injected to trap H2S followed by trichloroacetic acid (10% w/v, 250 µl) to stop the reaction Subsequently, N,N-dimethyl-p-phenylenediamine sulfate (20 mM; 133 µl) dissolved in 7.2 M hydrochloric acid (HCl) was added followed immediately by iron (III) chloride (FeCl3) (30 µM;

133 µl) dissolved in 1.2 M HCl Absorbance of samples (300 µl) was determined at an absorbance wavelength of 670 nm using a 96-well microplate reader (Tecan Systems Inc., CA, USA) All standards and samples were assayed in duplicate The H2S concentration of each sample was calculated against a calibration curve of NaHS standards (3.125–250 µM) and results are expressed as nm H2S formed per mg soluble protein as determined using the Bradford assay (Bio-Rad Ltd., CA, USA)

2.1.3 Measurement of plasma H 2 S concentration

Plasma H2S was measured by a high-performance liquid chromatography (HPLC) method as described [63] Mouse plasma (15 µl) and NaHS standards were derivatised for 30 min at room temperature with the fluorescent probe monobromobimane (MBB, 2 mM) in the dark

Thereafter, a standard curve of NaHS (0.018–1.5 µM) was prepared from the derivatised NaHS stock solution HPLC analysis of derivatized plasma or NaHS solution was carried out on a C18 column using an Agilent 1100 Series HPLC System (Agilent Technologies., CA, USA) with mobile phases comprising of 10% v/v methanol and 0.25% v/v acetic acid and 90% v/v methanol and 0.25% v/v acetic acid Excitation and emission wavelengths were 385 nm and 475 nm respectively Derivatised H2S has a retention time in this system of 24.4 ± 0.01 min.

2.1.4 Western blotting for H 2 S synthesizing enzymes

Lung, kidney and liver were homogenized (1:12.5 w/v) in ice cold lysis buffer comprising EDTA (5 mM) containing protease and phosphatase inhibitors (HaltTM Protease Inhibitor Cocktail and Halt™ Phosphatase Inhibitor Cocktail) and 1% v/v Triton-X 100 in phosphate buffered saline (PBS) The homogenates were incubated on ice for one hour before being centrifuged at 16,000g for 10 min at 4oC and the resulting supernatant were collected Protein concentration was quantified using the Bradford assay (Bio-Rad Ltd., CA, USA) Samples were resolved on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred onto nitrocellulose membranes (Bio-Rad Ltd., CA, USA) The membranes were incubated for one hour at room temperature with blocking buffer (PBS, containing 5% v/v skimmed milk and 0.1% v/v Tween-20) Membranes were then incubated overnight at 4oC with primary antibodies directed against CBS, CSE (Abcam Ltd., USA) or 3-MST or actin (Sigma–Aldrich Ltd., MO, USA) After overnight incubation, primary antibodies were removed, membranes were washed three times with PBS containing 0.1% v/v Tween-20 and thereafter incubated for one hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse

IgG and goat anti-rabbit IgG) (Thermo Fisher., MA, USA) The immunoreactive bands were visualized using LuminataTM Crescendo Western HRP Substrate (Merck Millipore Ltd., MA, USA) and exposed to X-ray film Resulting blots were scanned and quantified using ImageJ software

2.1.5 Measurement of plasma cytokine and chemokine levels

A range of cytokines and chemokines were assayed in mouse plasma using a Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (Bio-Rad Ltd., CA, USA) according to the manufacturer’s instructions Fluorescence was measured using the Luminex 100 system and results analysed using Bio-plex Manager™ software (Bio-Rad Ltd., CA, USA)

2.1.6 Measurement of plasma SAA and CRP levels

Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits (USCN Life Science Inc., HB, China and GenWay Biotech Inc., CA, USA) respectively Experiments were performed according to the manufacturers’ instructions

2.1.7 Immunohistochemistry (IHC) staining for CSE, CBS and 3-MST

Fixed tissue samples were sliced into 5 µm sections using microtome (Leica AG., HE, Germany) The sections were deparaffinised, rehydrated before antigen recall was performed Sections were blocked with blocking buffer (tris-buffered saline (TBS) containing 1% v/v bovine

serum albumin (BSA)) for one hour at room temperature Sections were then incubated overnight

at 4oC with primary antibodies directed against CBS, CSE or 3-MST.After overnight incubation, primary antibodies were removed; sections were washed with PBS containing 0.1% v/v Tween-

20 and thereafter incubated for one hour at room temperature with horseradish conjugated secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG) Photo-images were captured in a light microscope equipped with a digital camera (Olympus., TK, Japan)

peroxidase-2.1.8 Oil red O staining

Fixed tissue samples were sliced into 5 µm sections using microtome The sections were deparaffinised and rehydrated Sections were then rinsed with 60% v/v isopropanol and stained for 15 min with Oil Red O solution (Sigma–Aldrich Ltd.,MO, USA) Sections were rinsed with PBS, counterstained with haematoxylin (Sigma–Aldrich Ltd.,MO, USA) and rinsed again with PBS Photo-images were captured in a light microscope equipped with a digital camera

2.1.9 Statistics

Data is expressed as mean ± standard error of mean (SEM) with the number of observations shown in parenthesis Analysis was carried out using Student's t-test and statistical significance was set at p < 0.05

2.2 Results

2.2.1 Effect of a high fat diet on plasma H 2 S concentration

Plasma H2S concentration in control animals prior to administration of a high fat diet was 310.1

± 16.1 nM (n = 6) as determined by HPLC No significant change in plasma H2S concentration was detected in either control or fat fed animals at 8, 12 or 16 weeks (Fig 3)

Figure 3 Plasma H 2 S concentration Control (open columns) and high fat fed (black columns)

mice at 8, 12 and 16 weeks Results show H2S concentration (nM) and are mean ± SEM, n = 6–

7 Hatched column is mice at 0 weeks

2.2.2 Effect of a high fat diet on CSE, CBS and 3-MST expression

The expression of the three H2S synthesising enzymes in liver, kidney and lung from control and high fat fed animals were determined by Western blotting The effect of a high fat diet on expression of these enzymes was tissue-dependent In the liver, both CSE and 3-MST were down-regulated after 8, 12 or 16 weeks of high fat diet In contrast, an up-regulation of CBS in liver was observed at the 8 and 16 week time points (Fig 4A and D) Perhaps this up-regulation may be compensatory in nature In the kidney, the expression of CBS was only up-regulated at 8 weeks and not at the other two time points There was no significant effect on the expression of either CSE or 3-MST after high fat diet treatment (Fig 4B and D) In the lung, CSE expression was reduced at 8 and 16 weeks but not 12 weeks after feeding a high fat diet There was no significant effect on the expression of either CBS or 3-MST after high fat diet treatment (Fig 4C and D)

Figure 4 Expression of CBS, CSE and 3-MST (A) Liver, (B) Kidney, (C) Lung Control

(open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks Results show expression of each protein (c.f actin) and are mean ± SEM, n = 4–6, *p < 0.05 (D) Representative blots

2.2.3 Effect of a high fat diet on tissue H 2 S synthesising activity

Experiments were performed to monitor tissue H2S synthesising activity ex vivo using liver,

kidney and lung samples Either cysteine or 3MP was used as substrate to monitor enzyme activity of CSE/CBS or 3-MST respectively In the liver, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of high fat diet (Fig 5A) In the lung, H2S biosynthesis using cysteine was significantly reduced after 8 and 12 weeks of high fat diet Interestingly, no significant different was observed at 16 weeks (Fig 5B) In the kidney, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of high fat diet (Fig 5C) In the liver, H2S biosynthesis using 3MP was significantly reduced at all three time points after administration of high fat diet (Fig 5D) In the kidney, there was no significant effect on

H2S biosynthesis using 3MP at all three time points after administration of high fat diet (Fig 5E).The interpretation of the liver biosynthesis data may be complicated by the observed increased expression of CBS in livers of animals fed a high fat diet This increased expression of CBS might compensate for the decreased CSE expression during H2S biosynthesis Interestingly, H2S biosynthesis from cysteine was reduced in kidney even though there were no significant changes

in kidney CSE, CBS or 3-MST expression Whether this reflects synthesis of H2S from another source or perhaps enhanced catabolism or quenching of the synthesised gas is not clear

Figure 5 Biosynthesis of H 2 S (A) Liver, (B) Lung (B), (C) Kidney from cysteine with P5P, (D)

Liver and (E) Kidney from 3MP with DTT Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks Results show H2S synthesis from starting substrate expressed as nmol/mg protein and are mean ± SEM, n = 10–14, *p < 0.05

2.2.4 Effect of a high fat diet on vascular H 2 S synthesizing enzyme localisation

In aortic sections of control mice after 16 weeks, IHC staining for CSE revealed that the localisation of CSE was in the endothelial cell layer and not in vascular smooth muscle cells (Fig 6A) In aortic sections of high fat fed mice after 16 weeks, CSE was greatly reduced or absent in the endothelial cell layer (Fig 6B) IHC staining for CBS and 3-MST revealed that neither enzyme was present in aorta of either control nor mice fed a high fat diet for up to 16 weeks (Fig 6C-E)

Figure 6 Representative photographs of IHC staining in aorta.Representative sections from

3 animals in each group (A) control diet mice CSE, (B) high fat fed mice CSE, (C) control diet mice CBS, (D) high fat fed mice CBS (E) control diet mice 3-MST, (F) high fat fed mice 3-

MST Arrows highlight areas of brown CSE staining Scale shows dimension (20 µm)

2.2.5 Effect of a high fat diet on plasma cytokines and chemokines

Plasma of mice at the time points of 8, 12 or 16 weeks were screened for changes in profile of various inflammatory cytokines and chemokines No significant difference in plasma concentrations between control and high fat mice was detected for IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-10, IL-13, IL-17, interferon gamma (IFN-γ), monocyte chemotactic protein 1 (MCP-1), regulated on activation, normal T expressed and secreted (RANTES) or TNF-α (Fig 7G-R) Significant differences were observed at some time points for IL-6, granulocyte-colony stimulating factor (G-CSF), keratinocyte chemoattractant (KC), IL-12p40, macrophage inflammatory protein (MIP)-1β and IL-5 Plasma concentrations of IL-6 and G-CSF were significantly increased in the high fat diet mice at both 12 and 16 weeks (Fig 7A and C) At all three time points, plasma concentration of IL-12p40 was increased for high fat fed mice (7B) Plasma concentration of KC was only significant increased at 16 weeks for high fat fed mice (7D) Plasma MIP-1β levels were significantly lower at 8 weeks but become significantly increased at 12 weeks for high fat fed mice (Fig 7E) Plasma concentration of IL-5 was decreased at 8 weeks but not at other time points for high fat fed mice (Fig 7F)