Christos Tikellis

A Breaker of Advanced Glycation End Products Attenuates Diabetes-Induced Myocardial Structural Changes

&NA; The formation of advanced glycation end products (AGEs) on extracellular matrix components leads to accelerated increases in collagen cross linking that contributes to myocardial stiffness in diabetes. This study determined the effect of the crosslink breaker, ALT‐711 on diabetes‐induced cardiac disease. Streptozotocin diabetes was induced in Sprague‐Dawley rats for 32 weeks. Treatment with ALT‐711 (10 mg/kg) was initiated at week 16. Diabetic hearts were characterized by increased left ventricular (LV) mass and brain natriuretic peptide (BNP) expression, decreased LV collagen solubility, and increased collagen III gene and protein expression. Diabetic hearts had significant increases in AGEs and increased expression of the AGE receptors, RAGE and AGE‐R3, in association with increases in gene and protein expression of connective tissue growth factor (CTGF). ALT‐711 treatment restored LV collagen solubility and cardiac BNP in association with reduced cardiac AGE levels and abrogated the increase in RAGE, AGE‐R3, CTGF, and collagen III expression. The present study suggests that AGEs play a central role in many of the alterations observed in the diabetic heart and that cleavage of preformed AGE crosslinks with ALT‐711 leads to attenuation of diabetes‐associated cardiac abnormalities in rats. This provides a potential new therapeutic approach for cardiovascular disease in human diabetes. (Circ Res. 2003;92:785–792.)

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Characterization of renal angiotensin - converting enzyme 2 in diabetic nephropathy

Abstract—ACE2, initially cloned from a human heart, is a recently described homologue of angiotensin-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of angiotensin I to angiotensin 1–9 [Ang(1–9)] and is not inhibited by classic ACE inhibitors. It also converts angiotensin II to Ang(1–7). Although the role of ACE2 in the regulation of the renin-angiotensin system is not known, the renin-angiotensin system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by ≈50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various angiotensin peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications.

Connective Tissue Growth Factor Plays an Important Role in Advanced Glycation End Product–Induced Tubular Epithelial-to-Mesenchymal Transition: Implications for Diabetic Renal Disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

ACE2, a new regulator of the renin–angiotensin system

Abstract Angiotensin-converting enzyme (ACE) is a zinc metalloproteinase and a key regulator of the renin–angiotensin system (RAS). ACE2 is a newly described enzyme identified in rodents and humans with a more restricted distribution than ACE, and is found mainly in heart and kidney. ACE2 cleaves a single residue from angiotensin I (Ang I) to generate Ang 1–9, and degrades Ang II, the main effector of the RAS, to the vasodilator Ang 1–7. The importance of ACE2 in normal physiology and pathophysiological states is largely unknown. ACE2 might act in a counter-regulatory manner to ACE, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function.

Chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2

Background: Angiotensin converting enzyme (ACE) 2 is a recently identified homologue of ACE that may counterregulate the actions of angiotensin (Ang) II by facilitating its breakdown to Ang 1–7. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of cirrhosis but the role of ACE2 in liver disease is not known. Aims: This study examined the effects of liver injury on ACE2 expression and activity in experimental hepatic fibrosis and human cirrhosis, and the effects of Ang 1–7 on vascular tone in cirrhotic rat aorta. Methods: In sham operated and bile duct ligated (BDL) rats, quantitative reverse transcriptase-polymerase chain reaction was used to assess hepatic ACE2 mRNA, and western blotting and immunohistochemistry to quantify and localise ACE2 protein. ACE2 activity was quantified by quenched fluorescent substrate assay. Similar studies were performed in normal human liver and in hepatitis C cirrhosis. Results: ACE2 mRNA was detectable at low levels in rat liver and increased following BDL (363-fold; p<0.01). ACE2 protein increased after BDL (23.5-fold; p<0.05) as did ACE2 activity (fourfold; p<0.05). In human cirrhotic liver, gene (>30-fold), protein expression (97-fold), and activity of ACE2 (2.4 fold) were increased compared with controls (all p<0.01). In healthy livers, ACE2 was confined to endothelial cells, occasional bile ducts, and perivenular hepatocytes but in both BDL and human cirrhosis there was widespread parenchymal expression of ACE2 protein. Exposure of cultured human hepatocytes to hypoxia led to increased ACE2 expression. In preconstricted rat aorta, Ang 1–7 alone did not affect vascular tone but it significantly enhanced acetylcholine mediated vasodilatation in cirrhotic vessels. Conclusions: ACE2 expression is significantly increased in liver injury in both humans and rat, possibly in response to increasing hepatocellular hypoxia, and may modulate RAS activity in cirrhosis.

Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis.

BACKGROUND/AIMS The aim of this study was to investigate whether in the liver, as in other tissues, there is evidence that angiotensin II, acting via the angiotensin II type 1 receptor (AT1-R), plays a role in fibrogenesis. METHODS Sprague-Dawley rats were divided into three groups; sham, bile duct ligated (BDL) and BDL + AT1-R antagonist, irbesartan. Real time RT-PCR was utilised to assess gene expression of the AT1 receptor, TGF-beta1 and alpha1 (I) collagen in the liver. TGF-beta1 and alpha1 (I) collagen mRNA expression and localisation were also assessed by in situ hybridisation. TGF-beta1 activity was assessed by using the TGF-beta inducible gene product betaig-h3. Fibrosis was assessed by the Knodell scoring system, tissue hydroxyproline content and picro-sirius red staining. RESULTS Real time RT-PCR revealed that there was a 6-fold up-regulation in AT1 receptor expression in BDL animals compared with shams. This was associated with marked increases in TGF-beta1, betaig-h3 and alpha1 (I) collagen gene expression which were attenuated by AT1-RA treatment. However, AT1-RA therapy produced no significant change in liver histology or hydroxyproline content. CONCLUSIONS These results suggest that in the liver angiotensin II may play an important role in the fibrogenic response to injury. However, whether treatment with an AT1-RA will be of therapeutic benefit remains to be determined.

Interactions between Renin Angiotensin System and Advanced Glycation in the Kidney

Although hemodynamic and metabolic factors are individually implicated in the development of diabetic nephropathy, their interaction has not been defined clearly. In this study, the effects of angiotensin II (Ang II) and advanced glycation end products (AGE) both individually on each other are explored and compared. In the first study arm, Sprague-Dawley rats received a continuous infusion of AGE-modified rat serum albumin (RSA) or unmodified RSA for 4 wk with or without the angiotensin receptor type 1 antagonist valsartan. In the second arm, animals received a continuous infusion of Ang II (58.3 ng/kg per min) with or without the AGE inhibitor pyridoxamine. Components of the intrarenal renin-angiotensin system were measured using real time reverse transcription-PCR, immunohistochemistry, and standard angiotensin-converting enzyme (ACE) activity assays. Renal and serum AGE were quantified by immunohistochemistry, ELISA, and AGE-fluorescence. After an infusion of AGE-RSA, renal expression of angiotensinogen, ACE, renin, and angiotensin receptor type 1 were increased significantly (all P < 0.01), and ACE activity was elevated. This was associated with tubular and glomerular hypertrophy and AGE accumulation, which could be antagonized by valsartan. However, valsartan had no effect on increased filtration fraction associated with an AGE-RSA infusion. At the same time, an infusion of Ang II increased the serum and renal accumulation of AGE and advanced oxidation protein products and induced renal hypertrophy and salt retention that could be antagonized by pyridoxamine. However, pyridoxamine had no effect on renal vasoconstriction manifested by reduced renal blood flow. AGE and Ang II have overlapping activities in the kidney. The beneficial effects of blockade of either pathway underline the importance of this interaction in diabetic renal disease and the aging kidney.

Distinguishing Hyperglycemic Changes by Set7 in Vascular Endothelial Cells

Rationale: Epigenetic changes are implicated in the persisting vascular effects of hyperglycemia. The precise mechanism whereby chromatin structure and subsequent gene expression are regulated by glucose in vascular endothelial cells remain to be fully defined. Objective: We have studied the molecular and functional mechanism whereby the Set7 methyltransferase associates with chromatin formation and histone methylation in vascular cells in response to current and previous exposure to glucose. Methods and Results: To characterize the molecular and functional identity of the Set7 protein, we used vascular cells overexpressing or lacking Set7. Chromatin fractionation for mono-methylation of lysine 4 on histone H3 identified methyltransferase activity. Immunofluorescence experiments strongly suggest that Set7 protein accumulates in the nucleus in response to hyperglycemia. Moreover, activation of proinflammatory genes by high glucose is dependent on Set7 but distinguished by H3K4m1 gene patterns. We show that transient hyperglycemia regulates the expression of proinflammatory genes in vascular endothelial cells in vitro and the persistent increase in glucose-induced gene expression in the aorta of nondiabetic mice. Conclusions: This study uncovers that the response to hyperglycemia in vascular endothelial cells involves the H3K4 methyltransferase, Set7. This enzyme appears to regulate glucose-induced chromatin changes and gene expression not only by H3K4m1-dependent but also H3K4m1-independent pathways. Furthermore, Set7 appears to be responsible for sustained vascular gene expression in response to prior hyperglycemia and is a potential molecular mechanism for the phenomenon of hyperglycemic memory.

Heparanase Is Involved in the Pathogenesis of Proteinuria as a Result of Glomerulonephritis

The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by selectively degrading the negatively charged side chains of heparan sulfate proteoglycans within the glomerular basement membrane. A loss of negatively charged heparan sulfate proteoglycans may result in alteration of the permselective properties of the glomerular basement membrane, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors. In this study, therefore, the role of heparanase in passive Heymann nephritis (PHN) was examined. Normal glomeruli showed low-level heparanase expression as determined by immunohistochemistry and Western blot analysis. Days 5, 14, and 28 of PHN were associated with an increase in endothelial and glomerular epithelial cell heparanase. Reverse transcription-PCR confirmed a significant increase in mRNA at day 21 of disease (P < 0.0004). Furthermore, urinary and glomerular heparanase activities were significantly increased at days 5 and 21 of disease, respectively. Western blot analysis of isolated glomeruli separated into membrane- and cytosol-enriched protein fractions showed that the active 58-kD heparanase species was increased but restricted to the cytosol of diseased glomeruli at day 21. The inactive 65-kD precursor, however, was found in membrane and cytosol-diseased fractions, suggesting cell membrane processing. Complement depletion prevented glomerular heparanase expression; in addition, administration of a polyclonal anti-heparanase antibody significantly reduced urinary protein excretion at day 5 of disease to 62 +/- 11 mg/d compared with 203 +/- 43 and 159 +/- 18 mg/d in the normal rabbit serum- and normal saline-treated experimental groups, respectively (P < 0.002). Proteinuria was reduced in the absence of any altered glomerular C5b-9 activity, sheep IgG deposition, or rat anti-sheep antibody titers. These data suggest that heparanase contributes to the pathogenesis of proteinuria in PHN.