Smriti M. Krishna

Genetic and epigenetic mechanisms and their possible role in abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a common disease associated with significant cardiovascular morbidity and mortality. The pathogenesis of AAA is poorly defined, making targeting of new therapies problematic. Current evidence favours an interaction of multiple environmental and genetic factors in the initiation and progression of AAA. Epigenetics is the term used to define the properties of the genome that are not explained by the primary sequence, but are due to the modifications of DNA and/or associated proteins. Previous research indicates the association of gene specific promoter DNA hyper-methylation and global DNA hypo-methylation with atherosclerosis. Evidence also suggests an important role for epigenetic processes such as histone acetylation in cardiovascular diseases including atherosclerosis and restenosis. Altered DNA methylation or histone acetylation occur in inflammation, cellular proliferation and remodelling processes and therefore maybe relevant to the pathology of AAA. Important risk factors for AAA, including cigarette smoking, older age, male gender and hypertension, have been linked with epigenetic effects and thus could act in this way to promote AAA. In this review, we discuss the potential role of epigenetic mechanisms in AAA. Since epigenetic alterations are to some extent reversible, further study of this area may identify new treatment targets for AAA.

Fenofibrate increases high-density lipoprotein and sphingosine 1 phosphate concentrations limiting abdominal aortic aneurysm progression in a mouse model

There are currently no acceptable treatments to limit progression of abdominal aortic aneurysm (AAA). Increased serum concentrations of high-density lipoprotein (HDL) are associated with reduced risk of developing an AAA. The present study aimed to assess the effects of fenofibrate on aortic dilatation in a mouse model of AAA. Male low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were maintained on a high-fat diet for 3 weeks followed by 6 weeks of oral administration of vehicle or fenofibrate. From 14 to 18 weeks of age, all mice were infused with angiotensin II (AngII). At 18 weeks of age, blood and aortas were collected for assessment of serum lipoproteins, aortic pathology, aortic Akt1 and endothelial nitric oxide synthase (eNOS) activities, immune cell infiltration, eNOS and inducible NOS (iNOS) expression, sphingosine 1 phosphate (S1P) receptor status, and apoptosis. Mice receiving fenofibrate had reduced suprarenal aortic diameter, reduced aortic arch Sudan IV staining, higher serum HDL levels, increased serum S1P concentrations, and increased aortic Akt1 and eNOS activities compared with control mice. Macrophages, T lymphocytes, and apoptotic cells were less evident and eNOS, iNOS, and S1P receptors 1 and 3 were up-regulated in aortas from mice receiving fenofibrate. The present findings suggest that fenofibrate antagonizes AngII-induced AAA and atherosclerosis by up-regulating serum HDL and S1P levels, with associated activation of NO-producing enzymes and reduction of aortic inflammation.

Down regulation of adhesion protein E-cadherin in Epstein-Barr virus infected nasopharyngeal carcinomas

Nasopharyngeal carcinoma (NPC) is a unique tumour due to its aetiology, incidence pattern and its consistent association with Epstein-Barr virus (EBV). EBV encodes many viral proteins, which targets crucial cell cycle regulatory proteins. Cadherins are a family of transmembrane glycoproteins, which mediates Ca2+-dependent intercellular adhesion. Epithelial (E)-cadherin is an important member of this family, which is expressed predominantly on the surface of epithelial cells. E-cadherin acts as an invasion/metastasis-suppressor gene, hence knowledge of the molecular mechanism that controls its expression or function is of primary importance in understanding the process of tumor invasion. Loss of E-cadherin function has been shown to potentiate tumor cell invasion and interestingly, a large number of invasive tumors do not involve mutation of E-cadherin, but rather down regulation of expression. Hence in this study, an attempt was made to evaluate the protein level expression pattern of E-cadherin in relation to viral involvement and to correlate it with other clinico-pathological parameters. We observed a significant down regulation of E-cadherin in NPC and its histological subsets, when compared to the controls (p<0.001). Expression of E-cadherin ranged from mild to moderate and none of the NPC showed intense expression. E-cadherin expression showed a significant down-regulation in NPC lesions with EBV infection (r=-0.436, p<0.001). The down regulation of E-cadherin observed in NPC is in line with the previous reports in E-cadherin expression in various cancers. The total lack of E-cadherin expression in neoplastic cells might be due to hypermethylation of E-cadherin promoter or its down regulation by cellular transcription repressor. Our study also shows a significant down regulation of E-cadherin in the presence of EBV, which also might involve the cellular DNA methylation machinery.

The role of thrombospondin-1 in cardiovascular health and pathology

Cardiovascular diseases (CVDs) remain a leading cause of morbidity and mortality in the developed world and are becoming increasingly prevalent in the developing world. Although a range of therapies already exist for established CVDs, there is a significant interest in further understanding disease pathogenesis in order to improve treatment. Thrombospondin (TSP)-1 is an important extracellular matrix component that influences the function of vascular smooth muscle cells, endothelial cells, fibroblasts and inflammatory cells with important implications for CVDs. TSP-1 regulates matrix production and organisation thereby influencing tissue remodelling and promoting the generation of T regulatory cells that control the inflammatory response. Reported findings from in vitro and animal studies are conflicting and suggest differing effects of TSP-1 on various cellular mechanisms, depending on the experimental setting. Vascular cells express a number of TSP-1 receptors, such as CD36, proteoglycans and several integrins, which are regulated by specific contextual signals which may explain the varying effects that TSP-1 elicits in different environments. Different domains of TSP-1 activate distinct signalling pathways eventually resulting in quite different cellular phenotypes and tissue specific effects. The sum total of the various pathways activated likely determines the overall effect on angiogenesis or proliferation in a specific tissue. Hence defining a common mechanism of action of TSP-1 in CVD is complicated. Increasing the understanding of the role of TSP-1 in various CVDs will potentially provide new opportunities for therapeutic intervention using peptides derived from its various domains currently under evaluation in other diseases.

A Review of the Pathophysiology and Potential Biomarkers for Peripheral Artery Disease

Peripheral artery disease (PAD) is due to the blockage of the arteries supplying blood to the lower limbs usually secondary to atherosclerosis. The most severe clinical manifestation of PAD is critical limb ischemia (CLI), which is associated with a risk of limb loss and mortality due to cardiovascular events. Currently CLI is mainly treated by surgical or endovascular revascularization, with few other treatments in routine clinical practice. There are a number of problems with current PAD management strategies, such as the difficulty in selecting the appropriate treatments for individual patients. Many patients undergo repeated attempts at revascularization surgery, but ultimately require an amputation. There is great interest in developing new methods to identify patients who are unlikely to benefit from revascularization and to improve management of patients unsuitable for surgery. Circulating biomarkers that predict the progression of PAD and the response to therapies could assist in the management of patients. This review provides an overview of the pathophysiology of PAD and examines the association between circulating biomarkers and PAD presence, severity and prognosis. While some currently identified circulating markers show promise, further larger studies focused on the clinical value of the biomarkers over existing risk predictors are needed.

Impaired Acetylcholine-Induced Endothelium-Dependent Aortic Relaxation by Caveolin-1 in Angiotensin II-Infused Apolipoprotein-E (ApoE−/−) Knockout Mice

Objective The angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE−/−) mouse model is widely used to study atherosclerosis and abdominal aortic aneurysm. An increase in blood pressure has been reported in this model however the underlying mechanism has not been fully explored. In this study, we investigated whether vasomotor dysfunction develops in AngII-infused ApoE−/− mice and the underlying mechanism involved. Methods ApoE−/− mice were infused with vehicle (distilled water) or AngII subcutaneously for 14 days. Blood pressure and heart rate were measured using the non-invasive tail cuff method. Aortic vascular reactivity and expression of key proteins (endothelial nitric oxide synthase (eNOS), phospho-eNOS and caveolin-1) were assessed using tension myography and Western blotting respectively. Plasma nitric oxide (NO) level was estimated using a colorimetric assay. Results AngII infusion caused a time-dependent increase in blood pressure (P<0.001). Aortas from AngII-infused mice were significantly less responsive to acetylcholine-induced endothelium-dependent relaxation when compared to aortas from mice infused with vehicle control (P<0.05). Contractile responses to phenylephrine (P<0.01) and potassium chloride (P<0.001) were significantly enhanced in aortas from AngII-infused mice. eNOS phosphorylation was significantly decreased in the aorta of AngII-infused mice (P<0.05). Aortic caveolin-1 protein expression was significantly increased in AngII-infused mice (P<0.05). Plasma nitrate/nitrite level was significantly reduced in AngII-infused mice (P<0.05). Pharmacological disruption of caveolae using methyl-β-cyclodextrin (MβCD) in isolated aortas from AngII-infused mice caused a significant leftward shift of the acetylcholine-induced relaxation concentration-response curve when compared to vehicle control (P<0.05). Conclusion Upregulation of caveolin-1 protein expression and reduced NO bioavailability contributes to aortic endothelial dysfunction in AngII-infused ApoE−/− mice.

Transforming growth factor-β and abdominal aortic aneurysms

Abdominal aortic aneurysms (AAAs) are common problems in aged people which can be associated with severe complications including aortic rupture and death. Transforming growth factor-β (TGFβ) has been implicated as causative in the development of thoracic aortic aneurysms (TAAs). In contrast, current evidence suggests TGFβ inhibits AAA development. Polymorphisms in the TGFβ signaling components are associated with AAA in some human population studies. In experimental animals TGFβ protects against AAA formation, progression and rupture. In animal models of AAA TGFβ decreases aortic inflammatory cell infiltration, extracellular matrix degradation, and vascular smooth muscle cell apoptosis, all factors implicated in AAA pathogenesis. The TGFβ signaling pathway may provide a therapeutic target for AAA although better clarity is needed regarding the distinct roles of TGFβ in TAA and AAA.

A Peptide Antagonist of Thrombospondin-1 Promotes Abdominal Aortic Aneurysm Progression in the Angiotensin II–Infused Apolipoprotein-E–Deficient Mouse

Objective—Interaction of the activating sequence in thrombospondin-1 (TSP-1) with the conserved sequence (leucine-serine-lysine-leucine [LSKL]) in the latency-associated peptide region of latent transforming growth factor (TGF)-&bgr; complex is important in regulating TGF-&bgr;1 activity. We aimed to assess the effect of blocking peptide LSKL on the progression of pre-established abdominal aortic aneurysm in angiotensin II–infused apolipoprotein E-deficient (ApoE−/−) mice. Approach and Results—Abdominal aortic aneurysm was established in 3-month-old male ApoE−/− mice with subcutaneous infusion of angiotensin II for 28 days. After this, mice received LSKL peptide or control SLLK (serine–leucine–leucine–lysine) peptide (4 mg/kg) via daily intraperitoneal injection for an additional 2 weeks. Administration of LSKL peptide promoted larger suprarenal aortic diameter, as determined by ultrasound and morphometric analysis, and stimulated more severe atherosclerosis within the aortic arch. In addition, mice receiving LSKL peptide exhibited elevated circulating proinflammatory cytokine levels and greater inflammatory cells within the suprarenal aorta compared with controls. Mice receiving LSKL peptide showed low plasma TGF-&bgr;1 activity and low levels of aortic tissue phosphorylated to total Smad2/3. Aortic gene expression of TGF-&bgr; receptor 1 (TGFBRI) and receptor 2 (TGFBRII), but not TGF-&bgr;1 and thrombospondin-1, were lower in mice receiving LSKL peptide than controls. LSKL peptide administration was associated with greater aortic elastin fragmentation and lower expression and activity of the TGF-&bgr;1-target gene lysyl oxidase like 1 (LOXL1). Conclusions—Attenuation of thrombospondin-1-directed activation of TGF-&bgr;1 promotes abdominal aortic aneurysm and atherosclerosis progression in the angiotensin II–infused ApoE−/− mouse model.

Meta-analysis of the association between transforming growth factor-beta polymorphisms and complications of coronary heart disease.

Objective To investigate the association between common transforming growth factor beta (TGF-β) single nucleotide polymorphisms (SNP) and significant complications of coronary heart disease (CHD). Method We performed a meta-analysis of published case-control studies assessing the association of TGF-β SNPs with a range of CHD complications. A random effects model was used to calculate odds ratios and confidence intervals. Analyses were conducted for additive, dominant and recessive modes of inheritance. Results Six studies involving 5535 cases and 2970 controls examining the association of common SNPs in TGF-β1 with CHD were identified. Applying a dominant model of inheritance, three TGF-β1 SNPs were significantly associated with CHD complications: The T alleles of rs1800469 (OR = 1.125, 95% CI 1.016–1.247, p = 0.031) and rs1800470 (OR = 1.146, 95% CI 1.026–1.279, p = 0.021); and the C allele of rs1800471 (OR = 1.207, 95% CI 1.037–1.406, p = 0.021). Conclusion This meta-analysis suggests that common genetic polymorphisms in TGF-β1 are associated with complications of CHD.

Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis.

Objective— Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. Approach and Results— SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E–deficient (ApoE−/−) mice (SOSTTg.ApoE−/−) and administration of recombinant mouse Sost inhibited angiotensin II–induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg.ApoE−/− mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg.ApoE−/− mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/&bgr;-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg.ApoE−/− mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/&bgr;-catenin pathway was activated in human AA samples. The cytosine–phosphate–guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. Conclusions— This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.