Suko Adiarto

Endothelial Cell–Derived Endothelin-1 Promotes Cardiac Fibrosis in Diabetic Hearts Through Stimulation of Endothelial-to-Mesenchymal Transition

Background— Persistently high plasma endothelin-1 (ET-1) levels in diabetic patients have been associated with the development of cardiac fibrosis, which results from the deposition of extracellular matrix and fibroblast recruitment from an as-yet unknown source. The underlying mechanism, however, remains elusive. Here, we hypothesize that ET-1 might contribute to the accumulation of cardiac fibroblasts through an endothelial-to-mesenchymal transition in diabetic hearts. Methods and Results— We induced diabetes mellitus in vascular endothelial cell–specific ET-1 knockout [ET-1f/f;Tie2-Cre (+)] mice and their wild-type littermates using the toxin streptozotocin. Gene expression and histological and functional parameters were examined at 8, 24, and 36 weeks after the induction of diabetes mellitus. Diabetes mellitus increased cardiac ET-1 expression in wild-type mice, leading to mitochondrial disruption and myofibril disarray through the generation of superoxide. Diabetic mice also showed impairment of cardiac microvascularization and a decrease in cardiac vascular endothelial growth factor expression. ET-1 further promotes cardiac fibrosis and heart failure through the accumulation of fibroblasts via endothelial-to-mesenchymal transition. All of these features were abolished in ET-1f/f;Tie2-Cre (+) hearts. Targeted ET-1 gene silencing by small interfering RNA in cultured human endothelial cells ameliorated high glucose–induced phenotypic transition and acquisition of a fibroblast marker through the inhibition of transforming growth factor-&bgr; signaling activation and preservation of the endothelial cell-to-cell contact regulator VE-cadherin. Conclusions— These results provide new insights suggesting that diabetes mellitus–induced cardiac fibrosis is associated with the emergence of fibroblasts from endothelial cells and that this endothelial-to-mesenchymal transition process is stimulated by ET-1. Targeting endothelial cell–derived ET-1 might be beneficial in the prevention of diabetic cardiomyopathy.

Vascular endothelial cell-derived endothelin-1 mediates vascular inflammation and neointima formation following blood flow cessation

AIMS Although endothelin-1 (ET-1) has been suggested to contribute to the pathogenesis of neointima formation and atherosclerosis, the individual roles of ET-1 derived from certain cell types in this disease remain unclear. In this study, we determined the role of vascular endothelial ET-1 on vascular inflammation and neointima formation using vascular endothelial ET-1-knockout [ET-1(f/f); Tie2-Cre (+)] mice. METHODS AND RESULTS Intimal hyperplasia was induced by complete ligation of the left carotid artery in 12-week-old male ET-1(f/f);Tie2-Cre (+) mice (n = 35) and the wild-type (WT) littermates (n = 34). Following this intervention, neointima formation was reduced in ET-1(f/f);Tie2-Cre (+) mice compared with the WT mice, independent of the difference in blood pressure. This reduction was associated with a decrease in inflammatory cell recruitment to the vessel wall, which was accompanied by reduced expression levels of endothelial adhesion molecules as well as chemokines and a decrease in vascular smooth muscle cell proliferation. CONCLUSION The results of our study provide direct evidence for the role of vascular endothelial ET-1 in mediating vascular inflammation and neointima formation following vascular injury in addition to promoting vasoconstriction and cell proliferation. Furthermore, this study suggests a strategy for the efficient design of ET receptor antagonists with targeted inhibition of ET-1 signalling in vascular endothelial cells.

ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy.

AIMS Hypertensive patients develop cardiac hypertrophy and fibrosis with increased stiffness, contractile deficit and altered perfusion. Angiotensin II (AngII) is an important factor in the promotion of this pathology. The effects of AngII are partly mediated by endothelin-1 (ET-1) and transforming growth factor-β. The exact feature of these pathways and the intercellular communications involved remain unclear. In this study, we explored the role of endothelial cell-derived ET-1 in the development of AngII-induced cardiac fibrosis and hypertrophy. MAIN METHODS We used mice with vascular endothelial cell specific ET-1 deficiency (VEETKO) and their wild type littermates (WT). Mice were infused for one week with AngII (3.2mg/kg/day, n=12) or vehicle (0.15 mol/L NaCl and 1 mmol/L acetic acid, n=5), using subcutaneous mini-pumps. Hearts were stained with hematoxylin-eosin and massons trichrome for histology. Cardiac gene expression and protein abundance were measured by Northern Blot, real time PCR and Western Blot. KEY FINDINGS AngII-induced cardiac hypertrophy, interstitial and perivascular fibrosis were less pronounced in VEETKO mice compared to WT. Blood pressure increased similarly in both genotypes. Expression of connective tissue growth factor, tumor growth factor-β, collagen I and III in response to AngII required endothelial ET-1. Endothelial ET-1 was also necessary to the elevation in protein kinase C δ abundance and ERK1/2 activation. AngII-induced elevation in PKCε abundance was however ET-1 independent. SIGNIFICANCE This study underscores the significance of ET-1 from the vasculature in the process of AngII-induced cardiac hypertrophy and fibrosis, independently from blood pressure. Endothelial ET-1 represents therefore a possible pharmacological target.

Dual ECE/NEP Inhibition on Cardiac and Neurohumoral Function During the Transition From Hypertrophy to Heart Failure in Rats

CGS 26303 is a vasopeptidase inhibitor that simultaneously inhibits endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP). We compared the effects of chronic treatment with CGS 26303 to the selective inhibition of angiotensin-converting enzyme (ACE) and NEP during the transition from left ventricular hypertrophy (LVH) to congestive heart failure (CHF) in hypertensive rats. LV geometry and function were assessed in Dahl salt-sensitive rats placed on a high-salt diet from age 6 weeks (hypertensive rats) and in control rats fed a low-salt diet. The hypertensive rats were randomized into groups that received no treatment or were treated with an ACE inhibitor (temocapril), an ECE/NEP inhibitor (CGS 26303), or a NEP inhibitor (CGS 24592) from the LVH stage (11 weeks) to the CHF stage (17 weeks). All treatments decreased the systolic blood pressure equally and significantly improved LV fractional shortening. Both temocapril and CGS 26303 ameliorated LV perivascular fibrosis, reduced mRNA levels of types I and III collagen, and decreased the heart weight/body weight ratio. CHF rats had increased plasma ET-1 levels compared with control rats. Only CGS 26303 reduced ET-1 to normal levels. ET-1 levels were found to correlate with heart/body weight, right ventricle/body weight and perivascular fibrosis ratios. During the transition to CHF, CGS 26303 produces effects that are comparable to temocapril and superior to CGS 24592. The beneficial effects of CGS 26303 are likely caused in part by the greater reduction of plasma ET-1. Dual ECE/NEP inhibitor may provide a new strategy for the treatment of human heart failure.

13 METHYLENETETRAHYDROFOLATE REDUCTASE C677T GENE POLYMORPHISM IS ASSOCIATED WITH HYPERTENSION IN RURAL SUNDANESE POPULATION OF GUNUNG SARI VILLAGE, BOGOR-INDONESIA

Background: Methylenetetrahydrofolate Reductase (MTHFR) C677T polymorphism is associated with hypertension depending on ethnic and geographic region. Several studies found that the affordable and widely available riboflavin or vitamin B2 as a cofactor of MTHFR could lower blood pressure in patients with MTHFR variants. The benefits of risk stratification and therapeutic potential made it interesting to be studied. No study of MTHFR C677T polymorphism on hypertension is available in Indonesia. Objective: This study sought to determine the association of MTHFR C677T gene polymorphism and hypertension in rural Sundanese population of Gunung Sari Village, Bogor-Indonesia. Methods: A total of 415 subjects consisting of 213 hypertensive subjects and 202 non-hypertensive subjects as a control group were enrolled in this case-control study. The genotypes of the MTHFR C677T were determined by a TaqMan assay. Results: There was a significant difference of MTHFR C677T polymorphism between hypertensive group and control group; 62,9% CC; 34,3% CT; 2,8% TT vs 77,7% CC; 20,8% CT; 1,5% TT with p value of 0,004; and mutant (TT and CT) vs wild type genotype (CC) with p value of 0,001 respectively. After adjustment of age, body mass index, waist circumference, and diabetes mellitus, there was an association between MTHFR C677T polymorphism with hypertension (OR 2,1; 95% CI 1,3–3,5) Conclusion: There is an association between MTHFR C677T gene polymorphism and hypertension in rural Sundanese population of Gunung Sari Village, Bogor-Indonesia.