Wenze Wang

LOX-1 abrogation reduces cardiac hypertrophy and collagen accumulation following chronic ischemia in the mouse

We hypothesized that lectin-like oxidized LDL receptor-1 (LOX-1) deletion may inhibit oxidative stress signals, reduce collagen accumulation and attenuate cardiac remodeling after chronic ischemia. Activation of LOX-1 plays a significant role in the development of inflammation, apoptosis and collagen signals during acute ischemia. Wild-type and LOX-1 knockout (KO) mice were subjected to occlusion of left coronary artery for 3 weeks. Markers of cardiac hypertrophy, fibrosis-related signals (collagen IV, collagen-1 and fibronectin) and oxidant load (nicotinamide adenine dinucleotide phosphate oxidase expression, activity of mitogen-activated protein kinases and left ventricular (LV) tissue thiobarbituric acid reactive substances) were analyzed. In in vitro experiments, HL-1 cardiomyocytes were transfected with angiotensin II (Ang II) type 1 receptor (AT1R) or type 2 receptor (AT2R) genes to determine their role in the cardiomyocyte hypertrophy. LOX-1 KO mice had 25% improvement in survival over the 3-week period of chronic ischemia. LOX-1 deletion reduced collagen deposition and cardiomyocyte hypertrophy (∼75%) in association with a decrease in oxidant load and AT1R upregulation (all P<0.05). The LOX-1 KO mice hearts exhibited a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 17 (ADAM17) expression and matrix metalloproteinase 2 activity, and increased AT2R expression (P<0.05). Attenuation of cardiac remodeling was associated with improved cardiac hemodynamics (LV ±dp/dt and cardiac ejection fraction). In vitro studies showed that it is AT1R, and not AT2R overexpression that induces cardiomyocyte hypertrophy. We demonstrate for the first time that LOX-1 deletion reduces oxidative stress and related intracellular signaling, which leads to attenuation of the positive feedback loop involving AT1R and LOX-1. This results in reduced chronic cardiac remodeling.

Statins and angiogenesis: is it about connections?

Statins, inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, have been shown to induce both angiogenic and angiostatic responses. We attempted to resolve this controversy by studying the effects of two different statins, rosuvastatin and simvastatin, in two different assay systems. In the matrigel angiogenesis assay, both statins enhanced tube formation by human umbilical vein endothelial cells (HUVECs, p<0.01 vs. control). In the ex vivo mouse aortic ring sprouting assay, both statins virtually abolished new vessel formation (p<0.01). As a basic difference between the two models of angiogenesis is dispersed state of endothelial cells vs. compact monolayer, we analyzed influence of statins on endothelial junction proteins. RT-PCR analysis and cytoimmunostaining of HUVECs treated with simvastatin revealed increased expression of VE-cadherin (p<0.05). The blockade of VE-cadherin with a specific antibody reversed simvastatin-induced tube formation (p<0.002). These data suggest that statins through VE-cadherin stimulation modulate cell-cell adhesion and diminish the ability of cells to proliferate and migrate. The observations of reduced angiogenesis in the intact vessel may relate to anti-atherosclerotic and anti-cancer effects of statins, and provide a feasible explanation for conflicting data under different experimental conditions.

Cardiac Hypertrophy During Hypercholesterolemia and Its Amelioration With Rosuvastatin and Amlodipine

Hypercholesterolemia is a common accompaniment of atherosclerosis and may be associated with cardiac hypertrophy. To define the mechanistic basis of cardiac hypertrophy in hypercholesterolemia, we fed low-density lipoprotein receptor knockout (LDLR KO) mice regular diet or high cholesterol (HC) diet for 26 weeks. There was clear evidence of cardiomyocyte hypertrophy and collagen deposition in the hearts of LDLR KO mice fed with HC diet, confirmed by histopathology (hematoxylin and eosin and Picrosirius staining) and upregulation of genes for brain natriuretic peptide, α-tubulin, transforming growth factor β1, and connective tissue growth factor (CTGF). These changes were independent of change in blood pressure. The hypercholesterolemic mice hearts showed an upregulation of LOX-1, an oxidized low-density lipoprotein receptor, and angiotensin II type 1 receptor (AT1R) at messenger RNA level. In addition, there was a marked upregulation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor κB (NF-κB) messenger RNA, indicating overexpression of markers of oxidant stress. A separate group of LDLR KO mice were fed HC diet along with a potent 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor rosuvastatin or a dihydropyridine calcium channel blocker amlodipine. Administration of rosuvastatin or amlodipine reduced the overexpression of genes for LOX-1 and AT1R and associated NADPH oxidase and NF-κB. These phenomena were associated with a marked decrease in cardiomyocyte hypertrophy and collagen deposits in and around the cardiomyocytes. In conclusion, this study provides evidence of cardiac hypertrophy and fibrosis in hypercholesterolemia independent of blood pressure change LOX-1 and AT1R act as possible signals for oxidant stress leading to alterations in cardiac structure during hypercholesterolemia. Most importantly, rosuvastatin and amlodipine ameliorate cardiomyocyte hypertrophy and fibrosis.

Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (CaL) current and vascular tone is increased because of increased expression of the noncardiac form of the CaL (Cav1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Cav1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Cav1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Cav1.2 expression by 61% and decreased the CaL current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Cav1.2, it did not affect the CaL expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Cav1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Cav1.2 siRNA without similarly affecting cardiac CaL expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension.

Angiotensin II causes endothelial-dependent increase in expression of CaV1.2 protein in cultured arteries

Examination was made of the direct vascular effects of the hypertension-inducing pressor hormone angiotensin II on expression and activity of the voltage-gated calcium channel Ca(V)1.2. Freshly dissected rat superior mesenteric artery beds were maintained in organ culture unpressurized for 24 h in the presence or absence of angiotensin II. Relative to controls, angiotensin II increased Ca(V)1.2 protein expression and tension-inducing activity but not Ca(V)1.2 message. The increase in Ca(V)1.2 protein expression by angiotensin II was abrogated by damaging the endothelium. Thus, the endothelium is involved in regulating Ca(V)1.2 expression in the vascular wall.

Angiotensin II upregulates Ca(V)1.2 protein expression in cultured arteries via endothelial H(2)O(2) production.

Background: We previously reported that angiotensin II caused an endothelial-dependent increase in L-type voltage-dependent Ca²⁺ channel (Ca_V1.2) in cultured arteries, but the signaling pathways are not clear. Methods: Endothelial damage was generated by brief intra-arterial perfusion with 0.3% CHAPS. Ca_V1.2 expression, function and H₂O₂ were measured by Western blot, tension recording and Amplex Red H₂O₂ assay kit, respectively. Results: Angiotensin II dose-dependently upregulated Ca_V1.2 expression in endothelium-intact arteries. The angiotensin II upregulation of Ca_V1.2 expression in endothelium-intact arteries was blocked by NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI), apocynin, a more specific NAD(P)H oxidase inhibitor gp91ds-tat and also by catalase. H₂O₂ similarly upregulated Ca_V1.2 expression in endothelium-intact and endothelium-damaged arteries, and the latter effect was also blocked by DPI and apocynin. Angiotensin II increased H₂O₂ production by endothelium-intact but not by endothelium-damaged arteries, and this effect was blocked by apocynin, catalase and gp91ds-tat. The upregulation of Ca_V1.2 by angiotensin II and H₂O₂ is accompanied by an increased tension response to KCl and the Ca²⁺ channel activator FPL 64176, and this effect was also attenuated by gp91ds-tat. Conclusion: These results suggest that angiotensin II stimulates endothelial NAD(P)H oxidase-produced H₂O_2, which may additionally act through vascular smooth muscle NAD(P)H oxidase, to upregulate vascular Ca_V1.2 protein.