Cell adhesion molecules and eNOS expression in aorta of normocholesterolemic mice with different predispositions to atherosclerosis.

C57BL/6J (B6) mice were demonstrated to be the most susceptible and C3H/HeJ (C3H) mice the most resistant to development of atherosclerosis. We hypothesized, whether pro-atherogenic (P-selectin, VCAM-1, and ICAM-1) and anti-atherogenic (endoglin and eNOS) proteins are expressed differently in aorta before the onset of atherosclerosis in these two mouse strains. B6 mice (n = 16) and C3H mice (n = 16) sustained on either chow or cholesterol (1 %) diet for 8 weeks. Biochemical analysis of lipoprotein profile and Western blot analysis of P-selectin, VCAM-1, ICAM-1, eNOS, endoglin, peNOS and TGF-ßRII in aorta were performed. Western blot analysis revealed a lower expression of P-selectin by 7 %, VCAM-1 by 51 %, ICAM-1 by 6 %, and a higher expression of eNOS (by 18 %) in C3H mice in comparison with B6 mice after cholesterol diet. Further analysis revealed that cholesterol diet significantly increased the expression of endoglin (by 97 %), TGF-ßRII (by 50 %), eNOS (by 21 %) and peNOS (by 122 %) in C3H mice, but not in B6 mice. We propose that lower expression of P-selectin, VCAM-1 and ICAM-1 and higher expression of eNOS in vivo in aorta of C3H mice might represent another potential mechanism for C3H mice being less susceptible to atherosclerosis when compared to B6 mice. In addition, endoglin seems to be involved in an upregulation of eNOS only in C3H mice. Thus, we propose that aorta of C3H mice is less prone to the expression of pro-inflammatory and endothelial dysfunction markers when compared to B6 mice, regardless of lipoprotein profile and before any signs of atherosclerotic process.

Endoglin as a possible marker of atorvastatin treatment benefit in atherosclerosis.

Endoglin (a type III TGF-ß receptor) is able to modulate ligand binding and signaling by association with the TGF-ß type I receptors (ALK-1 and ALK-5). In this study, we hypothesized whether atorvastatin treatment affects endoglin/ALK-1/p-Smad1/VEGF expression in the aorta and endoglin levels in serum in ApoE/LDLR double knockout mice. ApoE/LDLR double knockout mice were fed with a diet containing either 1% of cholesterol (CHOL) or cholesterol with atorvastatin (ATV) at a dose of 50mg/kg/day. Biochemical analysis of cholesterol levels and ELISA analysis of endoglin levels in serum, lesion area size, immunohistochemistry and Western blot analysis in mice aorta were performed. Atorvastatin treatment resulted in a significant decrease of total, VLDL and LDL cholesterol, atherosclerotic lesion size and endoglin serum levels in comparison with CHOL mice. On the other hand, atorvastatin treatment significantly increased the expressions of endoglin by 1431%, ALK-1 by 310%, p-Smad1 by 135% and VEGF by 62% in aorta when compared to CHOL mice. In conclusion, it has been demonstrated that atorvastatin increases endoglin/ALK-1/p-Smad1/VEGF expression in aorta and decreases the size of atherosclerotic lesions, suggesting that activation of this endothelial-protective pathway might support the antiatherogenic effects of atorvastatin. Moreover, atorvastatin concurrently decreased serum levels of endoglin suggesting that monitoring of endoglin levels in blood might represent an important marker of the progression and/or treatment of atherosclerosis.

Cholesterol effects on endoglin and its downstream pathways in ApoE/LDLR double knockout mice.

BACKGROUND: The aim of the study was to evaluate whether cholesterol-rich diet affects transforming growth factor-ß-RIII (endoglin) levels in blood and 2 endoglin-related pathways in the aorta of ApoE/LDLR double knockout mice. METHODS AND RESULTS: Mice were fed either chow diet (CHOW) (n=8) or by 1% cholesterol-rich diet (CHOL) (n=8). Biochemical analysis of cholesterol and endoglin levels in blood, lesion size area, immunohistochemistry and Western blot analysis in mice aortas were performed. Biochemical analysis showed that cholesterol-rich diet resulted in a significant increase of cholesterol and endoglin levels in serum, and increased plaque size in the aorta. In addition, a cholesterol-rich diet significantly decreased the expressions of endoglin by 92%, activin receptor-like kinase (ALK)-1 by 71%, p-Smad2 by 21%, and vascular endothelial growth factor (VEGF) by 37% when compared to CHOW mice, but ALK-5, p-Smad1, and endothelial nitric oxide synthase were not significantly affected. CONCLUSIONS: Hypercholesterolemia increases endoglin levels in blood and simultaneously decreases its expression in aorta, together with atherosclerosis protective markers p-Smad2 and VEGF, followed by increased plaque size. Inhibition of endoglin signaling might be one of the mechanisms responsible for the promoting of endothelial dysfunction and atherogenesis. Moreover, the monitoring of endoglin serum levels might represent an attractive blood marker of progression of disease; however, the precise source and role of endoglin in blood serum remains to be elucidated.

Spirulina platensis and phycocyanobilin activate atheroprotective heme oxygenase-1: a possible implication for atherogenesis.

Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.

The role of endoglin in atherosclerosis.

Endoglin (CD 105, TGF-ß receptor III) is a homodimeric transmembrane glycoprotein that plays a regulatory role in TGF-ß signaling. Its functional role in the context of atherosclerosis has yet to be defined and should be stated here. Therefore, we focused on the role of endoglin in atherosclerosis in both humans and experimental animals. Endoglin expression was demonstrated in atherosclerotic vessels predominantly in endothelial cells and smooth muscle cells in various types of blood vessels in mice and humans, suggesting its participation in atherogenesis. Endoglin expression was also related to the expression of eNOS in endothelium, repair of the vessel wall, plaque neoangiogenesis, production of collagen and stabilization of atherosclerotic lesions. In addition, increased levels of soluble endoglin were associated with hypercholesterolemia, atherosclerosis, acute myocardial infarction and were related to inhibition of TGF-ß signaling in the vessel wall. Moreover, soluble endoglin levels were significantly lowered after a series of extracorporeal eliminations in patients with familial hypercholesterolemia. Additionally, statin treatment decreased levels of soluble endoglin and increased its expression in aorta, which was related to reduced atherosclerosis in mice. In conclusion, we propose that measurement of soluble endoglin might give information about progression of the atherosclerotic process or the efficacy of therapeutic interventions, which is the task that must be answered in clinical trials.

Activation of TGF-ß receptors and Smad proteins by atorvastatin is related to reduced atherogenesis in ApoE/LDLR double knockout mice.

AIM: Transforming growth factor-beta (TGF-ß) plays important role in atherogenesis via TGF-ß receptors and Smad proteins, which determine its signaling activity. In this study, we hypothesized, whether non-lipid related effects of atorvastatin, affect both endoglin/ALK-5/Smad2/eNOS and/or endoglin/ALK-1/Smad1/VEGF previously proposed pathways in ApoE/LDLR double knockout mice. METHODS: ApoE/LDLR double knockout mice were divided into two groups. The chow group (CHOW) (n =8) was fed with chow diet, while in the atorvastatin group (ATV) (n =8) atorvastatin was added to the chow diet at dose 50 mg/kg/day. Biochemical analyses of lipid profile, lesion area measurement, immunohistochemistry and Western blot analysis of endoglin, ALK-1, 5, phosphorylated and non-phosphorylated forms Smad-1, 2, VEGF and eNOS proteins in mice aorta were performed. RESULTS: Biochemical analysis of blood serum and morphometric analysis of aortic lesion size showed that atorvastatin treatment resulted in a significant increase of cholesterol levels and simultaneously in reduced lesion size in aortic sinus when compared to CHOW mice. Western blot analysis revealed that atorvastatin treatment significantly increase the expressions of endoglin by 102%, ALK-1 by 113%, ALK-5 by 296%, pSmad-1 by 202%, pSmad-2 by 34%, VEGF by 68% and eNOS by 687% as compared with CHOW mice. Immunofluorescence staining revealed endoglin coexpression with all studied markers that were increased by atorvastatin treatment mainly in endothelial cells covering atherosclerotic plaques. CONCLUSION: This study shows that atorvastatin treatment increases the expression of endoglin, ALK-1, ALK-5, phosphorylated forms of Smad1 and Smad2, VEGF and eNOS and reduces atherosclerotic lesion size beyond its lipid lowering effects. Therefore, we propose that endoglin related receptors and signal transducers might play protective role in atherogenesis.