BackgroundAngiopoietin like‐2 (angptl2), a proinflammatory protein, is overexpressed in endothelial cells (ECs) from patients with coronary artery disease (CAD). Whether angptl2 contributes to atherogenesis is unknown. We tested the hypothesis that angptl2 promotes inflammation and leukocyte adhesion onto ECs, thereby accelerating atherogenesis in preatherosclerotic dyslipidemic mice.Methods and ResultsIn ECs freshly isolated from the aorta, basal expression of TNF‐α and IL‐6 mRNA was higher in 3‐month‐old severely dyslipidemic mice (LDLr−/−; hApoB100+/+ [ATX]) than in control healthy wild‐type (WT) mice (P<0.05) and was increased in both groups by exogenous angptl2 (100 nmol/L). Angptl2 stimulated the adhesion of leukocytes ex vivo on the native aortic endothelium of ATX, but not WT mice, in association with higher expression of ICAM‐1 and P‐selectin in ECs (P<0.05). Antibodies against these endothelial adhesion molecules prevented leukocyte adhesion. Intravenous administration of angptl2 for 1 month in preatherosclerotic 3‐month‐old ATX mice increased (P<0.05) total cholesterol and LDL‐cholesterol levels, strongly induced (P<0.05) the expression of endothelial proinflammatory cytokines and adhesion molecules while accelerating atherosclerotic lesion formation by 10‐fold (P<0.05). Plasma and aortic tissue levels of angptl2 increased (P<0.05) with age and were higher in 6‐ and 12‐month‐old ATX mice than in age‐matched WT mice. Angptl2 accumulated to high levels in the atherosclerotic lesions (P<0.05). Finally, angptl2 was greatly expressed (P<0.05) in ECs cultured from CAD patients, and circulating angptl2 levels were 6‐fold higher in CAD patients compared with age‐matched healthy volunteers.ConclusionsAngptl2 contributes to the pathogenesis of atherosclerosis.

The cardiac cycle imposes a mechanical stress that dilates elastic carotid arteries, while shear stress largely contributes to the endothelium-dependent dilation of downstream cerebral arteries. In the presence of dyslipidemia, carotid arteries stiffen while the endothelial function declines. We reasoned that stiffening of carotid arteries would be prevented by reducing resting heart rate (HR), while improving the endothelial function would regulate cerebral artery compliance and function. Thus we treated or not 3-mo-old male atherosclerotic mice (ATX; LDLr(-/-):hApoB(+/+)) for 3 mo with the sinoatrial pacemaker current inhibitor ivabradine (IVA), the β-blocker metoprolol (METO), or subjected mice to voluntary physical training (PT). Arterial (carotid and cerebral artery) compliance and endothelium-dependent flow-mediated cerebral dilation were measured in isolated pressurized arteries. IVA and METO similarly reduced (P < 0.05) 24-h HR by ≈15%, while PT had no impact. As expected, carotid artery stiffness increased (P < 0.05) in ATX mice compared with wild-type mice, while cerebral artery stiffness decreased (P < 0.05); this paradoxical increase in cerebrovascular compliance was associated with endothelial dysfunction and an augmented metalloproteinase-9 (MMP-9) activity (P < 0.05), without changing the lipid composition of the wall. Reducing HR (IVA and METO) limited carotid artery stiffening, but plaque progression was prevented by IVA only. In contrast, IVA maintained and PT improved cerebral endothelial nitric oxide synthase-dependent flow-mediated dilation and wall compliance, and both interventions reduced MMP-9 activity (P < 0.05); METO worsened endothelial dysfunction and compliance and did not reduce MMP-9 activity. In conclusion, HR-dependent mechanical stress contributes to carotid artery wall stiffening in severely dyslipidemic mice while cerebrovascular compliance is mostly regulated by the endothelium.

The present study tested the hypothesis that vessel remodeling secondary to hypertension was characterized by nestin upregulation in vascular smooth muscle cells. Two weeks after suprarenal abdominal aorta constriction of adult male Sprague-Dawley rats, elevated mean arterial pressure increased the media area and thickness of the carotid artery and aorta and concomitantly upregulated nestin protein levels. In the normal adult rat carotid artery, nestin immunoreactivity was observed in a subpopulation of vascular smooth muscle cells, and the density significantly increased following suprarenal abdominal aorta constriction. Filamentous nestin was detected in cultured rat carotid arteryand aorta-derived vascular smooth muscle cells and an analogous paradigm observed in human aorta-derived vascular smooth muscle cells. ANG II and EGF treatment of vascular smooth muscle cells stimulated DNA and protein synthesis and increased nestin protein levels. Lentiviral short-hairpin RNA-mediated nestin depletion of carotid artery-derived vascular smooth muscle cells inhibited peptide growth factor-stimulated DNA synthesis, whereas protein synthesis remained intact. These data have demonstrated that vessel remodeling secondary to hypertension was characterized in part by nestin upregulation in vascular smooth muscle cells. The selective role of nestin in peptide growth factor-stimulated DNA synthesis has revealed that the proliferative and hypertrophic responses of vascular smooth muscle cells were mediated by divergent signaling events. nestin; carotid artery; aorta; hypertension; vascular remodeling DURING THE DEVELOPMENT of the central nervous system (CNS), a population of neuroepithelial stem cells was initially identified via expression of the intermediate filament protein nestin (8,18). Nestin is a 240-kDa protein and a member of the class VI family of intermediate filament proteins, and, in contrast to other classes, it is unable to self-assemble and form homodimers because of a short NH 2 terminus (37, 39). Therefore, nestin will form heterodimers with other intermediate filament proteins, including vimentin and desmin (37). The promoter region upstream of exon 1 of the nestin gene does not contain any identifiable elements regulating expression. However, the nestin gene does contain regulatory elements in the various intron regions that drive expression in a cell-specific manner (37). In neural progenitor/stem cells, nestin expression is independently regulated by restricted enhancer elements identified in the second intron (43). In humans, a highly conserved region that directed expression was also identified in the second intron of the nestin gene (21). However, nestin expression driven by the second intron was not limited to CNSresident stem cells, since a transgenic mouse containing the 5.8-kb fragment of the promoter region and the 1.8-kb fragment of the second intron of the rat nestin gene linked to the reporter green fluorescent protein (GFP) identified progenitor/ stem cell populations in the skin, skeletal mus...

Recent studies have revealed the existence of multipotent nestin-immunoreactive cells in the adult mammalian heart. These cells were recruited to infarct site following ischemic injury and differentiated to a vascular lineage leading to de novo blood vessel formation. Here, we show that a sub-population of cardiac resident nestin((+)) cells can further differentiate to a neuronal-like fate in vivo following myocardial infarction. In the ischemically damaged rat heart, neurofilament-M((+)) fibres were detected innervating the peri-infarct/infarct region and the preponderance of these fibres were physically associated with processes emanating from nestin((+)) cells. One week after isogenic heterotopic cardiac transplantation, the beating transplanted rat heart was devoid of neurofilament-M((+)) fibre staining. The superimposition of an ischemic insult to the transplanted heart led to the de novo synthesis of neurofilament-M((+)) fibres by cardiac resident nestin((+)) cells. Nerve growth factor infusion and the exposure of normal rats to intermittent hypoxia significantly increased the density of neurofilament-M((+)) fibres in the heart. However, these newly formed neurofilament-M((+)) fibres were not physically associated with nestin((+)) processes. These data highlight a novel paradigm of reparative fibrosis as a subpopulation of cardiac resident nestin((+)) cells directly contributed to neural remodelling of the peri-infarct/infarct region of the ischemically damaged rat heart via the de novo synthesis of neurofilament-M fibres.

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-β1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-β1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.

Endothelial dysfunction and oxidative stress contribute to the atherosclerotic process that includes stiffening of large peripheral arteries. In contrast, our laboratory previously reported a paradoxical increase in cerebrovascular compliance in LDLr(-/-):hApoB(+/+) atherosclerotic (ATX) mice (7). We hypothesized that prevention of cerebral artery endothelial dysfunction with a chronic dietary antioxidant intake would normalize the changes in cerebral artery wall structure and biomechanics and prevent the decline in basal cerebral blood flow associated with atherosclerosis. Three-month-old ATX mice were treated, or not, for 3 mo with the polyphenol (+)-catechin (CAT; 30 mg·kg(-1)·day(-1)) and compared with wild-type controls. In isolated, pressurized cerebral arteries from ATX mice, CAT prevented endothelial dysfunction (deterioration of endothelium-dependent, flow-mediated dilations; P < 0.05), the inward hypertrophic structural remodeling (increase in the wall-to-lumen ratio; P < 0.05), and the rise in cerebrovascular compliance (rightward shift of the stress-strain curve measured in passive conditions, reflecting mechanical properties of the arterial wall; P < 0.05). Doppler optical coherence tomography imaging in vivo confirmed these findings, showing that cerebral compliance was higher in ATX mice and normalized by CAT (P < 0.05). CAT also prevented basal cerebral hypoperfusion in ATX mice (P < 0.05). Active remodeling of the cerebrovascular wall in ATX mice was further suggested by the increase (P < 0.05) in pro-metalloproteinase-9 activity, which was normalized by CAT. We conclude that, by preserving the endothelial function, a chronic treatment with CAT prevents the deleterious effect of severe dyslipidemia on cerebral artery wall structure and biomechanical properties, contributing to preserving resting cerebral blood flow.

BackgroundAngiopoietin‐like‐2 (angptl2) is produced by several cell types including endothelial cells, adipocytes and macrophages, and contributes to the inflammatory process in cardiovascular diseases. We hypothesized that angptl2 impairs endothelial function, and that lowering angptl2 levels protects the endothelium against high‐fat diet (HFD)‐induced fat accumulation and hypercholesterolemia.Methods and ResultsAcute recombinant angptl2 reduced (P<0.05) acetylcholine‐mediated vasodilation of isolated wild‐type (WT) mouse femoral artery, an effect reversed (P<0.05) by the antioxidant N‐acetylcysteine. Accordingly, in angptl2 knockdown (KD) mice, ACh‐mediated endothelium‐dependent vasodilation was greater (P<0.05) than in WT mice. In arteries from KD mice, prostacyclin contributed to the overall dilation unlike in WT mice. After a 3‐month HFD, overall vasodilation was not altered, but dissecting out the endothelial intrinsic pathways revealed that NO production was reduced in arteries isolated from HFD‐fed WT mice (P<0.05), while NO release was maintained in KD mice. Similarly, endothelium‐derived hyperpolarizing factor (EDHF) was preserved in mesenteric arteries from HFD‐fed KD mice but not in those from WT mice. Finally, the HFD increased (P<0.05) total cholesterol–to–high‐density lipoprotein ratios, low‐density lipoprotein–to–high‐density lipoprotein ratios, and leptin levels in WT mice only, while glycemia remained similar in the 2 strains. KD mice displayed less triglyceride accumulation in the liver (P<0.05 versus WT), and adipocyte diameters in mesenteric and epididymal white adipose tissues were smaller (P<0.05) in KD than in WT fed an HFD, while inflammatory gene expression increased (P<0.05) in the fat of WT mice only.ConclusionsLack of angptl2 expression limits the metabolic stress induced by an HFD and maintains endothelial function in mice.

Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.