Tetsuaki Hirase

Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is regarded as a protective factor against atherosclerosis. Therefore, augmentation of eNOS expression or NO production by pharmacological intervention is postulated to inhibit atherosclerosis. We crossed eNOS-overexpressing (eNOS-Tg) mice with atherogenic apoE-deficient (apoE-KO) mice to determine whether eNOS overexpression in the endothelium could inhibit the development of atherosclerosis. After 8 weeks on a high-cholesterol diet, the atherosclerotic lesion areas in the aortic sinus were unexpectedly increased by more than twofold in apoE-KO/eNOS-Tg mice compared with apoE-KO mice. Also, aortic tree lesion areas were approximately 50% larger in apoE-KO/eNOS-Tg mice after 12 weeks on a high-cholesterol diet. Expression of eNOS and NO production in aortas from apoE-KO/eNOS-Tg mice were significantly higher than those in apoE-KO mice. However, eNOS dysfunction, demonstrated by lower NO production relative to eNOS expression and enhanced superoxide production in the endothelium, was observed in apoE-KO/eNOS-Tg mice. Supplementation with tetrahydrobiopterin, an NOS cofactor, reduced the atherosclerotic lesion size in apoE-KO/eNOS-Tg mice to the level comparable to apoE-KO mice, possibly through the improvement of eNOS dysfunction. These data demonstrate that chronic overexpression of eNOS does not inhibit, but accelerates, atherosclerosis under hypercholesterolemia and that eNOS dysfunction appears to play important roles in the progression of atherosclerosis in apoE-KO/eNOS-Tg mice.

HMG-CoA Reductase Inhibitor Has Protective Effects Against Stroke Events in Stroke-Prone Spontaneously Hypertensive Rats

Background and Purpose— Recent clinical studies suggest that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert protective effects against nonhemorrhagic stroke. In a murine cerebral ischemia model produced by occlusion of the middle cerebral artery, statins were shown to reduce infarct size. However, the effect of statins on hypertension-based stroke is unknown. The purpose of this study is to clarify the effect of a statin on stroke in stroke-prone spontaneously hypertensive rats (SHR-SP), in which both cerebral hemorrhage and infarction occur. Methods— We treated SHR-SP chronically from 4 weeks of age with cerivastatin (2 mg/kg per day by gavage) or vehicle. The physiological parameters, the incidence of stroke-associated symptoms, and mortality were assessed. Results— At 14 weeks of age, the incidence (13±3% versus 37±8%;P <0.01) and the size of stroke (1.6±0.2 versus 2.2±0.1 arbitrary units;P <0.01) were significantly decreased by cerivastatin, although blood pressure and plasma cholesterol levels were not different. Moreover, stroke-associated symptoms and early mortality of SHR-SP were markedly reduced in the statin-treated group (mortality at the age of 15 weeks: 15% versus 50%;P <0.05). Statin treatment significantly reduced superoxide production from nonstroke parenchyma of brain and infiltration of inflammatory cells to the stroke lesions. Conclusions— Our data show that a high dose of statin exerts protection against hypertension-based stroke and ameliorates the disease severity via inhibition of superoxide production and modulation of inflammation in brain.

Overexpression of Endothelial Nitric Oxide Synthase in Endothelial Cells Is Protective against Ischemia- Reperfusion Injury in Mouse Skeletal Muscle

Microvascular injury has been proposed to be a main cause of ischemia-reperfusion (I/R) injury. The roles of endothelial nitric oxide synthase (eNOS)-derived NO, a key regulator of vascular function, in I/R injury are incompletely understood. We used transgenic mice overexpressing eNOS in endothelial cells (eNOS-Tg) and their littermates wild-type mice (WT) to investigate the roles of eNOS in I/R injury in skeletal muscle. Superoxide levels in the affected muscles were reduced by approximately 50% in eNOS-Tg compared with WT during reperfusion. In WT, the disassembly of endothelial junctional proteins seen in the early period of reperfusion was recovered in the later phase. These findings were correlated with the increased vascular permeability in vivo. In contrast, eNOS-Tg maintained the endothelial junction assembly as well as vascular permeability during reperfusion. Leukocyte extravasation into tissue and up-regulated expression of adhesion molecules in the reperfused vessels were significantly inhibited in eNOS-Tg. Tissue viability of the affected muscle was decreased in WT time-dependently after reperfusion, whereas eNOS-Tg showed no significant reduction. NOS inhibition completely reversed these protective effects of eNOS overexpression in I/R injury. Thus, eNOS overexpression appears to prevent the I/R injury in skeletal muscle by maintaining vascular integrity.

A calcium channel blocker, benidipine, inhibits intimal thickening in the carotid artery of mice by increasing nitric oxide production

Objective Recent studies suggest that several calcium channel blockers exert their protective effects against vascular disorders by increasing nitric oxide (NO) production from the endothelium. The purpose of this study was to clarify the effects of a long-lasting calcium channel blocker, benidipine, on vascular remodeling. Methods The left common carotid arteries of mice were completely ligated just proximal to the carotid bifurcation. Treatment with benidipine (3 mg/kg per day) or vehicle was started 1 week before the carotid ligation, and continued throughout the experiments. Four weeks after the carotid ligation, these mice were killed and vascular remodeling was analyzed. Moreover, NO production and endothelial NO synthase (eNOS) expression were assessed. Results At 4 weeks after ligation, the neointimal area in the vehicle-treated mice was 39 400 ± 4900 μm2 (n = 8), whereas that in the drug-treated mice was reduced to 18 300 ± 3800 μm2 (n = 10). Consequently, the luminal area was 35% larger in the drug-treated mice. Benidipine increased the basal as well as agonist-induced NO production from the endothelium, detected by Griess method or NOx analyzer. Endothelial NOS expression in vessels of the drug-treated mice was increased compared with that of the vehicle-treated mice. Conclusion Our data provide evidence that benidipine increases NO production via increment of eNOS protein in vessels and prevents intimal thickening in mice. These results show the possibility of benidipine as a protective tool against vascular remodeling independent of its effect on blood pressure.

Simvastatin Stimulates Vascular Endothelial Growth Factor Production by Hypoxia-inducible Factor-1α Upregulation in Endothelial Cells

Objective: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and plays an important pathophysiological role in the maintenance of tissue structure as well as regeneration after ischemic injury. Three-hydroxy-3methylglutaryl-CoA reductase inhibitors reduce vascular inflammation and induce angiogenesis. This study examined whether simvastatin stimulates VEGF expression in endothelial cells as well as the nature of its underlying mechanism. Methods and Results: Simvastatin induced mRNA expression and protein secretion of VEGF in endothelial cells that were reversed by pretreatment with mevalonate and geranylgeranylpyrophosphate but not by farnesylpyrophosphate. Adenovirus-mediated expression of the dominant-negative mutant of RhoA induced VEGF mRNA and protein. Simvastatin increased hypoxia-inducible factor-1α (HIF-1α) protein level without changing its mRNA expression. Inhibition of RhoA had similar effects to simvastatin on VEGF expression. Inhibition of RhoA caused the translocation of HIF-1α to the nuclear fraction. Depletion of HIF-1α by RNA interference blocked simvastatin-induced VEGF mRNA expression. Conclusions: Simvastatin stimulates VEGF expression by RhoA downregulation and HIF-1α upregulation in endothelial cells. These data indicate a novel role for RhoA as a negative regulator of HIF-1α.

Angiotensin II induces microtubule reorganization mediated by a deacetylase SIRT2 in endothelial cells

Angiotensin II has been implicated in vascular remodeling. Microtubule composed of tubulins regulates cell shape, migration and survival. Tubulin acetylation has an important role in the control of microtubule structure and microtubule-based cellular functions. In this study, angiotensin II induced disassembly and deacetylation of α-tubulin, which were blocked by pretreatment with an angiotensin II type 1 receptor blocker losartan and a sirtuin class deacetylase inhibitor sirtinol, and by depletion of a deacetylase SIRT2 using RNA interference. We investigated the involvement of SIRT2 in angiotensin II-induced endothelial cell migration using the Boyden chamber method. Angiotensin II caused a significant increase in cell migration, which was blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported that angiotensin II is involved in cytoskeletal reorganization stimulated by mechanical stretch in endothelial cells. We also demonstrated that endothelial cells subjected to a 10% uniaxial stretch showed vertical alignment to the direction of tension and tubulin deacetylation in the peripheral side of cells, in comparison with control static cells. The mechanical stretch-induced changes of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion. Immunofluorescence microscopy showed that acetylated tubulin was decreased in platelet-endothelial cell adhesion molecule-1-positive cells in the intima of the aortic walls in mice loaded with angiotensin II, in comparison with mice loaded with control vehicle. These data show that angiotensin II and mechanical stretch stimulate microtubule redistribution and deacetylation via SIRT2 in endothelial cells, suggesting the emerging role of SIRT2 in hypertension-induced vascular remodeling.

Pentraxin-3 regulates the inflammatory activity of macrophages

Background and aims Pentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aimed to investigate the function of PTX3 in macrophages. Methods PMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis. Results In the presence of PTX3, macrophage interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and MCP-1 levels were reduced significantly (−39%, P=0.007; −21%, P=0.008; and −67%, P=0.0003, respectively), whilst activated transforming growth factor-β (TGF−β) was detected in the THP-1 macrophages (P=0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced nuclear factor-kappa B (NF-κB) activation by 35% (P=0.002), which was induced by TNF-α in THP-1 macrophages. Furthermore, silencing of PTX3 in apoptotic cells resulted in increased macrophage binding, elevated expression rate of HLA-DR (+30%, P=0.015) and CD86 (+204%, P=0.004) positive cells, and induction of IL-1β (+36%, P=0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner. Conclusions We identified PTX3 as a novel regulator of macrophage activity, and this function suggests that PTX3 acts to resolve inflammation.

Tight junctions and adherens junctions in endothelial Cells: structure and regulation

Endothelial cells lining the vasculature provide a crucial interface between plasma and tissue environments, separating the solute, macromolecular and cellular composition of the plasma from that of the interstitial fluid [1–3]. This chapter will focus on the role of endothelial cell-cell adhesion as relates to this function. In particular, the tight junction plays the most important role in the separation of plasma and tissue environments. The tight junction also plays a crucial role in the establishment and maintenance of cellular polarity, the separation of the plasma membrane into apical and basolateral compartments, important for the vectorial transport of essential nutrients and factors across endothelia with especially well developed tight junctions [4]. An example of such a differentiated endothelium is the blood-brain barrier, where tight junctions are so well developed that even ionic permeability is severely limited [5]. In epithelial cells [6] and endothelial cells [3, 7, 8], the establishment of tight junctions is dependent on the prior formation of intercellular adherens junctions. For this reason, the adherens junction will also be discussed. Both tight and adherens junctions are composed of transmembrane proteins with associated cytoplasmic components. Also, both junctions are linked to the actin-based cytoskeleton which in itself is subject to complex regulation.

PS 13-05 PENTRAXIN-3 REGULATES THE INFLAMMATORY ACTIVITY OF MACROPHAGES

Objective: Pentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aims to investigate the function of PTX3 in macrophages. Design and Method: PMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis. Results: In the presence of PTX3, macrophage interleukin-1b (IL-1b), tumor necrosis factor-alpha (TNF-a) and MCP-1 levels were reduced significantly (−39%, P = 0.007; -21%, P = 0.008; and-67%, P = 0.0003, respectively), whilst the activated transforming growth factor-b (TGF-b) was detected in the THP-1 macrophages (P = 0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced the nuclear factor-kappa B (NF-&kgr;B) activation by 35% (P = 0.002), which was induced by TNF-a in THP-1 macrophages. Furthermore, the silencing of PTX3 in apoptotic cells resulted in the increased macrophage binding, elevated expression rate of HLA-DR (+30%, P = 0.015) and CD86 (+204%, P = 0.004) positive cells, and induction of IL-1b (+36%, P = 0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner. Conclusions: We identified PTX3 as a novel regulator of macrophage activity, which suggests that PTX3 acts to resolve inflammation.