Citations

Abstract—Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10−9to 10−5mol/L) and Ca2+ionophore (10−9to 10−6mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10−10to 10−5mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O2−) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn–superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10−5mol/L), reduced O2−production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [14C]l-Citrulline assay showed a decrease of Ca2+-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O2−and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O2−and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.

We previously reported that small mesenteric arteries from hypertensive rats have increased NOS-derived H2O2 and reduced NO/cGMP signaling. We hypothesized that antihypertensive therapy lowers blood pressure through a tetrahydrobiopterin (BH4)-dependent mechanism restoring NO/cGMP signaling and endothelial NOS (NOS3; eNOS) phosphorylation in small arteries. To test this hypothesis, small mesenteric arteries from normotensive rats (NORM), angiotensin II-infused rats (ANG), ANG rats with triple therapy (reserperine, hydrochlorothiazide, and hydralazine), or ANG rats with oral BH4 therapy were studied. Both triple therapy and oral BH4 therapy attenuated the rise in systolic blood pressure in ANG rats and restored NO/cGMP signaling in small arteries similarly. Triple therapy significantly increased vascular BH4 levels and BH4-to-BH2 ratio similar to ANG rats with BH4 supplementation. Furthermore, triple therapy (but not oral BH4 therapy) significantly increased GTP cyclohydrolase I (GTPCH I) activity in small arteries without a change in expression. NOS3 phosphorylation at Ser1177 was reduced in small arteries from ANG compared with NORM, while NOS3 phosphorylation at Ser633 and Thr495 were similar in ANG and NORM. NOS3 phosphorylation at Ser1177 was restored with triple therapy or oral BH4 in ANG rats. In conclusion, antihypertensive therapy regulates NO/cGMP signaling in small arteries through increasing BH4 levels and NOS3 phosphorylation at Ser1177.

Previous studies have shown that treatment with erythropoietin (EPO) exerts vascular protective effects. The exact mechanisms responsible for these effects are not completely understood. In the present study, we hypothesized that EPO stimulates expression and activity of copper- and zinc-containing superoxide dismutase (SOD1), thus protecting vascular tissue from oxidative stress induced by excessive concentrations of superoxide anions. EPO treatment of wild-type mice for 2 weeks (1000 U/kg, SC, biweekly) significantly increased aortic expression of SOD1. This effect resulted in a significant reduction of superoxide anion concentrations in aorta of treated mice. The ability of EPO to reduce vascular production of superoxide anions was abolished in SOD1-deficient mice. In a mouse model of wire-induced injury of the common carotid artery, treatment of wild-type mice with EPO prevented pathological remodeling, whereas the vascular effect of EPO was absent in SOD1-deficient mice. Our findings demonstrate that treatment with EPO increases vascular expression of SOD1. This effect appears to be an important molecular mechanism underlying vascular protection by EPO.

Tetrahydrobiopterin is an essential cofactor required for the synthesis of NO. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrated that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial NO synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a 2-fold increase in GTPCH I activity was detected in the aortas of caveolin-1–deficient mice, suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by the disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells, and tetrahydrobiopterin production occurs in close proximity to endothelial NO synthase. In addition, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.

Tetrahydrobiopterin (BH 4 ) is an essential cofactor required for enzymatic activity of endothelial NO synthase. Recently, it has been shown that vascular protective effects of erythropoietin (EPO) are dependent on activation of endothelial NO synthase. Therefore, our objective was to characterize the effect of EPO on the biosynthesis of BH 4 in the vascular wall. Incubation of isolated C57BL/6J mouse aortas for 18 hours with recombinant human EPO (1 to 50 U/mL) caused a concentration-dependent increase in intracellular BH 4 levels and activity of GTP-cyclohydrolase I. Maximal biosynthesis of BH 4 was detected at therapeutic concentrations of 5 U/mL. Removal of the endothelium abolished EPO-induced biosynthesis of BH 4 demonstrating that the vascular endothelium is a major source of BH 4 . Treatment with a selective phosphatidylinositol 3-kinase inhibitor wortmannin significantly reduced BH 4 biosynthesis stimulated by EPO. The stimulatory effect of EPO on vascular GTP-cyclohydrolase I activity, BH 4 production, and phosphorylation of endothelial NO synthase was also detected in vivo in mice treated with recombinant human EPO. These effects of EPO were abolished in protein kinase Bα/Akt1-deficient mice. In addition, EPO significantly increased systolic blood pressure and the number of circulating platelets in Akt1-deficient mice. Our results demonstrate that EPO stimulates biosynthesis of BH 4 in vascular endothelium and that the increase in BH 4 levels is caused by de novo biosynthesis of BH 4 via the phosphatidylinositol 3-kinase/Akt1 pathway. This effect is most likely designed to provide optimal intracellular concentration of the cofactor necessary for EPO-induced elevation of endothelial NO synthase activity.

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI 2 ) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI 2 biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-α. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI 2 analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI 2 synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR)δ agonist GW501516 but not by the selective PGI 2 receptor agonist cicaprost. Downregulation of PPARδ by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPARδ siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI 2 synthase siRNA, COX-1 siRNA, or PPARδ siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI 2 /PPARδ pathway is an important mechanism underlying proangiogenic function of EPCs.

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine. In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Injured arteries exhibited impairment of endothelium-dependent relaxations to acetylcholine ( P <0.05). This was associated with increased medial cross-sectional area ( P <0.05). EPO upregulated expression of phosphorylated Ser1177-eNOS and normalized the vasodilator response to acetylcholine ( P <0.05). Furthermore, EPO prevented the injury-induced increase in medial cross-sectional area ( P <0.05). The vascular protective effects of EPO were abolished in eNOS-deficient mice. Most notably, EPO significantly increased systolic blood pressure and enhanced medial thickening of injured carotid arteries in eNOS-deficient mice ( P <0.05). Our results demonstrate that EPO prevents aberrant remodeling of the injured carotid artery. The protective effects of EPO are critically dependent on activation of eNOS.

Background and Purpose— Aging is a major risk for stroke and a highly complex biological process believed to involve multiple mechanisms. Mutant mice that express low levels of the spindle assembly checkpoint protein BubR1 are known to develop several aging-associated phenotypes at a very young age, including cataracts, lordokyphosis, loss of subcutaneous fat, and impaired wound healing. However, whether BubR1 acts to prevent vascular aging has not yet been established. The present study was designed to investigate the vascular phenotype of mutant mice with low levels of BubR1. Methods— Morphological, functional, and biochemical analyses were performed on aortas and carotid arteries of 3- to 5-month-old BubR1 mutant mice and wild-type littermates. Results— Arterial wall thickness and inner diameter were significantly reduced in BubR1 mutant mice. Arterial walls of BubR1 mutant mice had low numbers of medial smooth muscle cells. Masson trichrome staining showed profound fibrosis in arterial walls of BubR1 mutant. In agreement with these morphological changes, functional analysis of pressurized isolated carotid arteries of BubR1 mutant mice demonstrated reduced elastic properties. Endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to the nitric oxide donor DEA-NONOate were significantly reduced in carotid arteries of BubR1 mutant mice. Furthermore, enzymatic activity of nitric oxide synthase and levels of cyclic GMP were significantly reduced in aortas of mutant mice, but production of superoxide anions was significantly increased. Conclusions— These findings demonstrate that BubR1 insufficiency in mice results in phenotypic changes reminiscent of vascular aging in humans and suggest a role for BubR1 in suppressing the vascular aging process.

Objective— Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic cytokines. Ability of VEGF to stimulate formation of superoxide anion in vivo has not been studied. We hypothesized that in vivo expression of recombinant VEGF in the rabbit carotid artery increases production of superoxide anion. Methods and Results— Plaque-forming units (10 9 ) of adenovirus-encoding human VEGF 165 (AdVEGF) or β-galactosidase (AdLacZ) were delivered into the lumen of rabbit carotid arteries. Three days after gene delivery, expression of recombinant proteins was detected in endothelium and smooth muscle cells. Endothelium-dependent relaxations to acetylcholine were impaired in AdVEGF-transduced arteries ( P <0.01; n=5). Treatment with superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10 −5 mol/L), improved relaxations to acetylcholine ( P <0.01; n=5). Western blot analysis demonstrated increased expression of p47 phox in AdVEGF-transduced arteries ( P <0.05; n=8). Lucigenin chemiluminescence showed significantly higher production of superoxide anion in AdVEGF-transduced arteries ( P <0.05; n=5 to 10). Conclusions— Our results suggest that in vivo expression of recombinant VEGF in the vascular endothelium increases local production of superoxide anion. Superoxide anion appears to be an important mediator of vascular effects of VEGF in vivo.