Smith, Leslie

The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor–δ (PPARδ) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPARδ agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPARδ significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with N G -nitro- l -arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPARδ stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.

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.

Abstract The coronary vasoconstrictor effects of endothelins, mediated by both endothelin ETA and ETB receptors, may be differentially altered in pathophysiological states associated with endothelial dysfunction and elevated endothelin levels. Experimental hypercholesterolemia is associated with coronary endothelial dysfunction and increased circulating endothelin concentrations. These studies were designed to test the hypothesis that experimental hypercholesterolemia is characterized by a differentially altered coronary contractile response to ETA- and ETB-receptor stimulation, in vitro. Pigs were fed either a normal or a high-cholesterol diet for 10 to 13 weeks. Changes in the intraluminal diameter of pressurized small coronary arteries (<481±25 μm in diameter) to cumulative concentrations (10 −10 to 10 −6 mol/L) of endothelin-1 (ET-1), and sarafotoxin 6c (S6c), a specific ETB-receptor agonist, were measured using a video dimension analyzer. The maximal contraction attained with ET-1 was greater than with S6c in both normal (86±7% versus 47±7%, P =.001) and hypercholesterolemic (77±6% versus 37±7%, P <.001) pigs. At 10 −10 mol/L, vessels from hypercholesterolemic pigs manifested greater contraction to both ET-1 (23±6% versus 8±3%, P =.02) and S6c (17±5% versus 4±2%, P =.02). Incubation of arteries from hypercholesterolemic pigs with BQ-788 (ETB-receptor antagonist), but not FR-139317 (ETA-receptor antagonist), altered the contractile response to ET-1 at 10 −10 mol/L. Removal of the endothelium abolished the difference in response to S6c between normal and hypercholesterolemic pigs. These studies demonstrate that experimental hypercholesterolemia is characterized by enhanced coronary vasoconstriction to endothelins in vitro, the mechanism of which is mediated mainly through the ETB receptor. Thus, the ETB receptor has a role in regulation of coronary artery tone in both the steady-state and pathophysiological states.

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.

Background and Purpose— The role of brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B, in control of cerebral circulation is poorly understood. The present study was designed to investigate the cerebral vascular effects of BDNF in vivo. Methods— Replication incompetent adenovirus encoding either rat BDNF (AdBDNF) or green fluorescent protein was injected intracisternally into rabbits. Forty-eight hours later, animals were euthanized. Plasma and cerebrospinal fluid levels of BDNF were measured by enzyme-linked immunosorbent assay, vasomotor function of isolated basilar arteries was studied in organ chambers, protein expression in the basilar arteries was studied by Western blotting, prostanoid levels were measured by enzyme-linked immunosorbent assay, and cyclic adenosine 3′,5′-monophosphate levels were measured by radioimmunoassay. Results— The levels of BDNF in the cerebrospinal fluid were significantly elevated in AdBDNF-treated rabbits as compared with adenovirus encoding green fluorescent protein-treated rabbits (37±5 ng/mL versus 0.006±0.003 ng/mL, respectively; P <0.05; n=14). Western blotting studies revealed that in basilar arteries, AdBDNF increased protein expression of prostacyclin synthase, whereas expression of endothelial nitric oxide synthase and phosphorylated (Ser 1177) endothelial nitric oxide synthase remained unchanged. During incubation with arachidonic acid (1 μmol/L), PGI 2 production and levels of cyclic adenosine 3′,5′-monophosphate were significantly elevated only in AdBDNF-treated rabbit basilar arteries ( P <0.05, n=6). Relaxations to acetylcholine (10 −9 to 10 −5 mol/L) and arachidonic acid (10 −9 to 10 −5 mol/L) were significantly potentiated in basilar arteries from rabbits injected with AdBDNF. Potentiation of relaxations to acetylcholine in AdBDNF-treated basilar arteries was inhibited by the nonselective cyclooxygenase inhibitor, indomethacin (10 −5 mol/L, P <0.05, n=6) and constitutive phospholipase A 2 inhibitor, AACOCF3 (2×10 −5 mol/L, P <0.05, n=5). Conclusion— Our results demonstrate that in cerebral arteries, BDNF-induced activation of tropomyosin receptor kinase B receptor signaling in vivo promotes prostacyclin biosynthesis. These findings provide novel mechanistic insight into the vascular protective effect of BDNF in cerebral circulation.

In the present study we hypothesized that endothelial progenitor cells (EPCs) enhance production of vasoprotective substances in cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM-2) for 7 days to yield EPCs. Rabbit basilar arteries were exposed to autologous EPCs (≈5×10 5 cells) in vitro or in vivo. Twenty-four hours after intracisternal delivery of autologous EPCs, basilar arteries were isolated and expression of vasoregulatory proteins, production of prostacyclin (PGI 2 ), and cAMP were determined. Arteries transplanted with EPCs demonstrated increased protein expression of cyclooxygenase-2 and PGI 2 in adventitia, media, and endothelium. Furthermore, production of PGI 2 and arterial content of cAMP, second messenger for PGI 2 , were significantly augmented after transplantation of EPCs. In contrast, production of thromboxane A 2 was significantly reduced, whereas production of prostaglandin E 2 remained unchanged. The increased production of PGI 2 and arterial content of cAMP were inhibited only by a selective cyclooxygenase-2 inhibitor, NS-398. In vitro or in vivo treatment of basilar artery with conditioned media from EPCs also caused increase in cyclooxygenase-2 and PGI 2 synthase protein expression associated with elevation of cAMP. Our results suggest that in cerebral arteries, paracrine effect of EPCs promotes vasoprotection by increasing PGI 2 production and intracellular concentration of cAMP. This effect appears to be mediated by activation of arachidonic acid metabolism via stimulation of cyclooxygenase-2/PGI 2 synthase pathway.

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— In the present study, the effect of subarachnoid hemorrhage (SAH) on the phosphorylation of endothelial NO synthase (eNOS) and the ability of recombinant erythropoietin (Epo) to augment this vasodilator mechanism in the spastic arteries were studied. Methods— Recombinant adenoviral vectors (10 9 plaque-forming units per animal) encoding genes for human Epo (AdEpo), and β-galactosidase were injected immediately after injection of autologous arterial blood into the cisterna magna (day 0) of rabbits. Cerebral angiography was performed on day 0 and day 2, and basilar arteries were harvested for Western blots, measurement of cGMP levels, and analysis of vasomotor functions. Results— Injection of autologous arterial blood into cisterna magna resulted in significant vasospasm of the basilar arteries. Despite the narrowing of arterial diameter and reduced expression of eNOS, expressions of phosphorylated protein kinase B (Akt) and phosphorylated eNOS were significantly increased in spastic arteries. Gene transfer of AdEpo reversed the vasospasm. AdEpo-transduced basilar arteries demonstrated significant augmentation of the endothelium-dependent relaxations to acetylcholine, whereas the relaxations to an NO donor, 2-( N , N -diethylamino)diazenolate-2-oxide sodium salt, were not affected. Transduction with AdEpo further increased the expression of phosphorylated Akt and eNOS and elevated basal levels of cGMP in the spastic arteries. Conclusions— Phosphorylation of eNOS appears to be an adaptive mechanism activated during development of vasospasm. The vascular protective effect of Epo against cerebral vasospasm induced by SAH may be mediated in part by phosphorylation of Akt/eNOS.

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.

Background and Purpose— Mutation of serine 1179 to aspartate on the endothelial NO synthase (eNOS) increases NO production in the absence of stimulation by agonists. The present study was designed to determine the effect of recombinant S1179DeNOS gene expression on the vasomotor function of human pial arteries. Methods— Pial arteries were isolated from 28 patients undergoing temporal lobectomy for intractable seizures. Adenoviral vectors (10 10 pfu/mL) encoding β-galactosidase (AdCMVLacZ) or S1179DeNOS (AdCMVS1179DeNOS) were used for ex vivo gene transfer, and vasomotor function was evaluated in control and transduced arteries. Results— Contractions to cumulative additions of U46619 were not affected by expression of LacZ or S1179DeNOS. Endothelium-dependent relaxations to bradykinin or endothelium-independent relaxations to Diethylaminodiazen-1-ium-1,2-dioate were significantly reduced in arteries expressing S1179DeNOS. A superoxide dismutase mimetic, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, failed to improve the reduced relaxations to bradykinin. The levels of cGMP were significantly elevated in arteries expressing S1179DeNOS. Conclusions— Our results support the concept that high local production of NO in pial arterial wall causes adaptive reduction of vasodilator reactivity to NO.