Vascular endothelial growth factor attenuates leukocyte-endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide.

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that induces vasodilation and increases endothelial release of nitric oxide (NO). NO is also reported to modulate leukocyte-endothelium interaction. Therefore, we hypothesized that VEGF might inhibit leukocyte-endothelium interaction via increased release of NO from the vascular endothelium. We used intravital microscopy of the rat mesenteric microcirculation to measure leukocyte-endothelium interactions 2, 4, and 24 h after systemic administration of VEGF to the rat (120 microg/kg, i.v., bolus). Superfusion of the rat mesentery with either 0.5 U/ml thrombin or 50 microM L-NAME consistently increased the number of rolling, adhering, and transmigrated leukocytes (P<0.01 vs. control mesenteries superfused with Krebs-Henseleit buffer). At 4 and 24 h posttreatment, VEGF significantly attenuated thrombin-induced and L-NAME-induced leukocyte rolling, adherence, and transmigration in rat mesenteric venules. In addition, adherence of isolated rat PMNs to thrombin-stimulated mesenteric artery segments in vitro was significantly reduced in mesenteric arteries isolated from VEGF-treated rats (P<0.001 vs. control rats). Direct measurement of NO demonstrated a threefold increase in basal NO release from aortic tissue of rats injected with VEGF, at 4 and 24 h posttreatment (P<0. 01 vs. aortic tissue from control rats). Finally, systemic administration of VEGF to ecNOS-deficient mice failed to inhibit leukocyte-endothelium interactions observed in peri-intestinal venules. We concluded that VEGF is a potent inhibitor of leukocyte-endothelium interaction, and this effect is specifically correlated to augmentation of NO release from the vascular endothelium.--Scalia, R., Booth, G., Lefer, D. J. Vascular endothelial growth factor attenuates leukocyte-endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide.     

L-Arginine inhibits xanthine oxidase-dependent endothelial dysfunction in hypercholesterolemia

Xanthine oxidase (XO)-derived superoxide contributes to endothelial dysfunction in humans and animal models of hypercholesterolemia (HC). Since L-arginine supplementation prevents defects in NO signaling, we tested the hypothesis that L-arginine blunts the inhibitory effect of XO on vascular function. Acetylcholine-mediated relaxation was significantly impaired in ring segments of HC rabbits, a response that was associated with an increase in plasma XO activity. L-Arginine treatment of HC rabbits reduced plasma XO and improved endothelial function. L-Arginine also modestly prolonged the lag time for oxidation in isolated lipoprotein samples. These results reveal that the principal action of L-arginine is to protect against the XO-dependent inactivation of NO in arteries of HC rabbits.     

HD-PTP inhibits endothelial migration through its interaction with Src

Endothelial migration, early step in angiogenesis, is tightly regulated by the coordinated action of tyrosine kinases and tyrosine phosphatases. HD-PTP contributes to endothelial motility, since endothelial cells silencing HD-PTP after transfection with iRNA acquire a scattered and spindle-shaped phenotype and migrate faster than controls. Since (i) the proto-oncogene Src contributes to the regulation of cell motility and (ii) HD-PTP has a potential binding site for Src, we investigated whether an interplay exists between these two proteins. We found that Src binds HD-PTP and this interaction is enhanced after exposure to basic fibroblast growth factor. While HD-PTP does not modulate the levels of Src phosphorylation both in vitro and in vivo, we found that Src phosphorylates HD-PTP on tyrosine residues. Here we show for the first time that (i) HD-PTP has a tyrosine phosphatase activity; (ii) HD-PTP phosphorylation by Src inhibits its enzymatic activity. Interestingly, pharmacological and genetic inhibition of Src abrogates the migratory phenotype of endothelial cells silencing HD-PTP. On these bases, and because we have previously demonstrated that HD-PTP binds and dephosphorylates focal adhesion kinase (FAK), another crucial regulator of cell migration, we hypothesize that HD-PTP participates to the regulation of endothelial motility through its interactions with Src and FAK.     

Mechanisms of endothelial dysfunction with development of type 1 diabetes mellitus: role of insulin and C-peptide

Complications associated with insulin-dependent diabetes mellitus (type-1diabetes) primarily represent vascular dysfunction that has its origin in the endothelium. While many of the vascular changes are more accountable in the late stages of type-1diabetes, changes that occur in the early or initial functional stages of this disease may precipitate these later complications. The early stages of type-1diabetes are characterized by a diminished production of both insulin and C-peptide with a significant hyperglycemia. During the last decade numerous speculations and theories have been developed to try to explain the mechanisms responsible for the selective changes in vascular reactivity and/or tone and the vascular permeability changes that characterize the development of type-1diabetes. Much of this research has suggested that hyperglycemia and/or the lack of insulin may mediate the observed functional changes in both endothelial cells and vascular smooth muscle. Recent studies suggest several possible mechanisms that might be involved in the observed decreases in vascular nitric oxide (NO) availability with the development of type-1 diabetes. In addition more recent studies have indicated a direct role for both endogenous insulin and C-peptide in the amelioration of the observed endothelial dysfunction. These results suggest a synergistic action between insulin and C-peptide that facilitates increase NO availability and may suggest new clinical treatment modalities for type-1 diabetes mellitus.     

Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells induced by high glucose concentration

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.     

PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 micro M forskolin plus 1 micro M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of (32)P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of approximately 23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.     

Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation.

Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current (I(Ca)). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20-24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries ( approximately 200 microm), and large arterioles ( approximately 100 microm). I(Ca) was determined by whole cell voltage clamp. L-type I(Ca) was reduced approximately 30% by HC compared with controls in the right coronary artery (-5.29 +/- 0.42 vs. -7.59 +/- 0.41 pA/pF) but not the microcirculation (small artery, -8.39 +/- 0.80 vs. -10.13 +/- 0.60; arterioles, -10.78 +/- 0.93 vs. -11.31 +/- 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca(v)1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I(Ca) by HC was reversed in vitro by the cholesterol scavenger methyl-beta-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-beta-cyclodextrin. These data indicate that CASM L-type I(Ca) is decreased in large coronary arteries in early stages of atherosclerosis, whereas I(Ca) in the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.     

A natural squamosamide derivative FLZ inhibits homocysteine-induced rat brain microvascular endothelial cells dysfunction

Hyperhomocysteinemia is believed to induce endothelial dysfunction, which is an independent risk factor for atherosclerosis and vascular diseases. Compound FLZ is a novel synthetic squamosamide cyclic analog with several phenolic hydroxy groups, and exhibits strong anti-oxidative and neuroprotective activities in Alzheimer's and Parkinson's models. In the present study, we examined the actions of FLZ against homocysteine-induced injury to primary cultured rat brain microvascular endothelial cell (rBMECs). Cell survival was measured by MTT assay. Cell nuclei were observed by Hoechst 33342 staining. Senescent cells were detected by senescence-associated β-galactosidase (SA-β-gal) staining. Reactive oxygen species (ROS) were measured by 2',7'-dichlorofluorescein (DCF) fluorescent microscopy. Homocysteine-induced expression of NF-κB, p53, Noxa and Fas, and the release of mitochondrial cytochrome c, were measured by Western blotting. We found that FLZ treatment antagonized homocysteine-induced cell death and apoptosis and increased numbers of senescent cells. These changes were correlated with decreased ROS accumulation. FLZ treatment inhibited activation of NF-κB, the upregulation of p53, Noxa, and Fas, and blocked mitochondrial cytochrome c release. These data suggest that FLZ has a protective action against homocysteine-induced toxicity in rBMECs, suggesting that FLZ may have therapeutic potential for the prevention of cardiovascular diseases.     

Improvement of the microcirculation in the acute ischemic rat limb during intravenous infusion of drag-reducing polymers

Drag-reducing polymers (DRPs) are blood-soluble macromolecules that can increase blood flow and reduce vascular resistance. The purpose of the present study is to examine the effects of DRPs on microcirculation in rat hind limb during acute femoral artery occlusion. Two groups of 20 male Wistar rats were subjected to either hemodynamic measurement or contrast enhanced ultrasound (CEU) imaging during peripheral ischemia. Both groups were further subdivided into a DRP-treated group or a saline-treated group. Polyethylene oxide (PEO) was chosen as the test DRP, and rats were injected with either 10 ppm PEO solution or saline through the caudal vein at a constant rate of 5 ml/h for 20 min. Abdominal aortic flow, iliac artery pressure, iliac vein pressure, heart rate, carotid artery pressure and central venous pressure (CVP) were monitored, and vascular resistance was calculated by (iliac artery pressure-iliac vein pressure)/abdominal aortic blood flow. Flow perfusion and capillary volume of skeletal muscle were measured by CEU. During PEO infusion, abdominal aortic blood flow increased (p<0.001) and vascular resistance decreased (p<0.001) compared to rats that received saline during peripheral ischemia. There was no significant change in ischemic skeletal capillary volume (A) with DRP treatment (p>0.05), but red blood cell velocity (β) and capillary blood flow (A×β) increased significantly (p<0.05) during PEO infusion. In addition, A, β and A×β all increased (p<0.05) in the contralateral hind limb muscle. In contrast, PEO had no significant influence on heart rate, mean carotid artery blood pressure or CVP. Intravenous infusion of drag reducing polymers may offer a novel hydrodynamic approach for improving microcirculation during acute peripheral ischemia.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.     

IL-10 deficiency increases superoxide and endothelial dysfunction during inflammation

Little is known about the role of interleukin-10 (IL-10), an anti-inflammatory cytokine, in blood vessels. We used IL-10-deficient mice (IL-10 -/-) to examine the hypothesis that IL-10 protects endothelial function after lipopolysaccharide (LPS) treatment. The responses of carotid arteries were studied in vitro 6 h after injection of a relatively low dose of LPS (10 microgram ip). In IL-10 -/- mice, the maximum relaxation to ACh (3 microM) was 56 +/- 6% (means +/- SE) after LPS injection and 84 +/- 4% after vehicle injection (P < 0.05). Thus endothelium-dependent relaxation was impaired in carotid arteries from IL-10 -/- mice after LPS injection. In contrast, this dose of LPS did not alter relaxation to ACh in vessels from wild-type (IL-10 +/+) mice. Relaxation to nitroprusside and papaverine was similar in arteries from both IL-10 -/- and IL-10 +/+ mice after vehicle or LPS injection. Because inflammation is associated with increased levels of reactive oxygen species, we also tested the hypothesis that superoxide contributes to the impairment of endothelial function by LPS in the absence of IL-10. Results using confocal microscopy and hydroethidine indicated that levels of superoxide are elevated in carotid arteries from IL-10 -/- mice compared with IL-10 +/+ mice after LPS injection. The impaired relaxation of arteries from IL-10 -/- mice after LPS injection was restored to normal by polyethylene glycol-suspended superoxide dismutase (50 U/ml) or allopurinol (1 mM), an inhibitor of xanthine oxidase. These data provide direct evidence that IL-10 protects endothelial function after an acute inflammatory stimulus by limiting local increases in superoxide. The source of superoxide in this model may be xanthine oxidase.     

Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury

We hypothesized that neutralization of TNF-alpha at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-alpha neutralizing antibodies at the time of reperfusion. I/R elevated TNF-alpha expression (mRNA and protein), whereas administration of anti-TNF-alpha before reperfusion attenuated TNF-alpha expression. We detected TNF-alpha expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-alpha at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-alpha before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-alpha are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-alpha expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.     

Asymmetric dimethylarginine inhibits HSP90 activity in pulmonary arterial endothelial cells: role of mitochondrial dysfunction

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.     

Leptin-induced endothelial dysfunction in obesity

Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) nanosensors were placed near the surface (5+/-2 microm) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, O(2)(-), ONOO(-) were recorded. Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, O(2)(-), or ONOO(-) release from HUVEC. However, a 12-h exposure of HUVEC to leptin increased eNOS expression and CaI-stimulated NO (625+/-30 vs. 500+/-24 nmol/l control) and dramatically increased cytotoxic O(2)(-) and ONOO(-) levels. The [NO]-to-[ONOO(-)] ratio ([NO]/[ONOO(-)]) decreased from 2.0+/-0.1 in normal to 1.30+/-0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration coincided with 100% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and a reduced l-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250+/-10 vs. 420+/-12 nmol/l control), and an elevated concentration of O(2)(-) (240%) and ONOO(-) (70%). L-Arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[ONOO(-)] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular l-arginine, resulting in eNOS an uncoupling and depletion of endothelial NO and an increase of cytotoxic ONOO(-). Hyperleptinemia triggers an endothelial NO/ONOO(-) imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and diabetes.     

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.     

Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction.

The effect of glutathione (GSH) depletion by L-buthionine-[S,R]-sulphoximine (BSO) on tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression and mononuclear leukocyte adhesion to human umbilical vein endothelial cells (HUVECs) was investigated. Cells with marked depletion of cytoplasmic GSH, but with an intact pool of mitochondrial GSH, only slightly enhanced TNF-alpha-induced E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression, compared with the control. However, TNF-a-induced expression of both molecules was markedly enhanced when the mitochondrial GSH pool was diminished to <15% of the control. In contrast, TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression was not affected by the depletion of either cytoplasmic or mitochondrial GSH. Marked enhancement of TNF-alpha-induced adhesion molecule expression by the depletion of mitochondrial GSH resulted in increased in mononuclear leukocyte adhesion to treated HUVECs, compared with the control. These effects parallel reactive oxygen species (ROS) formation by the depletion of mitochondrial but not cytoplasmic GSH. Our findings demonstrate that depletion of mitochondrial GSH renders more ROS generation in HUVECs, and mitochondrial GSH modulates TNF-alpha-induced adhesion molecule expression and mononuclear leukocyte adhesion in HUVECs.