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Several studies have demonstrated that the endocrine disruptor bisphenol A (BPA) negatively affects animal and human health. An angiogenic process has been suggested among the events disrupted by this molecule, but the underlying mechanisms have not yet been clarified. The effect of BPA on angiogenesis was investigated by means of a bioassay previously validated in our laboratory. Using immortalized swine aortic endothelial cell line (AOC), the development of new blood vessels through a three-dimensional in vitro angiogenesis assay was evaluated. Subsequently, since vascular endothelial growth factor (VEGF) and nitric oxide (NO) are key players in the regulation of the angiogenic process, the effect of BPA on the production of these molecules by AOC was examined. BPA (10 [micro]mol/L) stimulated AOC growth (p < 0.05) and VEGF production (p < 0.05), but did not modify NO levels. Our data suggest that the endocrine-disrupting effects of BPA could also be associated with the promotion of vascular growth, thus interfering with a physiologically finely tuned process resulting from a delicate balance of numerous molecular processes. The stimulatory effects of BPA on VEGF production may have negative implications, potentially switching the balance toward uncontrolled neovascularization. Moreover, since angiogenesis is involved in several pathologies, including cancer growth and progression, potential health risks of BPA exposure should be carefully monitored. Key words: AOC, angiogenesis, VEGF, NO. Plusieurs etudes montrent que le bisphenol A (BPA), un perturbateur endocrinien, a des effets deleteres sur la sante animale et humaine. Le processus d'angiogenese a ete cite parmi les evenements pouvant etre perturbes par cette molecule, mais les mecanismes sous-jacents demeurent a clarifier. Nous avons etudie l'effet du BPA sur l'angiogenese a l'aide d'une methode de dosage biologique deja validee dans notre laboratoire. A l'aide d'un modele tridimensionnel d'angiogenese in vitro, nous avons etudie le developpement de nouveaux vaisseaux sanguins dans une lignee de cellules endotheliales d'aorte (CAO) immortalisees chez le porc. Par la suite, puisque le facteur de croissance endothelial vasculaire (ou VEGF pour <>) et l'oxyde nitrique (NO) jouent des roles preponderants dans le processus de regulation de l'angiogenese, nous avons evalue l'effet du BPA sur la production de ces molecules par les CAO. Nous avons observe que le BPA (a 10 [micro]mol/L) entrainait la stimulation de la croissance des CAO (p < 0,05) et la production de VEGF (p < 0,05), mais sans modification des taux de NO. Nos donnees laissent entendre que les effets perturbateurs endocriniens du BPA pourraient aussi etre associes a la stimulation de la croissance vasculaire, et par le fait meme a la perturbation d'un processus physiologique fin a partir d'un equilibre delicat dans de nombreux processus moleculaires. En effet, les effets stimulants de la production de VEGF par le BPA pourraient avoir des consequences negatives, eventuellement en faisant pencher la balance vers une neovascularisation anarchique. De surcroit, puisque l'angiogenese joue un role dans plusieurs phenomenes pathologiques, dont l'evolution et la croissance du cancer, il convient de surveiller attentivement les risques pour la sante de l'exposition au BPA. [Traduit par la Redaction] Mots-cles : cellules endotheliales d'aorte, angiogenese, VEGF, NO., Introduction The heterogeneous family of endocrine disruptors includes the organic compound of industrial origin 2,2-bis(4-hydroxyphenyl) propane ([C.sub.15][H.sub.16][O.sub.2]; molecular weight 228.0 Da), known as bisphenol A (BPA). BPA can be polymerized [...]

Unraveling the fate specification of resident stem cells during lung regeneration is of clinical importance. It has been reported that [c-kit.sup.+] progenitor cells resident in the human lung regenerate epithelial lineages upon transplantation into injured mouse lung. Here we test the lineage potential of [c-kit.sup.+] cells by inducible genetic lineage tracing. We find that [c-kit.sup.+] cells do not contribute to lung epithelium during homeostasis and repair, and instead maintain a vascular endothelial cell fate. These findings call attention to the clinical application of [c-kit.sup.+] stem cells as lung epithelial progenitors for the treatment of pulmonary disease., Lung disease is among the leading causes of death in the industrial world. The identification of lung stem cells during homeostasis, repair and regeneration are of clinical importance (1,2). The [...]

Cancer cells have been at the centre of cell metabolism research, but the metabolism of stromal and immune cells has received less attention. Nonetheless, these cells influence the progression of malignant, inflammatory and metabolic disorders. Here we discuss the metabolic adaptations of stromal and immune cells in health and disease, and highlight how metabolism determines their differentiation and function., Stromal cells are connective cells that support parenchymal cells. Aside from endothelial cells (ECs), which build blood vessels to supply oxygen and nutrients (1,2), other stromal cells include fibroblasts, which [...]

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkuhn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality., Introduction Characterized clinically by anorexia, vomiting, diarrhea, dehydration, systemic infection, and, in extreme cases, septic shock and death, the radiation gastrointestinal (GI) syndrome involves destruction of crypt/villus units, loss of [...]

Hyperhomocysteinemia (HHcy) has been associated with impaired vascular endothelial function. Our previous study demonstrated significantly higher secretion of the chemokine monocyte chemoattractant protein-1 from monocytes in response to lipopolysaccharide in patients with HHcy. In the present study, we investigated whether coronary endothelial function was damaged in patients with chronic HHcy (plasma level of homocysteine >15 [micro]mol/l) and, if so, whether this impaired endothelial function is induced by the uncoupling of endothelial nitric oxide synthase (eNOS). When tetrahydrobiopterin levels are inadequate, eNOS is no longer coupled to L-arginine oxidation, which results in reactive oxygen species rather than nitric oxide production, thereby inducing vascular endothelial dysfunction. The 71 participants were divided into two groups, control (n = 50) and HHcy (n = 21). Quantification of coronary flow velocity reserve (CFVR) was after rest and after adenosine administration done by noninvasive Doppler echocardiography. Plasma levels of nitric oxide and tetrahydrobiopterin were significantly lower in patients with HHcy than in controls (99.54 [+ or -] 32.23 vs. 119.50 [+ or -] 37.68 [micro]mol/l and 1.43 [+ or -] 0.46 vs. 1.73 [+ or -] 0.56 pmol/ml, all P < 0.05). Furthermore, CFVR was significantly lower in the HHcy than the control group (2.76 [+ or -] 0.49 vs. 3.09 [+ or -] 0.52, P < 0.05). In addition, plasma level of homocysteine was negatively correlated with CFVR. Chronic HHcy may contribute to coronary artery disease by inducing dysfunction of the coronary artery endothelium. The uncoupling of eNOS induced by HHcy in patients with chronic HHcy may explain this adverse effect in part. nitric oxide doi: 10.1152/ajpendo.00367.2010.

T cell egress from the thymus is essential for adaptive immunity, yet the requirements for and sites of egress are incompletely understood. We have shown that transgenic expression of sphingosine-1-phosphate receptor-1 (S1P1) in immature thymocytes leads to their perivascular accurnulation and premature release into circulation. Using an intravascular procedure to label emigrating cells, we found that mature thymocytes exit via blood vessels at the corticomedullary junction. By deleting sphingosine kinases in neural crest-derived pericytes, we provide evidence that these specialized vessel-ensheathing cells contribute to the S1P that promotes thymic egress. Lymphatic endothelial cell-derived S1P was not required. These studies identify the major thyrnic egress route and suggest a role for pericytes in promoting reverse transmigration of celts across blood vessel endothelium. doi: 10.1126/science.1188222

Vascular endothelium is covered with an extensive mesh of glycocalyx constituents, which acts like an effective barrier up to several micrometers thick that shields the luminal surface of the vasculature from direct exposure to flowing blood. Many studies report that various enzymatic and pharmaceutical challenges are able to increase glycocalyx porosity, resulting in farther permeation of plasma macromolecules and greater access of red blood cells into glycocalyx domain. Attenuation of glycocalyx barrier properties therefore potentially increases the amount of blood that effectively occupies available microvascular volume. We tested in the present study whether attenuation of coronary glycocalyx barrier properties actually increases coronary blood volume and whether such changes would be noticeable during measurements of coronary flow reserve using adenosine. In anesthetized goats (n = 6) with cannulated left main coronary artery that were perfused under controlled pressure, coronary blood volume was measured via the indicator-dilution technique using high-molecular-weight (2,000 kDa) dextrans as plasma tracer and labeled red blood cells as red blood cell tracer. Coronary blood volume was determined at baseline and during intracoronary infusion of adenosine causing maximal vasodilation (0.2-0.6 mg'*[kg.sup.-]*[h.sup.-1]) before and after intracoronary hyaluronidase treatment (170,000 units) of the glycocalyx. With an intact glycocalyx, coronary blood volume was 18.9 [+ or -] 1.1 ml/100 g heart tissue at baseline, which increased to 26.3 [+ or -] 2.7 ml/100 g after hyaluronidase treatment of the coronary glycocalyx. Maximal vasodilation by administration of adenosine further increased coronary blood volume to 33.9 [+ or -] 6.8 ml/100 g, a value not different from the maximal coronary blood volume of 33.2 [+ or -] 5.3 ml/100 g obtained by administration of adenosine in the absence of hyaluronidase treatment. Adenosine-induced increases in coronary conductance were not affected by hyaluronidase treatment. We conclude that acute attenuation of glycocalyx barrier properties increases coronary blood volume by ~40%, which is of similar magnitude as additional changes in coronary blood volume during subsequent maximal vasodilation with adenosine. Furthermore, maximal coronary blood volume following administration of adenosine was similar with and without prior hyaluronidase degradation of the glycocalyx, suggesting that adenosine and hyaluronidase potentially increase glycocalyx porosity to a similar extent. Hyaluronidase-mediated changes in coronary blood volume did not affect baseline and adenosine-induced increases in coronary conductance, demonstrating that measurements of coronary flow reserve are insufficient to detect impairment of coronary blood volume recruitment in conditions of damaged glycocalyx. adenosine; blood flow; coronary circulation; indicator-dilution doi: 10.1152/ajpheart.01306.2008.

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-[beta]1 (TGF-[beta]1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-[beta]1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-[beta]1, indicating the involvement of ALK-5 receptor subtype for TGF-[beta]1. Sphingosine 1-phosphate (SIP) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Racl, ultimately to promote migration. Because SIP receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-[beta]1 enhances effects of SIP on BAEC. Whereas SIP alone evokes robust BAEC responses to S1P, pretreatment with TGF-[beta]1 leads to even higher magnitudes of SIP-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-[beta]1-induced promotion of BAEC responses to SIP. Collectively, these data demonstrate that TGF-[beta]1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-[beta]1 promotes subsequent heterologous receptor signaling by SIP, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries. cytokines; sphingolipids; G protein-coupled receptors; endothelium; signal transduction doi: 10.1152/ajpcell.00109.2009.

The production of hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) is a key event in the development of diabetic complications. Because resveratrol, a naturally occurring polyphenol, has been reported to confer vasoprotection, improving endothelial function and preventing complications of diabetes, we investigated the effect of resveratrol on mtROS production in cultured human coronary arterial endothelial cells (CAECs). The measurement of MitoSox fluorescence showed that resveratrol attenuates both steady-state and high glucose (30 mM)-induced mtROS production in CAECs, an effect that was prevented by the knockdown of the protein deacetylase silent information regulator 2/sirtuin 1 (SIRT 1), an intracellular target of resveratrol. An overexpression of SIRT1 mimicked the effects of resveratrol, attenuating mtROS production. Similar results were obtained in CAECs transfected with mitochondria-targeted [H.sub.2][O.sub.2]-sensitive HyPer-Mito fluorescent sensor. Amplex red assay showed that resveratrol and SIRT1 overexpression significantly reduced cellular [H.sub.2][O.sub.2] levels as well. Resveratrol upregulated MnSOD expression and increased cellular GSH content in a concentration-dependent manner (measured by HPLC coulometric analysis). These effects were attenuated by SIRT1 knockdown and mimicked by SIRT1 overexpression. We propose that resveratrol, via a pathway that involves the activation of SIRT1 and the upregulation of antioxidant defense mechanisms, attenuates mtROS production, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. vasoprotection; histone deacetylase; sirtuin 1 doi: 10.1152/ajpheart.00375.2009.

Pulmonary vascular endothelial cells express a variety of ion channels that mediate [Ca.sup.2+] influx in response to diverse environmental stimuli. However, it is not clear whether [Ca.sup.2+] influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the [[alha].sub.1G] T-type [Ca.sup.2+] channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic [Ca.sup.2+], immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-[K.sup.+] perfusate induces [Ca.sup.2+] entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, [Ca.sup.2+] entry in septal endothelium evoked by the selective TRPV4 activator 4[alpha]-phorbol-12,13-didecanoate (4[alpha]-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of [[alpha].sub.1G] abolishes depolarization-induced [Ca.sup.2+] entry and surface expression of P-selectin but does not prevent 4[alpha]-PDD-activated [Ca.sup.2+] entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4[alpha]-PDD-activated [Ca.sup.2+] entry and the increase in permeability, while not impacting depolarization-induced [Ca.sup.2+] entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries [Ca.sup.2+] entry through [[alpha]-G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively. T-type calcium channel; transient receptor potential vanilloid 4; vascular permeability doi:10.1152/ajplung.00015.2009.

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 rain after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine- 123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 [micro]M). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration. sepsis; systemic inflammation; oxidative stress; leukocyte adhesion/ migration; proteolytic enzymes; carbon monoxide; carbon monoxide-releasing molecules; tricarbonylchloroglycinate-ruthenium(II)

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1[alpha], nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRTl-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. vasoprotection; histone deacetylase; endothelial dysfunction; diabetes; obesity

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. [Heg1.sup.-/-] mice showed defective integrity of the heart, blood vessels and lymphatic vessels. [Heg1.sup.-/-]; [Ccm2.sup.lacZ/+] and [Ccm2.sup.lacZ/lacZ] mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity., CCMs are a common vascular malformation, with a prevalence of 0.1%-0.5% in the human population (1). CCMs arise primarily in the brain as thin-walled, dilated blood vessels that cause seizures, [...]

Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and central nervous system blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We show that CCM2 regulates endothelial cytoskeletal architecture, cell-to-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. On the basis of our biochemical studies indicating that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice with simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacological treatment of a human vascular dysplasia with a widely available and safe drug., ********** Cerebral cavernous malformations are common vascular malformations that affect the systemic and central nervous system (CNS) vasculature with a prevalence of I in 200 to 250 individuals (1,2) in [...]

The cardiovascular system is the life providing task of delivering oxygen and any problem in the system can be dangerous so extensive studies are conducted to elucidate the mechanism behind this cardiovascular development. Summary is presented on the factors determining vascular integrity and suitability of the zebrafish to study this phenomenon.

[beta]-Catenin plays an important role in the regulation of vascular endothelial cell-cell adhesions and barrier function by linking the VE-cadherin junction complex to the cytoskeleton. The purpose of this study was to evaluate the effect of [beta]-catenin and VE-cadherin interactions on endothelial permeability during inflammatory stimulation by histamine. We first assessed the ability of a [beta]-catenin binding polypeptide known as inhibitor of [beta]-catenin and T cell factor (ICAT) to compete [beta]-catenin binding to VE-cadherin in vitro. We then overexpressed recombinant FLAG-ICAT in human umbilical vein endothelial cells (HUVECs) to study its impact on endothelial barrier function controlled by cell-cell adhesions. The binding of [beta]-catenin to VE-cadherin was quantified before and after stimulation with histamine along with measurements of transendothelial electrical resistance (TER) and apparent permeability to albumin ([P.sub.a]) under the same conditions. The results showed that ICAT bound to [beta]-catenin and competitively inhibited binding of the VE-cadherin cytoplasmic domain to [beta]-catenin in a concentration-dependent manner. Overexpression of FLAG-ICAT in endothelial cell monolayers did not affect their basal permeability properties, as indicated by unaltered TER and [P.sub.a]; however, the magnitude and duration of histamine-induced decreases in TER were significantly augmented. Likewise, the increase in [P.sub.a] in the presence of histamine was exacerbated. Overexpression of FLAG-ICAT also significantly decreased the level of [beta]-catenin-associated VE-cadherin following histamine stimulation. Taken together, these data suggest that inflammatory agents like histamine cause a transient and reversible disruption of binding between [beta]-catenin and VE-cadherin, during which endothelial permeability is elevated. endothelial barrier; cell-cell junction; signal transduction; inflammation

We hypothesized that in preeclampsia (PE), contribution of endothelium-derived hyperpolarizing factor (EDHF) and the mechanism/s of its action differ from that in normal pregnancy (NP). We aimed to assess endothelial function and morphology in arteries from NP and PE with particular focus on EDHF. Arteries ([approximately equal to] 200 [micro] m) were dissected from subcutaneous fat biopsies obtained from women undergoing cesarean section. With the use of wire myography, responses to the endothelium-dependent agonist bradykinin (BK) were determined before and after inhibition of pathways relevant to EDHF activity. The overall responses to BK in arteries from PE (n = 13) and NP (n = 17) were similar. However, in PE, EDHF-mediated relaxation was reduced (P < 0.05). All women within the PE group were divided into two subgroups: with more (group 1) or less (group 2) than 50% reduction of EDHF-typed responses after 18-[alpha]-glycyrrhetinic acid (an inhibitor of myoendothelial gap junctions, MEGJs). The division showed that 1) MEGJs are principally involved when the EDHF contribution is reduced; and 2) when the EDHF contribution is similar to that in NP, the H202 and/or cytochrome P-450 epoxygenase products of arachidonic acid (AA), along with MEGJs, confer EDHF-mediated relaxation. In contrast, MEGJs were the main pathway for EDHF in NP. The abundant presence of MEGJs in arteries from NP but deficiency of them in PE was observed using transmission electron microscopy. We conclude that PE is associated with heterogeneous contribution of EDHF, and the mechanism behind EDHF-typed responses is mediated either by MEGJs alone or in combination with [H.sub.2][O.sub.2] or cytochrome P-450 epoxygenase metabolites of AA. gap junctions; small arteries; pregnancy

The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(-/-)], or nNOS gene [nNOS(-/-)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at [10.sup.-8] and [10.sup.-6] mol/1 reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at [10.sup.-8] and [10.sup.-6] mol/l. The response to angiotensin II was enhanced in nNOS(-/-) mice (41% and 25% at [10.sup.-8] and [10.sup.-6] mol/l) and even more enhanced in eNOS(-/-) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). L-NAME led to complete constriction of Af in these groups. Mediato-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types IA and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation. renin-angiotensin system; eNOS knockout; nNOS knockout; [N.sup.G]nitro-L-arginine methyl ester; renal hemodynamics; juxtaglomerular apparatus

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin E[C.sub.50] = 3.3 x [10.sup.-6] mol/l, peak 21[DELTA]% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine E[C.sub.50] = 1.3 x [10.sup.-6] mol/l, peak 27[DELTA]% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16A% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with [N.sup.G]-monomethyl-L-arginine [1[DELTA]% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16[DELTA]% (SD 3) vs. 10[DELTA]% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats. nitric oxide synthase; endothelium-derived hyperpolarizing factor; rodents; age-dependent endothelial dysfunction

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20HETE-driven endothelial dysfunction, the interactions between 20HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by [N.sup.G]-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction. nitric oxide; vascular endothelium; vasodilator-stimulated phosphoprotein; cytochrome P-450; superoxide; endothelial nitric oxide synthase

Although changes in gene expression are necessary for arterial remodeling during hypertension, the genes altered and their mechanisms of regulation remain uncertain. The goal of this study was to identify cerebral artery genes altered by hypertension and define signaling pathways important in their regulation. Intact cerebral arteries from Dahl salt-sensitive normotensive and hypertensive high-salt (HS) rats were examined by immunostaining, revealing an increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERKI/2) and expression of the proliferative marker Ki-67 in arteries from hypertensive animals. Arterial RNA analyzed by microarray and validated with RT-quantitative PCR revealed that jun family member junB and matricellular genes plasminogen activator inhibitor-1 (PAI-1) and osteopontin (OPN) were significantly overexpressed in HS arteries. Fisher exact test and annotation-based gene subsets showed that genes upregulated by Jun and [Ca.sup.2+]/cAMP-response element-binding protein (CREB) were overrepresented. A model of cultured rat cerebrovascular smooth muscle cells was used to test the hypothesis that angiotensin II (ANG II), JunB, and CREB are important in the regulation of genes identified in the rat hypertension model. ANG II induced a transient induction of junB and a delayed induction of PAI-1 and OPN mRNA levels, which were reduced by ERK inhibition with U-0126. Silencing junB using small-interfering RNA reduced mRNA levels of OPN but not PAI-1. The silencing of CREB reduced PAI-1 induction by ANG II but enhanced the transcription of OPN. Together, these results suggest that salt-induced hypertensive disease promotes changes in matricellular genes that are stimulated by ANG II, regulated by ERK, and selectively regulated by JunB and CREB. microarray; Dahl rat; brain arteries; mitogen-activated protein kinase signaling; transcription factors

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 [micro]m in width at a constant depth of 1 [micro]m. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/[cm.sup.2]), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/ [cm.sup.2]) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow. endothelial cell alignment; shear stress; focal adhesion

The work herein represents a novel approach for the modeling of low-density lipoprotein (LDL) transport from the artery lumen into the arterial wall, taking into account the effects of local wall shear stress (WSS) on the endothelial cell layer and its pathways of volume and solute flux. We have simulated LDL transport in an axisymmetric representation of a stenosed coronary artery, where the endothelium is represented by a three-pore model that takes into account the contributions of the vesicular pathway, normal junctions, and leaky junctions also employing the local WSS to yield the overall volume and solute flux. The fraction of leaky junctions is calculated as a function of the local WSS based on published experimental data and is used in conjunction with the pore theory to determine the transport properties of this pathway. We have found elevated levels of solute flux at low shear stress regions because of the presence of a larger number of leaky junctions compared with high shear stress regions. Accordingly, we were able to observe high LDL concentrations in the arterial wall in these low shear stress regions despite increased filtration velocity, indicating that the increase in filtration velocity is not sufficient for the convective removal of LDL. low-density lipoprotein transport; lipid accumulation; atherosclerosis; leaky junction

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-I (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased [Ca.sup.2+] sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-1H augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent [Ca.sup.2+] sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 [micro]M) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 [micro]M) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor Go-6976 (1 [micro]M) had no effect on ET-1-mediated vasoconstriction in either group, but a PKC[delta]-selective inhibitor (rottlerin, 3 [micro]M) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKC[delta] phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by Go-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKC[delta] activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension. sleep apnea; intermittent hypoxia; hypercapnia; endothelin-1; protein kinase C [delta]; vascular smooth muscle cell; mesenteric arteries

Members of the claudin family constitute tight junction strands and are major determinants in specificity and selectivity of paracellular barriers. Transcriptional control of clandin gene expression is essential to establish individual claudin expression patterns and barrier properties. Using full genome expression profiling, we now identify sex-determining region Y-box (SOX)-18, a member of the SOX family of high-mobility group box transcription factors, as one of the most differentially induced genes during establishment of the endothelial barrier. We show that overexpression of SOX-18 and a dominant-negative mutant thereof, as well as SOX-18 silencing, greatly affect levels of claudin-5 (CLDN5). The relevance of an evolutionary conserved SOX-binding site in the CLDN5 promoter is shown using sequential promoter deletions, as well as point mutations. Furthermore, SOX-18 silencing abrogates endothelial barrier function, as measured by electric cell-substrate impedance sensing. Thus an obligatory role for SOX-18 in the regulation of CLDN5 gene expression in an endothelial-specific and cell density-dependent manner is established, as well as a crucial, nonredundant role for specifically SOX-18 in the formation of the endothelial barrier. endothelium; claudin; promoter; sox

The purpose of this study was to determine if hypoxia elicits different proliferative and apoptotic responses in systemic arterial smooth muscle cells incubated under conditions that do or do not result in cellular ATP depletion and whether these effects are relevant to vascular remodeling in vivo. Gene expression profiling was used to identify potential regulatory pathways. In human aortic smooth muscle cells (HASMCs) incubated at 3% [O.sub.2], proliferation and progression through the G1/S interphase are enhanced. Incubation at 1% [O.sub.2] reduced proliferation, delayed G1/S transition, increased apoptotic cell death, and is associated with mitochondrial membrane depolarization and reduced cellular ATP levels. In aorta and mesenteric artery from rats exposed to hypoxia (10% [O.sub.2], 48 h), both proliferation and apoptosis are increased, as are medial nuclear density and smooth muscle cell content. Although nuclear levels of hypoxia-inducible factor 1-[alpha] (HIF-1[alpha]) are increased to a similar extent in HASMCs incubated at 1 and 3% [O.sub.2], expression of tumor protein p53, its transcriptional target p21, as well as their regulatory factors and downstream effectors, are differentially affected under these two conditions, suggesting that the bidirectional effects of hypoxia are mediated by this pathway. We conclude that hypoxia induces a state of enhanced cell turnover through increased rates of both smooth muscle cell proliferation and death. This confers the ability to remodel the vasculature in response to changing tissue metabolic needs while avoiding the accumulation of mutations that may lead to malignant transformation or the formation of abnormal vascular structures. systemic circulation; cell cycle; apoptosis

We previously showed that Snitroso-N-acetylpenicillamine, a nitric oxide donor, decreased the levels and functions of [G.sub.i][alpha] proteins by formation of peroxynitrite ([ONOO.sup.-]) in vascular smooth muscle cells (VSMC). The present studies were undertaken to investigate whether [ONOO.sup.-] can modulate the expression of [G.sub.i][alpha] protein and associated adenylyl cyclase signaling in VSMC. Treatment of A-10 and aortic VSMC with [ONOO.sup.-] for 24 h decreased the expression of [G.sub.i][alpha]-2 and [G.sub.i][alpha]-3, but not [G.sub.s][alpha], protein in a concentration-dependent manner; expression was restored toward control levels by [sup.111]Mn-tetralis(benzoic acid porphyrin) and uric acid, hut not by 1H[1,2,4]oxadiazole[4,3-a]quinoxaline-l-one (ODQ) and KT-5823. cGMP levels were increased by ~50% and 150% by 0.1 and 0.5 mM [ONOO.sup.-] , respectively, and attenuated toward control levels by ODQ. In addition, 0.5 mM [ONOO.sup.-] attenuated the inhibition of adenylyl cyclase by ANG II and C-type atrial natriuretic peptide ([C-ANP.sup.4-23]), as well as the inhibition of forskolin-stimulated adenylyl cyclase activity by GTP[gamma]S, whereas, the [G.sub.s]-mediated stimulations were augmented. In addition, 0.5 mM [ONOO.sup.-] decreased phosphorylation of ERK1/2 and p38 MAP kinase and enhanced JNK phosphorylation but did not affect AKT1/3 phosphorylation. These results suggest that [ONOO.sup.-] decreased the expression of [G.sub.i] proteins and associated functions in VSMC through a cGMP-independent mechanism and may involve the MAP kinase signaling pathway. G protein; mitogen-activated protein kinase

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-I (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-I immuno-reactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-I enhanced NF-[kappa]B DNA-binding activity. CT- 1-mediated upregulation of ICAM- 1 and MCP- 1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-[kappa]B pathways and suggests that CT-1 plays an important role in the patho-physiology of vascular inflammation and atherosclerosis. atherosclerosis; vascular inflammation

We recently reported that increased vascular endothelial nitric oxide production could protect against the development of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and right ventricular hypertrophy (RVH) in rats (32). The present study investigated whether the pleiotropic action of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors in upregulating endothelial function could also protect against the MCT-induced end-organ damages. Rosuvastatin (2 mg*[kg.sup.-1] * [day.sup.1] via oral gavage) or placebo was initiated 1 wk before or 1 wk after MCT (60 mg/kg ip) administration. One month after MCT, significant PAH developed in the placebo rats, which were accompanied by histological evidence of pulmonary vascular thickening and right ventricular hypertrophy. The coronary endothelial vasodilatory function, assessed with endothelial/nitric oxide-dependent responses to acetylcholine and [N.sup.G]-nitro-L-arginine methyl ester (L-NAME), was depressed, while the constrictory responses to known coronary constrictors was enhanced. In rats that received rosuvastatin treatment 1 wk before MCT administration, a significantly reduced PAH and RVH was observed, as well as reduced pulmonary vascular and right ventricular remodelings. Rosuvastatin 1-wk posttreatment had no effect on PAH, but inhibited RVH. Right coronary endothelial dysfunction, which was shown in placebo rats, was effectively prevented by both pre- and postrosuvastatin treatment, while this effect was more dramatic in the pretreated group. Left coronary endothelial function, which was not affected by MCT, also showed an upregulation by rosuvastatin. Taken together, our results demonstrated the pleiotropic protection of rosuvastatin against the development of PAH and RVH and confirmed our previous finding that the targeted preservation of coronary endothelial function and vasoactivity may provide a novel approach to protect against cardiac remodeling. heart failure; endothelium-dependent relaxation; acetylcholine; nitric oxide; monocrotaline

The repair of the endothelium after inflammatory injury is essential to maintaining homeostasis. The link between inflammation-induced endothelial damage and repair has not been fully characterized in vivo. We have developed a rat model to evaluate the coupling of lipopolysaccharide (LPS)-induced endothelial injury and repair. Aortic endothelium injury was analyzed by both inmunohistochemistry and flow cytometry to quantify the number of endothelial cells and the percentage of apoptotic endothelial cells. We have also identified the percentage of circulating angiogenic cells capable of repairing the damaged endothelium. Erythropoietin was administered to inhibit LPS-induced endothelial apoptosis. Loss of the normal endothelial structure was observed in the aorta of the animals treated with LPS. Eight hours after LPS administration, the number of endothelial cells decreased by 40%, returning to normal after 24 h. There was a three-fold increase in the percentage of circulating angiogenic cells, which did not return to normal levels until 48 h after LPS administration. Circulating angiogenic cell levels did not change when LPS-induced endothelial damage was prevented by erythropoietin. The endothelial injury caused by inflammation activates the mobilization of circulating angiogenic cells, thus completing endothelial repair. Inflammation without endothelial injury does not trigger the mobilization of circulating angiogenic cells. endothelium; lipopolysaccharide; apoptosis; circulating angiogenic cells; erythropoietin

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with [greater than or equal to] 8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for [greater than or equal to] 1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF. fetal hemoglobin; erythrocyte adhesion

Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 [+ or -] 0.1, adult = 3.9 [+ or -] 0.1, old = 2.2 [+ or -] 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond ~20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation. endothelial; smooth muscle; gap junction; microcirculation; aging

Targeting cannabinoid-2 ([CB.sub.2]) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which [CB.sub.2] activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of [CB.sub.2]-receptor activation on TNF-[alpha]-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-[alpha] induced NF-[kappa]B and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF-[alpha] were attenuated by [CB.sub.2] agonists. [CB.sub.2] agonists also decreased the TNF-[alpha]- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. [CB.sub.1] and [CB.sub.2] receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF-[alpha]-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting [CB.sub.2] receptors on endothelial cells may offer a novel approach in the treatment of these pathologies. endothelial activation; inflammation; RhoA; adhesion molecules

Caveolin-1 (Car-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Car-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to [[alpha].sub.1]-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 [+ or -] 6%) compared with WT (17 [+ or -] 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.

Little is known of the vasomotor responses of skeletal muscle arterioles during and following muscle contraction. We hypothesized that aging leads to impaired arteriolar responses to muscle contraction and recovery. Nitric oxide (NO) availability, which is age dependent, has been implicated in components of these kinetics. Therefore, we also hypothesized that changes in the kinetics of vascular responses are associated with the NO pathway. Groups were young (3 mo), old (24 too), endothelial NO synthase knockout (eNOS-/-), and [N.sup.G]-nitro-L-arginine (L-NA)-treated male and female C57BL/6 mice. The kinetics of vasodilation during and following 1 min of contractions of the gluteus maximus muscle were recorded in second-order (regional distribution) and third-order (local control) arterioles. Baseline, peak (during contraction), and maximal diameters (pharmacological) were not affected by age or sex. The kinetics of dilation and recovery were not different between males and females at the young age. There was a significant slowing of vasodilation at the onset of contractions (~2-fold; P < 0.05) and a significant speeding of recovery (~5-fold; P < 0.05) in old males vs. old females and vs. young eNOS-/-, and L-NA did not affect the kinetics at the onset of muscle contraction. eNOS-/- mimicked the rapid recovery of old males in second-order arterioles; acute NO production (L-NA) explained ~50% of this effect. These data demonstrate fundamental age-related differences between the sexes in the dynamic function of skeletal muscle arterioles. Understanding how youthful function persists in females but not males may provide therapeutic insight into clinical interventions to maintain dynamic microvascular control of nutrient supply with age. muscle contraction; resistance vessel; gender; arteriole

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ER[alpha]-knockout (ER[alpha]-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ER[alpha]-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ER[alpha]-KO arteries, [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) inhibited FID by ~26%, whereas indomethacin inhibited dilations by ~50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/[cm.sup.2] shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ER[alpha]-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ER[alpha]-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ER[beta] in ER[alpha]-KO arteries. Treatment of ER[alpha]-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ER[alpha]-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone. estradiol; testosterone; receptors; nitric oxide; endothelium-derived hyperpolarizing factor

With most cardiovascular disease risk factors, endothelium-dependent dilation of skeletal muscle resistance arterioles is compromised, although with hypercholesterolemia, impairments to reactivity are not consistently observed. Using apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) gene deletion male mouse models of hypercholesterolemia at 20 wk of age, we tested the hypothesis that arteriolar dilation would be maintained due to an increased stimulus-induced production of dilator metabolites via cyclooxygenase and cytochrome P-450 epoxygenase pathways. Arterioles from both strains demonstrated mild reductions in dilation to hypoxia and acetylcholine versus responses in C57/B1/6J (C57) controls. However, although inhibition of nitric oxide synthase (NOS) attenuated dilation in arterioles from C57 controls, this effect was absent in ApoE or LDLR strains. In contrast, cyclooxygenase-dependent portions of dilator reactivity were maintained across the three strains. Notably, although combined NOS and cyclooxygenase inhibition abolished arteriolar responses to hypoxia and acetylcholine in C57 controls, significant reactivity remained in ApoE and LDLR strains. Whereas inhibition of cytochrome P-450 [omega]-hydroxylase and epoxygenases had no effect on this residual reactivity in ApoE and LDLR strains, inhibition of 12/15-lipoxygenase with nordihydroguaiaretic acid abolished the residual reactivity. With both hypoxic and methacholine challenges, arteries from ApoE and LDLR strains demonstrated an increased production of both 12(S)- and 15(S)-hydroxyeicosatetraenoic acid, end products of arachidonic acid metabolism via 12/15-lipoxygenase, a response that was not present in C57 controls. These results suggest that with development of hypercholesterolemia, mechanisms contributing to dilator reactivity in skeletal muscle arterioles are modified such that net reactivity to endothelium-dependent stimuli is largely intact. skeletal muscle microcirculation; mouse models of cardiovascular disease

Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic system and in the modulation of extracellular proteolysis. Increased PAI-1 was found in atherosclerotic lesions, and high PAI-1 plasma levels were associated with coronary heart disease. Smooth muscle cells (SMC) are a major source of PAI-1 within the vascular wall, and PAI-1 was implicated in SMC migration, proliferation, and apoptosis. We treated human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), or oncostatin M (OSM). Only OSM increased PAI-1 antigen and activity production significantly in these cells up to 20-fold. OSM up-regulated mRNA specific for PAI-1 up to 4.5-fold in these cells. HCASMC and HASMC express gpl30, OSM receptor, IL-6 receptor, and LIF receptor. OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylations in HASMC. A phosphatidylinositol 3-kinase inhibitor and a mitogen-activated protein/extracellular signal-regulated kinase inhibitor reduced Akt and ERK1/2 phosphorylation, respectively, and abolished OSM-induced PAI-1 upregulation. A janus kinase/signal transducer and activator of transcription inhibitor, a p38 mitogen-activated protein kinase inhibitor, or c-Jun NH2-terminal kinase inhibitor I did not inhibit the OSM-dependent PAI-1 induction. OSM enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. We hypothesize that, if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo, it could, via modulation of fibrinolysis and extracellular proteolysis, be involved in the development of vascular pathologies such as plaque progression, destabilization and subsequent thrombus formation, and restenosis and neointima formation. smooth muscle cells; oncostatin M; glycoprotein 130; plasminogen; plasminogen activator inhibitor

In a vulnerable plaque (VP), rupture often occurs at a site of high stress within the cap. It is also known that vessels do not become free of stress when all external loads are removed. Previous studies have shown that such residual stress/strain (RS/S) tends to make the stress distribution more uniform throughout the media of a normal artery. However, the influence of RS/S on the wall stress distribution in pathological coronaries remains unclear. The aim of this study was to investigate the effects of RS/S on the biomechanical stability of VPs. RS/S patterns were studied ex vivo in six human vulnerable coronary plaque samples. Because the existence of RS/S can only be assessed by releasing it, the opening angle technique was the experimental approach used to study the geometrical opening configurations of the diseased arteries, producing an arterial wall in a near-zero stress state. Reciprocally, these opening geometries were used in finite element simulations to reconstruct the RS/S distributions in closed arteries. It was found that the RS/S 1) is not negligible, 2) dramatically affects the physiological peak stress amplitude in the thin fibrous cap, 3) spotlights some new high stress areas, and 4) could be a landmark of the lipid core's developmental process within a VP. This study demonstrates that plaque rupture is not to be viewed as a consequence of intravascular pressure alone, but rather of a subtle combination of external loading and intraplaque RS/S. atherosclerosis; zero-stress state; lipid core; plaque growth; finite element analysis

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissueengineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the [[alpha].sub.5][[beta].sub.1]--integrin complex, whereas ECs used either [[alpha].sub.5][[beta].sub.1]- or [[alpha].sub.v][[beta].sub.3]-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC [[alpha].sub.5][[beta].sub.1]-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels. focal adhesions; fibrillar adhesions; tissue engineering; integrin; elastic modulus

Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of [[alpha].sub.2C]-adrenoceptors ([[alpha].sub.2C]-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17[beta]-estradiol regulates [[alpha].sub.2C]-AR expression and function in cutaneous VSMs. 17[beta]-Estradiol (0.01-10 nmol/1) increased expression of the [[alpha].sub.2C]-AR protein and the activity of the [[alpha].sub.2C]-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17[beta]-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17[beta]-estradiol-induced activation of the [[alpha].sub.2C]-AR gene promoter, whereas a constitutively active mutant of Rap2 increased [[alpha].sub.2C]-AR promoter activity. The effects of 17[beta]-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 [micro]mol/1), and were mimicked by a cellimpermeable form of the hormone (estrogen:BSA) or by the selective ER-[alpha] receptor agonist 4,4',4'-(4-propyl- [[sup.1]H]-pyrazole-1,3,5-triyl)trisphenol (PPT; 10 nmol/1) or the selective ER-[beta] receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17[beta]-estradiol increased expression of [[alpha].sub.2C]-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in [[alpha].sub.2C]-AR transcription. In mouse tail arteries, 17[beta]-estradiol (10 nmol/1) increased [[alpha].sub.2C]-AR expression and selectively increased the cold-induced amplification of [[alpha].sub.2]-AR constriction, which is mediated by [[alpha].sub.2C]-ARs. An estrogen-dependent increase in expression of coldsensitive [[alpha].sub.2C]-ARs may contribute to the increased activity of coldinduced vasoconstriction under estrogen-replete conditions. Raynaud's phenomenon; Rapl; Rap2; adenosine 3',5'-cyclic monophosphate; estrogen receptors

Cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells are the major cellular constituents of the heart. The aim of this study was to observe alterations in myocardial cell populations during early neonatal development in the adult animal and to observe any variations of the cardiac cell populations in different species, specifcally, the rat and mouse. Whole hearts were isolated from either mice or rats during the neonatal and adult stages of development, and single cell suspensions were prepared via sequential collagenase digestion. Heterogeneous cell populations were immunolabeled for specific cell types and analyzed using fluorescence-activated cell sorting (FACS). In addition, the left ventricle, fight ventricle, and septa were isolated, fixed, and sectioned for morphometric analyses. These same cardiac regions were also analyzed using FACS. We observed that the adult murine myocardium is composed of ~56% myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells. Moreover, our morphometric and FACS data demonstrated similar percentages in the three regions examined. During murine neonatal cardiac development, we observed a marked increase in numbers of cardiac fibroblasts and a resultant decrease in percentages of myocytes in late neonatal development (day 15). Finally, FACS analyses of the rat heart during development displayed similar results in relation to increases in cardiac fibroblasts during development; however, cell populations in the rat differed markedly from those observed in the mouse. Taken together, these data enabled us to establish a homeostatic model for the myocardium that can be compared with genetic and cardiac disease models. cardiac fibroblast; myocyte; cardiac remodeling; flow cytometry

Hemodynamic regulation of directional endothelial cell (EC) migration implies an essential role of shear stress in governing EC polarity. Shear stress induces reorientation of the microtubule organizing center toward the leading edge of migrating cells in a Cdc42-dependent manner. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to 4 days, but not shear stressinduced morphology. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor-free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and induced an increase in the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant-negative Par6, a major downstream effector of Cdc42-induced polarity. Shear stressinduced directed migratory polarity is modulated by exogenous growth factors and dependent on Par6 activity and shear stress direction. flow; Par6; motility

Oxyhemoglobin (OxyHb) can suppress voltage-dependent [K.sup.+] channel (Kv) currents through protein tyrosine kinase activation, which may contribute to cerebral vasospasm following subarachnoid hemorrhage. Here we have tested the hypothesis that shedding of heparin-binding EGF-like growth factor (HB-EGF) and the resulting activation of the tyrosine kinase EGF receptor (EGFR) underlie OxyHb-induced Kv channel suppression in the cerebral vasculature. With the use of the conventional whole cell patch-clamp technique, two EGFR ligands, EGF and HB-EGF, were found to mimic OxyHb-induced Kv suppression in rabbit cerebral artery myocytes. Kv current suppression by OxyHb or EGF ligands was eliminated by a specific EGFR inhibitor, AG-1478, but was unaffected by PKC inhibition. Compounds (heparin and CRM-197) that specifically interfere with HB-EGF signaling eliminated OxyHb-induced Kv suppression, suggesting that HB-EGF is the EGFR ligand involved in this pathway. HB-EGF exists as a precursor protein that, when cleaved by matrix metalloproteases (MMPs), causes EGFR activation. MMP activation was detected in OxyHb-treated arteries by gelatin zymography. Furthermore, the MMP inhibitor (GM-6001) abolished OxyHb-induced Kv current suppression. We also observed Kv current suppression due to EGFR activation in human cerebral artery myocytes. In conclusion, these data demonstrate that OxyHb induces MMP activation, causing HB-EGF shedding and enhanced EGFR activity, ultimately leading to Kv channel suppression. We propose that EGFR-mediated Kv suppression contributes to vascular pathologies, such as cerebral vasospasm, and may play a more widespread role in the regulation of regional blood flow and peripheral resistance. vascular smooth muscle; growth factors; subarachrloid hemorrhage; tyrosine kinase

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem ceils and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these ceils, which was inhibited by NOS inhibitor [N.sub.G]-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro. progenitor cells

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the singlechannel permeability ([P.sub.[gamma]]). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 ([AF.sup.350]), but mCx30.2 channels exhibited a significantly lower [P.sub.[gamma]] than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited [P.sub.[gamma]] values that were intermediate to the corresponding homotypic channels. [P.sub.[gamma]] values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for [AF.sup.350] were similar and approximately twofold higher than [P.sub.[gamma]] values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over ~400 Da. The ratio of single-channel conductance to permeability for [AF.sup.350] was ~40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication. connexin; gap junctions; dye; Lucifer yellow; 4,6-diamidino-2-phenylindole

The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines, and expression of E-selectin. For all different EC cultures, purities of [greater than or equal to] 99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and monocyte chemoattractant protein-1, especially EC from coronary arteries produced significantly more IL-6 compared with other EC types, independent of age, gender, and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank, enabling us to study tissue-specific aspects at the level of the endothelium. purification; cell growth; fluorescence-activated cell separation; function