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Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([[Ca.sup.2+]]i) influx and hyperactivation of [Ca.sup.2+]-dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical [Ca.sup.2+] channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [[Ca.sup.2+]]i was sustained at higher levels in an extracellular [Ca.sup.2+]-dependent manner, and normalization of TRCP-1 expression restored the abnormal [[Ca.sup.2+]]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1-mediated [Ca.sup.2+] entry to maintain endothelial integrity. endothelial cell | vascular calcification doi/ 10.1073/pnas.1008544107

Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, antiPECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti--PECAM-1 mAb inhibits the proliferation of PECAM-1--negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically, mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity. metastasis | endothelium | tumor microenvironment doi/ 10.1073/pnas.1004654107

Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions, which are regulated by changes in mitogen-activated protein (MAP) kinases, GTPases, and intracellular calcium. We previously showed that melanoma cells induce VE-cadherin disassembly through contact with human umbilical vein endothelial cells in coculture. However, the exact mechanism by which melanoma cells signal endothelial cells to induce VE-cadherin junction disassembly is not well understood. In this study, VE-cadherin junction disassembly was further examined under fluorescence microscopy. We found that melanoma-induced VE-cadherin junction disassembly and upregulation of p38 MAP kinase in endothelial cells is regulated by both soluble factors from melanomas, particularly interleukin (IL)-8, IL-6, and IL-1[beta], and through vascular cell adhesion molecule-1. Neutralizing melanoma-secreted soluble factors reduced endothelial gap formation. Endothelial cells transfected with MAP kinase kinase 6, a direct activator of p38 MAP kinase, increased VE-cadherin-mediated gap formation, facilitating melanoma transendothelial migration. In contrast, endothelial cells transfected with small-interfering RNA against p38 MAP kinase expression largely prevented melanoma transendothelial migration in Boyden chamber experiments. These findings indicate that p38 MAP kinase proteins regulate VE-cadherin junction disassembly, facilitating melanoma migration across endothelial cells. cytokines; chemokines; tumor conditioned medium; vascular cell adhesion molecule-1; mitogen-activated protein kinase; signaling; endothelial junctions; migration; vascular endothelial-cadherin doi: 10.1152/ajpcell.00242.2009.

We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% 02, 5% C[O.sub.2]) or hypoxia (1% [O.sub.2], 5% C[O.sub.2]). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced profiferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC. vascular remodeling; L-arginine; pulmonary hypertension doi: 10.1152/ajplung.00122.2009.

Endothelial nitric oxide synthase (eNOS) uncoupling is a mechanism that leads to endothelial dysfunction. Previously, we reported that shear stress-induced release of nitric oxide in vessels of aged rats was significantly reduced and was accompanied by increased production of superoxide (18, 27). In the present study, we investigated the influence of aging on eNOS uncoupling. Mesenteric arteries were isolated from young (3 mo) and aged (24 mo) C57 BL/6J mice. The expression of eNOS protein in young vs. aged mice was not significantly different. However, the aged mice had remarkable increases in the ratio of eNOS monomers to dimers and [N.sup.[omega]]-nitro-L-arginine methyl ester-inhibitable superoxide formation. The level of nitrotyrosine in the total protein and precipitated eNOS of aged vessels was increased compared with that in young vessels. HPLC analysis indicated a reduced level of tetrahydrobiopterin (BH4), an essential cofactor for eNOS, in the mesenteric arteries of aged mice. Quantitative PCR results implied that the diminished BH4 may result from the decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, enzymes involved in BH4 biosynthesis. When isolated and cannulated second-order mesenteric arteries (~150 [micro]m) from aged mice were treated with sepiapterin, acetylcholine-induced, endothelium-dependent vasodilation improved significantly, which was accompanied by stabilization of the eNOS dimer. These data suggest that eNOS uncoupling and increased nitrosylation of eNOS, decreased expressions of GTP cyclohydrolase I and sepiapterin reductase, and subsequent reduced BH4 bioavailability may be important contributors of endothelial dysfunction in aged vessels. tetrahydrobiopterin; nitrotyrosine; superoxide doi: 10.1152/ajpheart.00230.2009.

In diabetic states, altered plasma insulin is likely to play key roles in 3-phosphoinositide-dependent protein kinase (PDK)/Akt pathway activation, in insulin resistance and in endothelial dysfunction. Since the molecular mechanism(s) remains unclear, we examined the relationship between the PDK/Akt/endothelial nitric oxide synthase (NOS) pathway and endothelial function in aortas from diabetic rats that were either insulin deficient or hyperinsulinemic. Untreated diabetic (diabetic) rats exhibited hyperglycemia and hypoinsulinemia, whereas high-insulin-treated diabetic (HI-diabetic) rats exhibited hyperinsulinemia. Aortas from the diabetic group displayed impaired endothelium-dependent relaxation in response to ACh, whereas the insulin-induced relaxation was increased. In HI-diabetic aortas, the ACh-induced relaxation was normal, but that induced by insulin was impaired. The insulin-induced relaxation was inhibited by treatment with an Akt inhibitor in control and diabetic aortas, but not in the HI-diabetic aorta. This inhibitory effect on insulin-induced relaxation was greater in diabetic aortas than in control aortas. In all groups, ACh-induced relaxation was unaffected by the above inhibitor. In the diabetic group, various insulin-stimulated levels (nitric oxide production, phosphorylation of endothelial NOS at [Ser.sup.1177], of Akt at [Thr.sup.308], and of PDK-1 at [Ser.sup.241]) were significantly increased, whereas, in the HI-diabetic group, these levels were all decreased (vs. control aortas). These results suggest that the plasma insulin level has a close relation to the level of aortic PDK-1/Akt (at [Thr.sup.308])/NOS activities, and that reduced actions of the PDK-1/Akt (at [Thr.sup.308]) signal pathway may contribute to the impairments of insulin-induced endothelial functions seen in hyperinsulinemic diabetes. hyperinsulinemia; diabetes; nitric oxide; 3-phosphoinositide-dependent protein kinase doi: 10.1152/ajpheart.00536.2009.

Adenosine mediates vascular responses through four receptor subtypes: [A.sub.1], [A.sub.2A], [A.sub.2B], and [A.sub.3]. The role of [A.sub.2A] receptors in aortic vascular tone was investigated using [A.sub.2A] adenosine receptor (AR) knockout ([A.sub.2A]KO) and corresponding wild-type ([A.sub.2A]WT) mice. Isolated aortic rings from [A.sub.2A]WT and [A.sub.2A]KO mice were precontracted with phenylephrine ([10.sup.-7] M), and concentration responses for adenosine analogs and selective agonists/ antagonists were obtained. Nonselective adenosine analog (NECA; [EC.sub.50] = 6.78 [micro]M) and CGS-21680 ([A.sub.2A]AR selective agonist; [EC.sub.50] = 0.013 [micro]M) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at [10.sup.-5] M NECA and CGS-21680, respectively) in [A.sub.2A] WT aorta. In [A.sub.2A]KO aorta, NECA ([EC.sub.50] = 0.075 [micro]M) induced concentration-dependent contraction (maximum contraction of 47% at [10.sup.-6] M; P < 0.05 compared with [A.sub.2A] WT), whereas CGS-21680 produced no response. SCH-58261 ([10.sup.-6] M; [A.sub.2A]AR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in [A.sub.2A] WT (P < 0.05), whereas no change was observed in [A.sub.2A]KO. When DPCPX ([10.sup.-5] M; [A.sub.1] selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in [A.sub.2A]WT (50% vs. 25% in controls at [10.sup.-5] M; P < 0.05), whereas lower contraction was seen in [A.sub.2A]KO tissues (5% vs. 47% in controls at [10.sup.-6] M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60-6583 ([A.sub.2B] selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, [A.sub.2A]AR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the [A.sub.2A] AR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism. endothelium; mouse aorta; vasorelaxation; adenosine agonists; antagonists doi: 10.1152/ajpheart.00192.2009.

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPAR[gamma] ligands attenuate, whereas the loss of function mutations in PPAR[gamma] stimulate, endothelial dysfunction, suggesting that PPAR[gamma] may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPAR[gamma] in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPAR[gamma] gene to produce endothelial PPAR[gamma] null mice ([ePPAR[gamma].sup.-/-]). When compared with littermate controls, [ePPAR[gamma].sup.-/-] animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in [ePPAR[gamma].sup.-/-] mice. Similarly, intact aortic segments from [ePPAR[gamma].sup.-/-] mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and [ePPAR[gamma].sup.-/-] aortas. Reduced nitric oxide production in [ePPAR[gamma].sup.-/-] aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-[kappa]B in aortic homogenates. These findings demonstrate that endothelial PPAR[gamma] regulates vascular nitric oxide production and that the disruption of endothelial PPAR[gamma] contributes to endothelial dysfunction in vivo. endothelial nitric oxide synthase doi: 10.1152/ajpheart.00148.2009.

The potential physiological role of plasmalemmal large-conductance calcium-activated potassium channels ([BK.sub.Ca]) in vascular endothelial cells is controversial. Studies of freshly isolated and cultured vascular endothelial cells provide disparate results, both supporting and refuting a role for [BK.sub.Ca] in endothelial function. Most studies using freshly isolated, intact, healthy arteries provide little support for a physiological role for [BK.sub.Ca] in the endothelium, although recent work suggests that this may not be the case in diseased vessels. In isolated and cultured vascular endothelial cells, the autocrine action of growth factors, hormones, and vasoactive substances results in phenotypic drift. Such an induced heterogeneity is likely a primary factor accounting for the apparent differences, and often enhanced [BK.sub.Ca] expression and function, in isolated and cultured vascular endothelial cells. In a similar manner, heterogeneity in endothelial [BK.sub.Ca] expression and function in intact arteries may be representative of normal and disease states, [BK.sub.Ca] being absent in normal intact artery endothelium and upregulated in disease where dysfunction induces signals that alter channel expression and function. Indeed, in some intact vessels, there is evidence for the presence of [BK.sub.Ca], such as mRNA and/or specific BK subunits, an observation that is consistent with the potential for rapid upregulation, as may occur in disease. This perspective proposes that the disparity in the results obtained for [BK.sub.Ca] expression and function from freshly isolated and cultured vascular endothelial cells is largely due to variability in experimental conditions and, furthermore, that the expression of [BK.sub.Ca] in intact artery endothelium is primarily associated with disease. Although answers to physiologically relevant questions may only be available in atypical physiological conditions, such as those of isolation and culture, the limitations of these methods require open and objective recognition. expression; large-conductance calcium-activated potassium channel; methodology; smooth muscle

Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/ OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-[kappa]B was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability. microvascular permeability; endothelial cell; pulmonary edema

Plasminogen activator inhibitor-1 (PAI-1) is implicated in thrombogenesis, inflammation, and extracellular matrix remodeling. Previous studies indicated that oxidized low-density lipoprotein (LDL) stimulated the generation of PAI-1 in vascular endothelial cells (EC). The present study demonstrated that LDL oxidized by copper, iron, or 3-morpholinosydnonimine increased the expression of NADPH oxidase (NOX) 2, PAI-1, and heat shock factor-1 (HSF1) in human umbilical vein EC or coronary artery EC compared with LDL or vehicle. Diphenyleneiodonium, a NOX inhibitor, prevented the increases of the expression of HSF1 and PAI-1 in EC induced by oxidized LDLs. Small-interference RNA (siRNA) for [p22.sup.phox], an essential subunit of NOX, prevented oxidized LDL-induced expression of NOX2, HSF1, and PAI-1 in EC. HSF1 siRNA inhibited oxidized LDL-induced expression of PAI-1 and HSF1, but not NOX2, in EC. The binding of HSF1 to PAI-1 promoter and the activity of PAI-1 promoter in EC were enhanced by oxidized LDL. Butylated hydroxytulene, a potent antioxidant, inhibited oxidized LDL-induced release of hydrogen peroxide ([H.sub.2][O.sub.2]) and the expression of NOX2, HSF1, and PAI-1 in EC. Treatment with [H.sub.2][O.sub.2] increased the abundance of NOX2, HSF1, and PAI-1 in EC. The results of the present study indicate that oxidized LDL-induced expression of NOX may lead to the elevated release of reactive oxygen species, the activation of HSF1, and the enhancement of the transcription of PAI-1 gene in cultured vascular EC. reduced nicotinamide adenine dinucleotide phosphatase; oxidized low-density lipoprotein

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo. paracellular transport; ascorbate transport; anion channels; endothelial permeability

The hypothesis tested was PKC[alpha] mediates the phosphorylation of glycogen synthetase kinase 3[beta] (GSK3[beta]) and that the GSK3[beta] inhibition modulates the response to tumor necrosis factor-[alpha] (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKC[alpha] in the phosphorylation of (GSK3[beta] (i.e., an indicator of (GSK3[beta] inhibition), PMEC were pretreated with 1) nonsense-RNA-PKC[alpha], 2) siRNA-PKC[alpha], and 3) the PKC inhibitor Go6983. In the nonsense RNA-PKC[alpha]+TNF and TNF groups, there was increased phosphorylated (GSK3[beta]-Ser9 that did not occur in the Go6983+TNF group. In the TNF groups, there was a significant correlation between PKC[alpha] protein and phosphorylated (GSK3[beta]-Ser9 that did not occur in the groups without TNF. Second, to assess the role of (GSK3[beta] in [beta]-catenin activity, PMEC were pretreated with 1) wild-type (w) (GSK3[beta] plasmid to enhance (GSK3[beta] activity, 2) kinase dead (kd)-(GSK3[beta] plasmid, and 3) the (GSK3[beta] inhibitor SB-216763. In the TNF group, there was increased unphosphorylated [beta]-catenin-Ser37/33 compared with the control group. In the (GSK3[beta]-inhibited groups (i.e., SB-216763 and kd(GSK3[beta]) [+ or -] TNF, the unphosphorylated [beta]-catenin-Ser37/33 was similar to the TNF group. In the (GSK3[beta]-enhanced group [+ or -] TNF, the unphosphorylated [beta]-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a [beta]-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the (GSK3[beta]-inhibited groups (i.e., SB-216763 and kd(GSK3[beta])) [+ or -] TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the (GSK3[beta]-enhanced group [+ or -] TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKC[alpha]-dependent inhibition of (GSK3[beta]. glycogen synthetase kinase; permeability

Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real-time fluorescence imaging technique was developed to simultaneously track influx of Alexa 680-labeled albumin and genetically tagged enhanced green fluorescent protein-neutrophils [polymorphonuclear neutrophils (PMN)] within the wound bed. Vascular permeability increased approximately threefold more rapidly than the rate of PMN influx, reaching a maximum at 12 h, on the order of ~0.15% per minute versus ~0.05% per minute for PMN influx, which peaked at 18 h. Systemic depletion of PMN with antibody blocked their extravasation to the wound but did not alter the increase in vascular permeability. In contrast, pretreatment with antiplatelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelium-specific agonists VEGF or thrombin at 24 h postwounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 h and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling. enhanced green fluorescent protein-polymorphonuclear neutrophils; Rho-kinase; vascular endothelial growth factor; platelets

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF-[alpha] causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors ([N.sup.[omega]]-nitro-L-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF-[alpha]-induced superoxide ([O.sub.2.sup.*-]) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF-[alpha]-evoked [O.sub.2.sup.*-] generation and apoptosis. TNF-[alpha] rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal [O.sub.2.sup.*-] production, and accounts for a burst of oxidative stress in response to TNF-[alpha]. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF-[alpha] in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of berne oxygenase (HO), which is critical for endothelial protection against TNF-[alpha] toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-Al), inhibited Nox4-generated [O.sub.2.sup.*-] and apoptosis caused by TNF-[alpha] stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF-[alpha]-induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF-[alpha]-induced oxidative stress by inhibiting Nox4 activity and/or by [O.sub.2.sup.*-] scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease. inflammatory brain disease; brain endothelium; blood-brain barrier; cerebral vascular injury; heme oxygenase isoform-2; carbon monoxide-releasing molecule-Al; reduced nicotinamide adenine dinucleotide phosphatase oxidase isoforms 2 and 4; bilirubin; antioxidants

Type 5 phosphodiesterase (PDE5) inhibitors increase endothelial cell cGMP and promote angiogenesis. However, not all endothelial cell phenotypes express PDE5. Indeed, whereas conduit endothelial cells express PDE5, microvascular endothelial cells do not express this enzyme, and they are rapidly angiogenic. These findings bring into question whether PDE5 activity is a critical determinant of the endothelial cell angiogenic potential. To address this question, human full-length PDE5A1 was stably expressed in pulmonary microvascular endothelial cells, hPDE5A1 expression reduced the basal and atrial natriuretic peptide (ANP)stimulated cGMP concentrations in these cells, hPDE5Al-expressing cells displayed attenuated network formation on Matrigel in vitro and also produced fewer blood vessels in Matrigel plug assays in vivo; the inhibitory actions of hPDE5A1 were reversed using sildenafil. To examine whether endogenous PDE5 activity suppresses endothelial cell angiogenic potential, small interfering RNA (siRNA) constructs were stably expressed in pulmonary artery endothelial cells, siRNA selectively decreased PDE5 expression and increased basal and ANPstimulated cGMP concentrations in these conduit cells. PDE5 downregulation increased network formation on Matrigel in vitro and increased blood vessel formation in Matrigel plug assays in vivo. Collectively, our results indicate that PDE5 activity is an essential determinant of angiogenesis and suggest that PDE5 downregulation in microvascular endothelium imparts a stable, enhanced angiogenic potential to this cell type. vasculogenesis; migration; proliferation; nitric oxide; sildenafil

Endothelial cell (EC) activation plays a key role in vascular inflammation, thrombosis, and angiogenesis. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that has been implicated in the regulation of inflammation. The expression and function of AIF- 1 in EC is uncharacterized, and the purpose of this study was to characterize AIF-1 expression and function in ECs. AIF-1 expression colocalized with CD31-positive ECs in neointima of inflamed human arteries but not normal arteries. AIF-1 is detected at low levels in unstimulated EC, but expression can be increased in response to serum and soluble factors. Stable transfection of AIF-1 small interfering RNA (siRNA) in ECs reduced AIF-1 protein expression by 73% and significantly reduced EC proliferation and migration (P < 0.05 and 0.001). Rescue of AIF-1 expression restored both proliferation and migration of siRNA-expressing ECs, and AIF-1 overexpression enhanced both of these activities, suggesting a strong association between AIF-1 expression and EC activation. Activation of mitogen-activated protein kinase p44/42 and PAK1 was significantly reduced in siRNA ECs challenged with inflammatory stimuli. Reduction of AIF-1 expression did not decrease EC tube-like structure or microvessel formation from aortic rings, but overexpression of AIF-1 did significantly increase the number and complexity of these structures. These data indicate that AIF-1 expression plays an important role in signal transduction and activation of ECs and may also participate in new vessel formation. small interfering RNA; vascular inflammation; allograft inflammatory factor- 1

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial [O.sub.2.sup.x-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular [O.sub.2.sup.*-] and [H.sub.2][O.sub.2] production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress. senescence; vascular disease; atherosclerosis; [Prop1.sup.df/df] mice

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO4-oxidized LDL (FoLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAl-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC. oxidized lipoproteins; fibrinolytic inhibitor; lectin-like oxidized low-density lipoprotein receptor-1; H-Ras; Raf-1

Adherent cells remodel their cytoskeleton, including its directionality, in response to directional mechanical stimuli with consequent redistribution of intracellular forces and modulation of cell function. We analyzed the temporal and spatial changes in magnitude and directionality of the cytoplasmic creep compliance ([GAMMA]) in confluent cultures of bovine aortic endothelial cells subjected to continuous laminar flow shear stresses. We extended particle tracking microrheology to determine at each point in the cytoplasm the principal directions along which [GAMMA] is maximal and minimal. Under static condition, the cells have polygonal shapes without specific alignment. Although [GAMMA] of each cell exhibits directionality with varying principal directions, [GAMMA] averaged over the whole cell population is isotropic. After continuous laminar flow shear stresses, all cells gradually elongate and the directions of maximal and minimal [GAMMA] become, respectively, parallel and perpendicular to flow direction. This mechanical alignment is accompanied by a transition of the cytoplasm to be more fluid-like along the flow direction and more solid-like along the perpendicular direction; at the same time [GAMMA] increases at the downstream part of the cells. The resulting directional anisotropy and spatial inhomogeneity of cytoplasmic rheology may play an important role in mechanotransduction in adherent cells by providing a means to sense the direction of mechanical stimuli. anisotropy | microrheology | shear stress

The interaction of tumor necrosis factor (TNF)-[alpha] with the endothelium is a pivotal factor during endotoxemia. Inflammatory conditions are characterized by the activation of the transcription factor NF-[kappa]B and the expression of inflammatory mediators. Previous reports indicate that inhibition of NF-[kappa]B activation during sepsis may be beneficial to the microvasculature. In addition, the phosphatidylinositol-3-kinase/Akt signaling pathway (PI3-kinase/Akt) has been shown to be cytoprotective. In this study, we examined the effect of inhibition of NF-[kappa]B and PI3-kinase/ Akt on cell viability, cytokine production, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) generation by TNF-[alpha]-treated cultured microvascular endothelial cells. TNF-[alpha] induced significant cytotoxicity and was associated with increased inflammatory cytokines and NO and increased expression of iNOS. The NF-[kappa]B inhibitor, pyrrolidine dithiocarbamate (PDTC), prevented these increases and significantly attenuated the TNF-[alpha]-induced cytotoxicity. TNF-[alpha] also caused PI3-kinase/Akt activation, which was further increased by PDTC and prevented by the PI3-kinase inhibitor, LY294002. Inhibition of PI3-kinase/Akt also significantly potentiated TNF-[alpha]-mediated cytotoxicity. LY294002 treatment resulted in the appearance of increased apoptosis, compatible with the known antiapoptotic properties of PI3-kinase/Akt. The present results therefore demonstrate a cytotoxic effect of TNF-[alpha] in microvascular endothelial cells which can be attenuated by NF-KB inhibition. In addition, PI3kinase/Akt activation during TNF-[alpha] exposure may represent a compensatory anti-necrotic and anti-apoptotic pathway. The cytoprotective effects of NF-[kappa]B inhibition and PI3-kinase/Akt activation may have potential implications in the treatment of endotoxemia and septic shock. endotoxemia; sepsis; cell culture; PDTC; LY294002

Sepsis-associated encephalopathy is an early manifestation of sepsis, resulting in a diffuse dysfunction of the brain. Recently, nitric oxide (NO) has been proposed to be one of the key molecules involved in the modulation of inflammatory responses in the brain. The aim of this study was to assess the role of NO in cerebrovascular endothelial cell activation/ dysfunction during the early onsets of sepsis. To this end, we employed an in vitro model of sepsis in which cultured mouse cerebrovascular endothelial cells (MCVEC) were challenged with blood plasma (20% vol/vol) obtained from sham or septic (feces-induced peritonitis, FIP; 6 h) mice. Exposing MCVEC to FIP plasma for 1 h resulted in increased production of reactive oxygen species and NO as assessed by intracellular oxidation of oxidant-sensitive fluorochrome, dihydrorhodamine 123 (DHR 123), and nitrosation of NO-specific probe, DAF-FM, respectively. The latter events were accompanied by dissociation of tight junction protein, occludin, from MCVEC cytoskeletal framework and a subsequent increase in FITC-dextran (3-kDa mol mass) flux across MCVEC grown on the permeable cell culture supports, whereas Evans blue-BSA (65-kDa tool mass) or FITC-dextran (10-kDa mol mass) flux were not affected. FIP plasma-induced oxidant stress, occludin rearrangement, and MCVEC permeability were effectively attenuated by antioxidant, 1-pyrrolidinecarbodithioic acid (PDTC; 0.5 mM), or interfering with nitric oxide synthase (NOS) activity [0.1 mM nitro-L-arginine methyl ester (L-NAME) or endothelial NOS (eNOS)-deficient MCVEC]. However, treatment of MCVEC with PDTC failed to interfere with NO production, suggesting that septic plasma-induced oxidant stress in MCVEC is primarily a NO-dependent event. Taken together, these data indicate that during early sepsis, eNOS-derived NO exhibits proinflammatory characteristics and contributes to the activation and dysfunction of cerebrovascular endothelial cells. oxidant stress; vascular permeability; systemic inflammation; cell culture

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3 [+ or -] 3.8 to 467 [+ or -] 22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4 [+ or -] 0.3 x [10.sup.-6] to 8.5 [+ or -] 0.4 x [10.sup.-6] cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation. endothelial nitric oxide; endothelial cells; microvascular permeability; endothelial nitric oxide synthase translocation; acetylcholine; platelet-activating factor

Endothelial cells (ECs) from different vascular beds display a remarkable heterogeneity in both structure and function. Phenotypic heterogeneity among arterial ECs is particularly relevant to atherosclerosis since the disease occurs predominantly in major arteries, which vary in their atherosusceptibility. To explore EC heterogeneity between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of freshly harvested porcine coronary (CECs) and iliac artery (IECs) ECs. Statistical analysis revealed 51 genes that were differentially expressed in CECs relative to IECs at a false discovery rate of 5%. Seventeen of these genes are known to be involved in atherogenesis. Consistent with coronary arteries being more atherosusceptible, almost all putative atherogenic genes were overexpressed in CECs, whereas all atheroprotective genes were downregulated, relative to IECs. A subset of the identified genes was validated by quantitative polymerase chain reaction (PCR). PCR results suggest that the differences in expression levels between CECs and IECs for the HOXA10 and HOXA9 genes were > 100-fold. Gene ontology (GO) and biological pathway analysis revealed a global expression difference between CECs and IECs. Genes in twelve GO categories, including complement immune activation, immunoglobulin-mediated response, and system development, were significantly upregulated in CECs. CECs also overexpressed genes involved in several inflammatory pathways, including the classical pathway of complement activation and the IGF-1-mediated pathway. The in vivo transcriptional differences between CECs and IECs found in this study may provide new insights into the factors responsible for coronary artery atherosusceptibility. vascular endothelium; atherosclerosis; pathophysiology; genomics

Epidemiological and clinical studies provide compelling support for a causal relationship between Helicobacter pylori infection and endothelial dysfunction, leading to vascular diseases. However, clear biochemical evidence for this association is limited. In the present study, we have conducted a comprehensive investigation of endothelial injury in bovine aortic endothelial cells (BAECs) induced by H. pylori-conditioned medium (HPCM) prepared from H. pylori 60190 [vacuolating cytotoxin A ([Vac.sup.+])]. BAECs were treated with either unconditioned media, HPCM (0-25% vol/vol), or Escherichia coli-conditioned media for 24 h, and cell functions were monitored. [Vac.sup.+] HPCM significantly decreased BAEC proliferation, tube formation, and migration (by up to 44%, 65%, and 28%, respectively). Posttreatment, we also observed sporadic zonnula occludens-1 immunolocalization along the cell-cell border, and increased BAEC permeability to FD40 Dextran, indicating barrier reduction. These effects were blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (VacA inhibitor) and were not observed with conditioned media prepared from either VacA-deleted H. pylori or E. coli. The cellular mechanism mediating these events was also considered. [Vac.sup.+] HPCM (but not [Vac.sup.-]) reduced nitric oxide (NO) by >50%, whereas S-nitroso-N-acetylpenicillamine, an NO donor, recovered all [Vac.sup.+] HPCM-dependent effects on cell functions. We further demonstrated that laminar shear stress, an endothelial NO synthase/NO stimulus in vivo, could also recover the [Vac.sup.+] HPCM-induced decreases in BAEC functions. This study shows, for the first time, a significant proatherogenic effect of H. pylori-secreted factors on a range of vascular endothelial dysfunction markers. Specifically, the VacA-dependent reduction in endothelial NO is indicated in these events. The athero-protective impact of laminar shear stress in this context is also evident. vacuolating cytotoxin A; shear stress

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-L-arginine (L-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. L-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro. persistent pulmonary hypertension of the newborn; vasculogenesis; nitric oxide; endothelial nitric oxide synthase; lung vascular development

Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/ acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability. vascular endothelial growth factor; hepatocyte growth factor

The involvement of ecto-5'-nucleotidase (E-5'Nu) in the elevation of extracellular adenosine during inflammation is unclear. In the present study, the effect of lipopolysaccharide (LPS), an inflammation inducer, was investigated on E-5'Nu in human umbilical vein endothelial cells (HUVECs). E-5'Nu activity was enhanced after a 24 h exposure to LPS. This effect was dose dependent, with an [EC.sub.50] of 1.66 ng/ml. At 10 [micro]M, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 abolished the LPS-induced E-5'Nu activity. However, at 10 [micro]M, the NF-[kappa]B inhibitor ammonium pyrrolidine dithiocarbamate had no effect. LPS upregulated the protein expression but not the messenger RNA expression of E-5'Nu. The inhibition of E-5'Nu by 100 [micro]M [alpha],[beta]-methylene adenosine-5'-diphosphate increased the LPS-induced inflammation, suggesting that E-5'Nu plays a significant role in reducing inflammation, probably through the generation of adenosine. In conclusion, the experiments indicate that LPS upregulates E-5'Nu activity in HUVECs through a PI3K-dependent increase in the abundance of E-5'Nu on cell membranes. Since adenosine is an anti-inflammatory molecule, E-5'Nu upregulation may be crucial in protecting endothelial cells against inflammatory damage. adenosine; cytokines; inflammation

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis. vascular endothelial function; oxidative stress

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation. vascular endothelia growth factor; phosphatidylinositol 3-kinase; Rho-GTPase; cytoskeleton; endothelium; angiogenesis

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1-based mechanosensory complex in ECs.

Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of yon Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI. acute renal failure; confocal fluorescence microscopy; vascular smooth muscle cell; vasculature

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

The mechanisms by which bone marrow (BM)-derived stem cells might contribute to angiogenesis and the origin of neovascular endothelial cells (ECs) are controversial. Neovascular ECs have been proposed to originate from VEGF receptor 2-expressing (VEGFR2+) stem cells mobilized from the BM by VEGF or tumors, and it is thought that angiogenesis and tumor growth may depend on such endothelial precursors or progenitors. We studied the mobilization of BM cells to circulation by inoculating mice with VEGF polypeptides, adenoviral vectors expressing VEGF, or tumors. We induced angiogenesis by syngeneic melanomas, APCmin adenomas, adenoviral VEGF delivery, or matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models, and subsequently analyzed the contribution of BM-derived cells to endothelium in a wide range of time points. To study the existence of circulating ECs in a nonmyeloablative setting, pairs of genetically marked parabiotic mice with a shared anastomosed circulatory system were created. We did not observe specific mobilization of VEGFR-2+ cells to circulation by VEGF or tumors. During angiogenesis, abundant BM-derived perivascular cells were recruited close to blood vessel wall ECs but did not form part of the endothelium. No circulation-derived vascular ECs were observed in the parabiosis experiments. Our results show that no BM-derived VEGFR-2+ or other EC precursors contribute to vascular endothelium and that cancer growth does not require BM-derived endothelial progenitors. Endothelial differentiation is not a typical in vivo function of normal BM-derived stem cells in adults, and it has to be an extremely rare event if it occurs at all. angiogenesis | cancer | progenitor | stem cell | differentiation

Advanced glycation end products (AGEs), which are elevated in diabetic and uremic patients, may induce vascular dysfunctions, and calcitriol may improve the cardiovascular complications. Therefore, we examined whether calcitriol may modify the endothelial response to AGEs stimulation. Knowing the importance of nuclear factor-[kappa]B in endothelial inflammatory responses, the effect of AGEs and calcitriol on this pathway was also studied. Calcitriol was added to endothelial cells previously incubated with AGE-human serum albumin (HSA). AGE-HSA induced a decrease in endothelial nitric oxide synthase (eNOS) mRNA expression and enzyme activity. Addition of calcitriol to AGE-HSA-treated endothelial cells improved the decreased action of AGEs on the eNOS system. AGE-HSA increased the AGEs receptor mRNA and protein, which were both blunted by calcitriol. The parallel elevation of interleukin-6 mRNA in the presence of AGE-HSA was also blunted by calcitriol. The NF-[kappa]B-p65 DNA binding activity was enhanced and associated with a decrease in inhibitor [kappa]B[alpha] (I[kappa]B[alpha]) and an increase in phosphorylated (p)-I[kappa]B[alpha] levels. Addition of calcitriol blunted the AGEs-induced elevation of NF-[kappa]B-p65 DNA binding activity, a phenomenon related to an increased expression of I[kappa]B[alpha]. This increase was correlated to declined p-I[kappa]B[alpha] levels. The present results support the concept that calcitriol may act as a vascular protective agent counteracting the probable deleterious actions of AGEs on endothelial cell activities. vitamin D; advanced glycation end products; nuclear factor-[kappa]B; endothelial cells

Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating agerelated alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-[gamma]/coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells. vascular senescence

[Na.sup.+] cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the [Na.sup.+]/[Ca.sup.2+] exchanger, bumetanide for the [Na.sup.+]- [Ksup.+]-2C1- cotransporter, and amiloride for [Na.sup.+]/ [H.sup.+] cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanidesensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The [Na.sup.+]/[Ca.sup.+] exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive [Na.sup.+]/ [H.sup.+] cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to [Na.sup.+]-[K.sup.+]2Cl-cotransporter and [Na.sup.+]/ [Ca.sup.2+] exchanger functions are key to cerebral vascular oxygen responses. neuronal nitric oxide synthase; endothelial nitric oxide synthase; brain

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Racl, and a peptide-based inhibitor of NADPH oxidase subunit [p47.sup.phox] association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac 1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of [p47.sup.phox] and tyrosine phosphorylation of [p47.sup.phox] in a time-dependent manner as well as increased activated Racl/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species

Magnesium, an essential intracellular cation, is critically involved in many biochemical reactions involved in the regulation of vascular tone and integrity. Decreased magnesium concentration has been implicated in altered vascular reactivity, endothelial dysfunction, vascular inflammation, and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Until recently, very little was known about mechanisms regulating cellular magnesium homeostasis, and processes controlling transmembrane magnesium transport had been demonstrated only at the functional level. Two cation channels of the transient receptor potential melastatin (TRPM) cation channel family have now been identified as magnesium transporters, TRPM6 and TRPM7. These unique proteins, termed chanzymes because they possess a channel and a kinase domain, are differentially expressed, with TRPM6 being found primarily in epithelial cells and TRPM7 occurring ubiquitously. Vascular TRPM7 is modulated by vasoactive agents, pressure, stretch, and osmotic changes and may be a novel mechanotransducer. In addition to its magnesium transporter function, TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization, and migration, important processes involved in vascular remodeling associated with hypertension and other vascular diseases. Emerging evidence suggests that vascular TRPM7 function may be altered in hypertension. This review discusses the importance of magnesium in vascular biology and implications in hypertension and highlights the transport systems, particularly TRPM6 and TRPM7, which may play a role in the control of vascular magnesium homeostasis. Since the recent identification and characterization of [Mg.sup.2+]-selective transporters, there has been enormous interest in the field. However, there is still a paucity of information, and much research is needed to clarify the exact mechanisms of magnesium regulation in the cardiovascular system and the implications of aberrant transmembrane magnesium transport in the pathogenesis of hypertension and other vascular diseases. calcium; cations; signal transduction; mechanotransduction; vascular smooth muscle; ion channels

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Car-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice ([Car-1.sup.-/-]) and wild-type control mice (WT; [Cav-1.sup.+/+]) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with [N.sup.G]-nitro-L-arginine methyl ester and more so in [Cav-1.sup.-/-] than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in [Cav-1.sup.-/-] (maximum 0.25 [+ or -] 0.06 g/mg) compared with WT (0.75 [+ or -] 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in [Cav-1.sup.-/-] (0.27 [+ or -] 0.05 g/mg) compared with WT mice (0.53 [+ or -] 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine ([10.sup.-5] M) caused aortic relaxation in WT mice on HS (23.6 [+ or -] 3.5%) and LS (23.7 [+ or -] 5.5%) that was enhanced in [Cav-1.sup.-/-] HS (72.6 [+ or -] 6.1%) and more so in [Cav-1.sup.-/-] LS mice (93.6 [+ or -] 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of [Cav-1.sup.-/-] compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake. endothelium; nitric oxide; vascular smooth muscle; blood pressure; hypertension

Store-operated channels (SOC) and store-operated [Ca.sup.2+] entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type [Ca.sup.2+] ([Ca.sup.2+.sub.L]) channels are thought to be fully responsible for agonist-induced [Ca.sup.2+] influx and vasoconstriction. We present evidence that SOC and their activation via a [Ca.sup.2+] -independent phospholipase [A.sub.2] ([iPLA.sub.2])-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular [Ca.sup.2+] in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) [Ca.sup.2+] and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a [Ca.sup.2.sub.L+] inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and [Ca.sup.2.sub.L+] channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of [iPLA.sub.2][beta] totally inhibited PE-induced [Ca.sup.2+] influx and constriction in cerebral, mesenteric, and carotid arteries, whereas [K.sup.+]-induced [Ca.sup.2+] influx and vasoconstriction mediated by [Ca.sup.2+.sub.L] channels were not affected. Thus [iPLA.sub.2]-dependent activation of SOC is crucial for agonist-induced [Ca.sup.2+] influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of [Ca.sup.2+] stores, nonselective SOC are activated via an [iPLA.sub.2]-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of [Ca.sup.2+.sub.L] channels and lead to [Ca.sup.2+] entry and vasoconstriction. store-operated calcium entry; smooth muscle cells; constriction

Experiments in cultured endothelial cell monolayers demonstrate that increased intracellular cAMP strongly inhibits the acute permeability responses by both protein kinase A (PKA)-dependent and -independent pathways. The contribution of the PKA-independent pathways to the anti-inflammatory mechanisms of cAMP in intact mammalian microvessels has not been systematically investigated. We evaluated the role of the cAMP-dependent activation of the exchange protein activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPase Rap1, in rat venular microvessels exposed to the platelet-activating factor (PAF). The cAMP analog 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP), which stimulates the Epac/Rap1 pathway but has no effect on PKA, significantly attenuated the PAF increase in microvessel permeability as measured by hydraulic conductivity (Lp). We also demonstrated that PAF induced a rearrangement of vascular endothelial (VE)-cadherin seen as numerous lateral spikes and frequent short breaks in the otherwise continuous peripheral immunofluorescent label. Pretreatment with O-Me-cAMP completely prevented the PAF-induced rearrangement of VE-cadberin. We conclude that the action of the Epac/Rap1 pathway to stabilize cell-cell adhesion is a significant component of the activity of cAMP to attenuate an acute increase in vascular permeability. Our results indicate that increased permeability in intact microvessels by acute inflammatory agents such as PAF is the result of the decreased effectiveness of the Epac/Rap1 pathway modulation of cell-cell adhesion. capillaries; vascular permeability; adenosine 3',5'-cyclic monophosphate; inflammation; edema

Endothelial progenitor cells (EPCs) have been isolated postnatally from bone marrow, blood, and both the intima and adventitia of conduit vessels. However, it is unknown whether EPCs can be isolated from the lung microcirculation. Thus we sought to determine whether the microvasculature possesses EPCs capable of de novo vasculogenesis. Rat pulmonary artery (PAEC) and microvascular (PMVEC) endothelial cells were isolated and selected by using a single-cell clonogenic assay. Whereas the majority of PAECs (~60%) were fully differentiated, the majority of PMVECs (~75%) divided, with ~50% of the single cells giving rise to large colonies (>2,000 cells/colony). These highly proliferative cells exhibited the capacity to reconstitute the entire proliferative hierarchy of PMVECs, unveiling the existence of resident microvascular endothelial progenitor cells (RMEPCs). RMEPCs expressed endothelial cell markers (CD31, CD144, endothelial nitric oxide synthase, and yon Willenbrand factor) and progenitor cell antigens (CD34 and CD309) but did not express the leukocyte marker CD45. Consistent with their origin, RMEPCs interacted with Griffonia simplicifolia and displayed restrictive barrier properties. In vitro and in vivo Matrigel assays revealed that RMEPCs possess vasculogenic capacity, forming ultrastructurally normal de novo vessels. Thus the pulmonary microcirculation is enriched with EPCs that display vasculogenic competence while maintaining functional endothelial microvascular specificity. endothelial progenitor cells; pulmonary circulation; vasculogenesis

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), Factin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-[NH.sub.2] and TFLLRN-[NH.sub.2]) were used to confirm a role for the PAR-1 receptor. PAR-APs (25-250 [micro]M) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 p[micro]g/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation. proteinase-activated receptor; permeability

In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, closeproximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1[beta]), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-[alpha] IV) and cytokineslgrowth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms. human microvascular endothelial cells; human proximal tubular cells; transepithelial electrical resistance; gene expression; cell culture

Ion transporters of blood-brain barrier (BBB) endothelial cells play an important role in regulating the movement of ions between the blood and brain. During ischemic stroke, reduction in cerebral blood flow is accompanied by transport of Na and C1 from the blood into the brain, with consequent brain edema formation. We have shown previously that a BBB Na-K-Cl cotransporter (NKCC) participates in ischemia-induced brain Na and water uptake and that a BBB Na/H exchanger (NHE) may also participate. While the abrupt reduction of blood flow is a prominent component of ischemia, the effects of flow on BBB NKCC and NHE are not known. In the present study, we examined the effects of changes in shear stress on NKCC and NHE protein levels in cerebral microvascular endothelial cells (CMECs). We have shown previously that estradiol attenuates both ischemia-induced cerebral edema and CMEC NKCC activity. Thus, in the present study, we also examined the effects of estradiol on NKCC and NHE protein levels in CMECs. Exposing CMECs to steady shear stress (19 dyn/[cm.sup.2]) increased the abundance of both NKCC and NHE. Estradiol abolished the shear stress-induced increase in NHE but not NKCC. Abrupt reduction of shear stress did not alter NKCC or NHE abundance in the absence of estradiol, but it decreased NKCC abundance in estradiol-treated cells. Our results indicate that changes in shear stress modulate BBB NKCC and NHE protein levels. They also support the hypothesis that estradiol attenuates edema formation in ischemic stroke in part by reducing the abundance of BBB NKCC protein. estrogen; ischemia; mechanotransduction; flow reduction; endothelium

Endothelial monolayer integrity is required to maintain endothelial barrier functions and has found to be impaired in several disorders like inflammatory edema, allergic shock, or artherosclerosis. Under physiologic conditions in vivo, endothelial cells are exposed to mechanical forces such as hydrostatic pressure, shear stress, and cyclic stretch. However, insight into the effects of hydrostatic pressure on endothelial cell biology is very limited at present. Therefore, in this study, we tested the hypothesis that physiological hydrostatic pressure protects endothelial monolayer integrity in vitro. We investigated the protective efficacy of hydrostatic pressure in microvascular myocardial endothelial (MyEnd) cells and macrovascular pulmonary artery endothelial cells (PAECs) by the application of selected pharmacological agents known to alter monolayer integrity in the absence or presence of hydrostatic pressure. In both endothelial cell lines, extracellular [Ca.sup.2+] depletion by EGTA was followed by a loss of vascular-endothelial cadherin (VE-caherin) immunostaining at cell junctions. However, hydrostatic pressure (15 cm[H.sub.2]O) blocked this effect of EGTA. Similarly, cytochalasin D-induced actin depolymerization and intercellular gap formation and cell detachment in response to the [Ca.sup.2+]/calmodulin antagonist trifluperazine (TFP) as well as thrombin-induced cell dissociation were also reduced by hydrostatic pressure. Moreover, hydrostatic pressure significantly reduced the loss of VE-cadherin-mediated adhesion in response to EGTA, cytochalasin D, and TFP in MyEnd cells as determined by laser tweezer trapping using VE-cadherin-coated microbeads. In caveolin-1-deficient MyEnd cells, which lack caveolae, hydrostatic pressure did not protect mono-layer integrity compromised by EGTA, indicating that caveolae-dependent mechanisms are involved in hydrostatic pressure sensing and signaling. vascular-endothelial cadherin; actin

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 [micro]M) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; [Tyr.sup.576]) phosphorylation associated with sphingosine-1-phosphate receptor type 1 ([S1P.sub.1]) transactivation. [S1P.sub.1] protein depletion by the short interfering RNA technique completely abolished edaravoneinduced FAK ([Tyr.sub.576]) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1[P.sub.1] transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of [S1P.sub.1] transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder. focal adhesion; Rac; sphingosine-l-phosphate receptor type 1; transmonolayer electrical resistance

Dietary salt plays a major role in the regulation of blood pressure, and the mineralocorticoid hormone aldosterone controls salt homeostasis and extracellular volume. Recent observations suggest that a small increase in plasma sodium concentration may contribute to the pressor response of dietary salt. Because endothelial cells are (i) sensitive to aldosterone, (ii) in physical contact with plasma sodium, and (iii) crucial regulators of vascular tone, we tested whether acute changes in plasma sodium concentration, within the physiological range, can alter the physical properties of endothelial cells. The tip of an atomic force microscope was used as a nanosensor to measure stiffness of living endothelial cells incubated for 3 days in a culture medium containing aldosterone at a physiological concentration (0.45 nM). Endothelial cell stiffness was unaffected by acute changes in sodium concentration atomic force microscopy | cell stiffness | endothelial dysfunction | eplerenone