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Endothelial barrier dysfunction is typically triggered by increased intracellular [Ca.sup.2+] concentration. Membrane-permeable analogs of guanosine 3',5'-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential ([E.sub.m]), which influences the electrochemical gradient for [Ca.sup.2+] influx, is regulated by [K.sup.+] channels, we investigated the effect of 8-bromo-cGMP on [E.sub.m] and inwardly rectifying [K.sup.+] ([K.sub.IR]) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to -50 mV that were abolished by application of 10 [micro] M [Ba.sup.2+], consistent with [K.sub.IR] current. [Ba.sup.2+] also depolarized both cell types. 8-Bromo-cGMP ([10.sup.-3] M) depolarized BPAEC and BMVEC and inhibited [K.sub.IR] current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of [K.sub.IR] current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for [Ca.sup.2+] entry. protein kinase G; membrane potential; ion channels

In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels ate continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree. Keywords: Blood Flow, Mechanotransduction, Migration, eNOS

Circulating endothelial cells: tea leaves for renal disease. Am J Physiol Renal Physiol 283: F11-F19, 2002; 10.1152/ajprenal.00008.2002.--Fully differentiated endothelial cells and their precursors circulate in the bloodstream. Since their initial description more than 30 years ago, circulating endothelial cells have been quantified in a number of different clinical conditions that affect the endothelium. Only recently, however, have investigators begun to examine the protein expression and functionality of these cells. Because a number of diseases prevalent in the field of nephrology affect endothelial cells, the study of circulating endothelial cells may allow the direct examination of the state of the endothelium in these conditions. This review will discuss the endothelium and renal disease, the methods to quantify these circulating endothelial cells, their origins, and their therapeutic potential endothelial progenitor cells; endothelial activation

Interaction of albumin with the endothelial cell surface. Am J Physiol Heart Circ Physiol 283: H398-H405, 2002. First published March 7, 2002; 10.1152/ajpheart.00558.2001.--Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine-N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37[degrees]C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYLstearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances. electron spin resonance; molecular mobility; endothelial surface layer

We examined bovine aortic endothelial cells (BAECs) for the functional expression of P2X receptors, the ATP-gated cation channels. We identified the P2X subtypes present in BAECs using RT-PCR. mRNA was present for only three of seven family members: P2X4, P2X5, and P2X7. We then characterized agonist-activated currents in whole cell and outside-out patch recordings using 2-methyl-thio-ATP (MeSATP) as a P2X4 and P2X5 receptor agonist and 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP) as a P2X7 receptor agonist. MeSATP (10-20 [micro]M) produced current with characteristics of P2X4 receptors. The current was an inwardly rectifying current, reversed near 0 mV, slowly desensitized, was not blocked by suramin (300 [micro]M) or reactive blue (60 [micro]M), and had a single channel conductance of 36 pS. BzATP (10-100 [micro]M), on the other hand, activated a 9-pS channel with sustained activity in the continued presence of the agonist. BzATP-activated current was blocked by reactive blue (60 [micro]M) and by suramin (~50% block at 300 [micro]M). We confirmed, by immunocytochemistry, the presence of P2X4 and P2X7 protein. The agonists failed, however, to induce significant uptake of the large molecule YO-PRO, indicating, the lack of pore development that has been demonstrated for P2X7 and P2X4 in response to agonist in some cell types. P2X4; P2X7; P2X5; endothelial cells; cation channels

Endothelial cells (ECs) line the mammalian vascular system and respond to the hemodynamic stimulus of fluid shear stress, the frictional force produced by blood flow. When ECs are exposed to shear stress, one of the fastest responses is an increase of [K.sup.+] conductance, which suggests that ion channels are involved in the early shear stress response. Here we show that an applied shear stress induces a [K.sup.+] ion current in cells expressing the endothelial Kir2.1 channel. This ion current shares the properties of the shear-induced current found in ECs. In addition, the shear current induction can be specifically prevented by tyrosine kinase inhibition. Our findings identify the Kir2.1 channel as an early component of the endothelial shear response mechanism.

Studies over the last two decades have shown that mammalian nonmacrophagic liver endothelial cells clear the blood from numerous physiological and foreign waste macromolecules, such as polysaccharides and proteins released during extracellular matrix turnover, intracellular macromolecules, modified serum proteins, and bacterial and fungal proteins [Smedsrod, B., Pertoft, H., Gustafson, S. & Laurent, T. C. (1990) Biochem. J. 266, 313-327]. These macromolecules are released daily in gram-amounts in a normal human body and are effectively taken up and degraded by the liver endothelial cells. Recent studies show that bony fishes harbor a similar system of specialized nonmacrophagic scavenger endothelial cells in either kidney [Smedsrod, B., Gjoen, T., Sveinbjornsson, B. & Berg, T. (1993) J. Fish Biol. 42, 279-291] or heart [Sorensen, K. K., Melkko, J. & Smedsrod, B. (1998) J. Exp. Biol. 201, 1707-1718], but not in liver. Using specific and extremely effective endocytosis, these fish scavenger endothelial cells function as their mammalian counterpart to eliminate soluble waste macromolecules from the circulation. We show here that species from all seven vertebrate classes carry a population of nonmacrophagic scavenger endothelial cells that efficiently eliminate an array of circulating waste macromolecules. Thus representing an important part of the vertebrate innate immune system, these scavenger endothelial cells display the following distribution in the different vertebrate classes: Gills in Agnatha and Chondrichtyes; heart or kidney in Osteichtyes; and liver in Amphibia, Reptilia, Aves, and Mammalia.

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microcoscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD[34.sup.+] and CD[45.sup.-] fraction. Cells in this fraction were ~94% positive for Sca-1, and mostly negative (

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or [p130.sup.Cas] cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway. cell survival; paxillin; [p130.sup.Cas]; endothelium; focal adhesion complexes

Platelet endothelial cell adhesion molecule (PECAM)-1 has been implicated in angiogenesis, but a number of issues remain unsettled, including the independent involvement of human PECAM-1 (huPECAM-1) in tumor angiogenesis and the mechanisms of its participation in vessel formation. We report for tumors grown in human skin transplanted on severe combined immunodeficiency mice that antibodies against huPECAM-1 (without simultaneous treatment with anti-VE-cadherin antibody) decreased the density of human, but not murine, vessels associated with the tumors. Anti-huPECAM-1 antibody also inhibited tube formation by human umbilical vein endothelial cells (HUVEC) and the migration of HUVEC through Matrigel-coated filters or during the repair of wounded cell monolayers. The involvement of huPECAM-1 in these processes was confirmed by the finding that expression of huPECAM-1 in cellular transfectants induced tube formation and enhanced cell motility. These data provide evidence of a role for PECAM-1 in human tumor angiogenesis (independent of VE-cadherin) and suggest that during angiogenesis PECAM-1 participates in adhesive and/or signaling phenomena required for the motility of endothelial cells and/or their subsequent organization into vascular tubes. endothelial cells; platelet endothelial cell adhesion molecule-1; cell adhesion molecules

Glomerular epithelial cells (GEC) are a known site of vascular endothelial growth factor (VEGF) production. We established immortalized rat GEC, which retained the ability to produce VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured on GEC-conditioned matrix, an indicator of the permeability of monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed that VEGF results in a rapid appearance of transcellular elongated structures decorated with caveolin. Transmission electron microscopy of endothelial cells showed that caveolae undergo rapid internalization and fusion 30 min after application of VEGF-165. Later (36 h), endothelial cells pretreated with VEGF developed fenestrae and showed a decrease in electrical resistance. Immunoelectron microscopy of glomeruli confirmed VEGF localization to podocytes and in the basement membrane. In summary, immortalized GEC retain the ability to synthesize VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of caveolae expression, their fission and fusion, formation of elongated caveolin-decorated structures, and eventual formation of fenestrae, both responsible for the increase in endothelial permeability. vascular endothelial growth factor; podocyte; caveolin; fenestrae; endothelial permeability; green fluorescent protein

In skeletal muscle arterioles, the pathway leading to non-nitric oxide (NO), non-prostaglandin-mediated endothelium-derived hyperpolarizing factor (EDHF)-type dilations is not well characterized. To elucidate some of the steps in this process, simultaneous changes in endothelial intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and the diameter of rat gracilis muscle arterioles (~60 [micro]m) to acetylcholine (ACh) were measured by fura 2 microfluorimetry (in the absence of NO and prostaglandins). ACh elicited rapid increases in endothelial [[[Ca.sup.2+]].sub.i] (101 [+ or -] 7%), followed by substantial dilations (73 [+ or -] 2%, coupling time: 1.3 [+ or -] 0.2 s) that were prevented by endothelial loading of an intracellular [Ca.sup.2+] chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Arteriolar dilations to ACh were also inhibited by intraluminal administration of the [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channel blockers charybdotoxin plus apamin or by palmitoleic acid, an uncoupler of myoendothelial gap junctions without affecting changes in endothelial [[[Ca.sup.2+]].sub.i]. The presence of large conductance [K.sub.Ca] channels on arteriolar endothelial cells was demonstrated with immunohistochemisty. We propose that in skeletal muscle arterioles, EDHF-type mediation is evoked by an increase in endothelial [[[Ca.sup.2+]].sub.i], which by activating endothelial [K.sub.Ca] channels elicits hyperpolarization that is conducted via myoendothelial gap junctions to the smooth muscle resulting in decreases in [[[Ca.sup.2+]].sub.i] and consequently dilation. endothelium-dependent hyperpolarizing factor; arteriolar endothelium; potassium channels; charybdotoxin

Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

Although standard anticancer chemotherapeutic drugs have been designed to inhibit the survival or growth of rapidly dividing tumor cells, it is possible to enhance the efficacy of such drugs by targeting the proliferating host endothelial cells (ECs) of the tumor vasculature. A theoretical advantage of this strategy lies in the possibility of circumventing, or significantly delaying, acquired drug resistance driven by the genetic instability of tumor cells. Here, we show that both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor significantly reduce the pro-apoptotic potency of chemotherapy on both micro- and macrovascular ECs. This cytoprotection to drug toxicity was found to be phosphatidylinositol 3-kinase-dependent and could be recapitulated in the absence of VEGF by overexpressing the dominant-active form of the serine/threonine kinase protein kinase B/Akt. Downstream of phosphatidylinositol 3-kinase, we also show that survivin plays a pivotal role in VEGF-mediated EC protection by preserving the microtubule network. In this respect, its induction effectively protects ECs against chemotherapeutic damage, whereas overexpression of its dominant-interfering mutant (C84A) abrogates the protective effects of VEGF. Accordingly, the potency of VEGF as a chemoprotectant was more pronounced with drugs that interfere with microtubule dynamics than those that damage DNA. These studies implicate a role for survivin up-regulation as a novel mechanism of EC drug 'resistance' and support the notion that angiogenic factors that induce the expression of survivin may act to shield tumor ECs from the apoptotic effects of chemotherapy. Thus, exploiting chemotherapeutic drugs as antiangiogenics is likely to be compromised by the high concentrations of pro-angiogenic survival/growth factors present in the tumor micro-environment; targeting EC survival pathways should improve the antiangiogenic efficacy of antineoplastic agents, particularly microtubule-inhibitor drugs.

Ets-1, which stimulates metalloproteinase gene transcription, has a key role in angiogenesis. We first examined whether activated polymorphonuclear leukocytes (PMNs) enhanced angiogenesis through the induction of Ets-1. Addition of activated PMNs to endothelial cells stimulated both in vitro angiogenesis in collagen gel and Ets-1 expression. Both angiogenesis and Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide, suggesting that Ets-1 is an important factor in PMN-induced angiogenesis. Although intercellular adhesion molecule (ICAM)-I and E-selectin are involved in PMN-induced angiogenesis, the mechanisms underlying their roles in angiogenesis have yet to be elucidated. PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. Moreover, the stimulation of angiogenesis by [H.sub.2][O.sub.2] without PMNs was inhibited by a monoclonal antibody to E-selectin but not ICAM-i. These findings suggested that ICAM-1 in endothelial cells may act as a signaling receptor to induce Ets-1 expression, whereas E-selectin seems to function in the formation of tubelike structures in vascular endothelial cell cultures. endothelial cell; intercellular adhesion molecule-1; Ets-1

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Our aim was to investigate the interaction between human umbilical vein endothelial cells (HUVEC) and human bone marrow stromal cells (HBMSC). Cell differentiation analysis performed with different cell culture models revealed that alkaline phosphatase activity and type I collagen synthesis were increased only by the direct contact of HUVEC with HBMSC. This 'juxtacrine signaling' could involve a number of different heterotypic connexions that require adhesion molecules or gap junctions. A dye coupling assay with Lucifer yellow demonstrated a functional coupling between HUVEC and HBMSC. Immunocytochemistry revealed that connexin43 (Cx43), a specific gap junction protein, is expressed not only in HBMSC but also in the endothelial cell network and that these two cell types can communicate via a gap junctional channel constituted at least by Cx43. Moreover, functional inhibition of the gap junction by 18a-glycyrrhetinic acid treatment or inhibition of Cx43 synthesis with oligodeoxyri-bonucleotide antisense decreased the effect of HUVEC cocultures on HBMSC differentiation. This stimulation could be mediated by the intercellular diffusion of signaling molecules that permeate the junctional channel. coupling; intercellular messenger; connexin43; osteoblast; endothelial cells

HC[O.sup.-.sub.3]-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HC[O.sub.3] transport have not been reported. Immunoblotting for CFTR showed a single band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during [Cl.sup.-] influx in the absence and presence of forskolin (FSK). Apical and basolateral [Cl.sup.-] permeability increased 10- and 3-fold, respectively, in the presence of 50 [micro]M FSK. FSK-activated apical chloride permeability was unaffected by [H.sub.2]DIDs (250 [micro]M); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 [micro]M) and glibenclamide (100 [micro]M) inhibited activated [Cl.sup.-] fluxes by 45% and 30%, respectively. FSK-activated basolateral [Cl.sup.-] permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by [H.sub.2]DIDS. HC[O.sup.-.sub.3] permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HC[O.sup.-.sub.3]]. FSK (50 [micro]M) increased apical HC[O.sup.-.sub.3] permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HC[O.sup.-.sub.3] permeability was unaffected by FSK. Genistein (50 [micro]M) significantly increased apical HC[O.sup.-.sub.3] and [Cl.sup.-] permeability by 1.8- and 16-fold, respectively. When 50 [micro]M genistein was combined with 50 [micro]M FSK, there was no further increase in [Cl.sup.-] permeability; however, HC[O.sup.-.sub.3] permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial [Cl.sup.-] and HC[O.sup.-.sub.3] transport. Furthermore, there is a cAMP-activated [Cl.sup.-] pathway on the basolateral membrane that is not CFTR. cornea; endothelium; chloride permeability; MEQ; bicarbonate permeability; intracellular pH; BCECF; forskolin; cAMP; genistein

Go, Young-Mi, Anna-Liisa Levonen, Douglas Moellering, Anup Ramachandran, Rakesh P. Patel, Hanjoong Jo, and Victor M. Darley-Usmar. Endothelial NOS-dependent activation of c-Jun N[H.sub.2]-terminal kinase by oxidized low-density lipoprotein. Am J Physiol Heart Circ Physiol 281: H2705--H2713, 2001.--Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun N[H.sub.2]-terminal kinase (JNK), also known as stressactivated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL. nitric oxide synthase; NAD(P)H oxidase; nitric oxide; extracellular signal-regulated kinase Received 10 May 2001; accepted in final form 27 August 2001

There is extensive ultrastructural evidence in endothelium for the presence of chained vesicles or clusters of attached vesicles, and they are considered to be involved in specific transport mechanisms, such as the formation of trans-endothelial channels. However, few details are known about their mechanical characteristics. In this study, the formation mechanism and mechanical aspects of vascular endothelial chained vesicles are investigated theoretically, based on membrane bending strain energy analysis. The shape of the axisymmetric vesicles was computed on the assumption that the cytoplasmic side of the vesicle has a molecular layer or cytoskeleton attached to the lipid bilayer, which induces a spontaneous curvature in the resting state. The bending strain energy is the only elasticity involved, while the shear elasticity is assumed to be negligible. The surface area of the membrane is assumed to be constant due to constant lipid bilayer thickness. Mechanically stable shapes of chained vesicles are revealed, in addition to a cylindrical tube shape. Unfolding of vesicles into a more flattened shape is associated with increase in bending energy without a significant increase in membrane tension. These results provide insights into the formation mechanism and mechanics of the chained vesicle.

The hypothesis that endothelial cell regrowth, morphology and endothelium-dependent vasoreactivity after catheter injury are improved in the diabetic rabbit compared to the euglycemic rabbit was tested. The percent endothelial regrowth was significantly increased in diabetic animals compared with euglycemic animals two weeks after catheter injury. The results suggest improved endothelial cell regrowth and morphology in diabetic animals compared to euglycemic animals.

The hypothesis that hyaluronan might be involved in the establishment of the permeation properties of the apical surface glycocalyx was tested in vivo. Hamster microvessels in the cremaster muscle were visualized by means of video microscopy. The results showed that cell surface hyaluronan has a role in regulating permeation of the apical glycocalyx to macromolecules which suggest that hyaluronan and other glycoconjugates are needed for the assembly of the matrix on the endothelial surface.

A model of in vivo short-term hyperoxia was used to characterize the protective effects of substrate attachment. Reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic was shown by culture of hyperoxic type 2 alveolar epithelial cells (AEC2) on various biological adhesion substrates. Increased activation of extracellular signal-regulated kinase was seen in hyperoxic AEC2 as soon as the cells started to attach laminin and was sustained after 24 hours of culture in contrast to that in control AEC2.

The ability of hydrogen peroxide (H2O2) to stimulate tyrosine phosphorylation of focal adhesion proteins in vascular endothelial cells (ECs) was examined. The data gathered indicated that H2O2 induced both focal adhesion kinase (FAK) and paxillin tyrosine phosphorylation through tyrosine phosphatase inhibition. The results showed that reactive oxygen species stimulated tyrosine phosphorylation of FAK and paxillin in vascular ECs. However, the increase in tyrosine phosphorylation of FAK appeared to be insensitive to tyrosine kinase inhibition.

The successful transduction of human heme oxygenase (HO)-1 gene into rat lung microvessel endothelium using replication-defective retroviral vector was reported. A 2.1-fold increase in HO-1 protein level was exhibited by cells transduced with human HO-1 gene. The results showed that the induction of HO-1 in response to injurious stimuli represents an important mechanism for moderating the severity of cell damage. Clinical applications loom as a possibility if HO activity is regulated in this manner.

The hypothesis that differences in enzyme regulation of cyclic adenylic acid (cAMP) synthesis and degradation uniquely establish an elevated content in pulmonary microvascular endothelial cells (PMVECs) was tested. This was because the mechanism that enables PMVECs to form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells was unknown. The results showed that suppressed calcium entry and low adenosine triphosphate-to-cAMP conversion intrinsincally influenced calcium sensitivity.

Ovine fetal pulmonary artery endothelial cells were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16 in an effort to establish an immortalized cell line. This is because investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. The results showed that immortalized pulmonary artery endothelial cells will provide an excellent model for further studies of endothelial nitric oxide synthase gene expression and function in fetal pulmonary endothelium.

Oxidized low density lipoprotein (LDL) is a key factor in the pathogenesis of atherosclerosis and its thrombotic complications, such as stroke and myocardial infarction. It activates endothelial cells and platelets through mechanisms that are largely unknown. Here, we show that lysophosphatidic acid (LPA) was formed during mild oxidation of LDL and was the active compound in mildly oxidized LDL and minimally modified LDL, initiating platelet activation and stimulating endothelial cell stress-fiber and gap formation. Antagonists of the LPA receptor prevented platelet and endothelial cell activation by mildly oxidized LDL. We also found that LPA accumulated in and was the primary platelet-activating lipid of atherosclerotic plaques. Notably, the amount of LPA within the human carotid atherosclerotic lesion was highest in the lipid-rich core, the region most thrombogenic and most prone to rupture. Given the potent biological activity of LPA on platelets and on cells of the vessel wall, our study identifies LPA as an atherothrombogenic molecule and suggests a possible strategy to prevent and treat atherosclerosis and cardiocerebrovascular diseases.

A study was conducted to analyze whether low shear stress-induced nuclear factor-KB (NF-KB) in human aortic endothelial cells enhances the expression of vascular cell adhesion molecule-1 (VCAM-1) and whether the blockade of NF-KB activation inhibits low shear stress-induced VCAM-1 expression and related endothelial monocyte adhesion. Results indicated that NF-KB supports a critical function in low shear-induced VCAM-1 expression.

A study was conducted to analyze mechanisms surrounding the metabolic adaptation of endothelial cells to substrate deprivation. The cells were plated and cultured after isolation from slaughterhouse material. Reverse-phase high performance liquid chromatography was then carried out to measure nucleoside and nucleobase releases of perfused endothelial cells. Results indicated that porcine aortic endothelial cells are capable of recovering from extended periods of substrate deprivation.

The involvement of protein kinase C (PKC), phospholipase A2 (PLA2), calcium, and oxidants in cytokine induction of HO-1 were examined. Acute exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated HO-1 mRNA. However, prolonged exposure blocked induction of HO-1 mRNA by IL-1 alpha and TNF-alpha. The results showed that TNF-alpha and IL-1 alpha induction of HO-1 required PKC-mediated phosphorylation and PLA2 activation as well as oxidant generation.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin H and the angiostatin binding protein as the [Alpha]/[Beta]-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant [Alpha]-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-[Alpha]-subunit ATP synthase antibody. Binding of angiostatin to the [Alpha]/[Beta]-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

The hypothesis that uridine 5'-triphosphate (UTP) plays a more important role in endothelial mitogenesis and chemotaxis than does adenosine triphosphate (ATP) is and that UTP is angiogenic is studied. The results from cultured endothelial cells showed that both vascular endothelial growth factor and UTP were significant chemotactic and mitogenic factors compared to ATP which demonstrated no significant chemotaxis in pig cardiac vasculature.

The hypothesis that increased flow through coronary arterioles increases endothelial cell nitric oxide synthase (ecNOS) and copper/zinc superoxide dismutase (Cu/Zn SOD) mRNA expression is tested. Single porcine coronary arterioles were cannulated, perfused and exposed to intraluminal flow. The results showed that ecNOS and SOD mRNA expression is regulated by flow in porcine coronary arterioles. In addition, the shear stress and threshold level of flow must be sustained to elicit the upregulation of SOD mRNA expression and ecNOS.

The process by which glutamine is transported into sinusoidal endothelial cells was investigated by functionally characterizing the transport of L-glutamine in hepatic endothelial cells isolated from male rats. Functional analyses, kinetics, cation substitution and amino acid inhibition showed that a Na+-dependent carrier different from system N in parenchymal cells mediated most of the glutamine transport in hepatic endothelial cells.

The role of isometric contraction in initiating a signaling pathway similar to that elicited by fluid shear stress was studied. Whether the process had a role in enhanced endothelial NO production was also taken into account. The results showed that the application of a NOS inhibitor to endothelium-intact rabbit aortic rings elicited a major additional increase in tension dependent on the level of vascular smooth muscle precontraction. Isometric contraction was found to be a determinant for the synthesis of NO in arteries.

The possible role of activated extracellular signal-regulated kinases (ERK) 1 and 2 in enhanced proliferation and morphological change induced by strain is studied using bovine aortic endothelial cells subjected to an average of 6% or 10% strain at a rate of 60 cycles per minute for up to four hours. Findings suggest that ERK1 and ERK2 are not critically involved in the strain-induced proliferation and orientation and that strain-dependent activation of ERK1 and ERK2 is independent of intracellular and extracellular calcium mobilization.

The possibility that orosomucoid was synthesized in endothelial cells was explored using primary cultures of human microvascular endothelial cells from dermal tissue. Human liver controls were used as positive controls and total RNA was prepared from both cell types. Findings indicate that endothelial cells normally produce orosomucoid, which is essential for capillary charge selectivity. It is possible that orosomucoid exerts its effect by interacting with other components of the endothelial glycocalyx.

A study was conducted to analyze the influence of endogenous and exogenous adrenergic stimulation on endotoxin-induced lung neutrophil accumulation and activation. Experimental results indicated that lung neutrophils were activated to a greater extent after endotoxemia than peripheral blood neutrophils. Findings also showed increases in staining for interleukin-1-beta in lung neutrophils and endothelial cells.

A study was conducted to test whether vascular endothelial growth factor (VEGF) released from hypoxia-exposed Hep G2 cells changes expression of kinase insert domain-containing receptor and fms-like tyrosine kinase 1 in human umbilical venous endothelial cells. Cells were cultured in plastic culture dishes and flasks. Results indicated that hypoxia-exposed Hep G2 cells evolve paracrine production of flt-1 through VEGF-independent mechanisms.

A study was conducted to investigate limb reduction defects in endothelial nitric oxide synthase-deficient mice. Reverse transcription polymerase chain reaction was utilized to determine a cDNA fragment in kidney RNA. Southern blot analysis was then carried out to examine DNA from clones. Experimental results indicated that a deficiency in endothelial nitric oxide synthase is a necessary component to cause limb deficiency in the mouse embryo.

A study was conducted to analyze the time course of events associated in splanchnic artery occlusion and reperfusion (SAO/R). Results showed endothelial dysfunction as an early event in the pathophysiology of SAO/R. The endothelial dysfunction further supported increases in the surface expression of P-selectin and adherence of neutrophils to endothelium supporting the accumulation of neutrophil in the splanchnic viscera.

A study was conducted to examine heart and lung vascular endothelial growth factor (VEGF) mRNA expression in two rat models of pulmonary hypertension supporting exposure to chronic hypoxia. Pulmonary vascular remodeling and myocardial capillary density were also analyzed. Results indicated that a sustained increase in VEGF expression in the hypertrophied right ventricle during chronic hypoxia may support increases in the number of capillaries per myocyte.

Research was conducted to examine the role a cytoplasmic component (CT) of an adhesion receptor molecule plays in inflammatory responses. The data suggest that CT of the adhesion receptor P-selectin in endothelial cells is important to this process.

A study was conducted to bovine aortic endothelial cells (BAEC) support a G protein-dependent form of phosphatidylinositol 3-kinase, PI3K. BAEC were determined from thoracic aortas that were maintained in a growth medium while soluble lysates from BAEC were incubated with a vsv antibody. Experimental results indicated that shear stress influenced the activity of a G protein-sensitive form of PI3K, PI3K-gamma.

A study was conducted to test whether atrial natriuretic peptide clearance receptor participates in modulating endothelial permeability. Endothelial cell monolayer of rat coronary endothelium was utilized to determine albumin flux. Results indicated that the ANP-C receptor influenced permeability decreases in coronary artery endothelial cell monolayers. Findings also showed that isoproterenol had no effect on cGMP.

A study was conducted to characterize a unique dual-radiolabeled monoclonal antibody method that can be utilized to determine regional P-selectin expression in vivo in a murine framework of myocardial ischemia-reperfusion (MI/R). The Iodo-Gen method was utilized to determine radiolabeled P-selectin and control MAbs. Results indicated an in vivo endothelial expression of P-selectin after MI/R. Findings also suggested that a deficiency in P-selectin may not support cardioprotection during extended periods of coronary artery occlusion.

A study was conducted to develop a way of transfecting endothelial cells supporting proteins for functional analysis. Bovine coronary venular endothelial cells were determined from postcapillary venules while cells were lysed in a cell lysis buffer. Experimental results indicated that the microvascular endothelium associated with various chemical stimuli and physical stress through an alteration of its role and structure.

Results of a study showed that thrombin does not alter total type 1 phosphatase (PPase 1) activity in endothelial cell homogenates but rather decreases myosin-associated PPAse 1 activity. Similar to active myosin-associated PPase 1, thrombin-inducible Ca2+/calmodulin-dependent PPase 2B was found to be possibly involved in agonist-mediated endothelial cell activation. In contrast to smooth muscle contraction, thrombin-mediated endothelial cell contraction-relaxation could require activation of a complex PPase cascade consisting of PPases 1 and 2B.

It has been shown that plymorphonuclear leukocyte (PMN) adherence to endothelial cells leads to transient increases in EC cytosolic free calcium concentration (Ca(super2+)). Ca(super2+) was measured in Fura2-loaded human EC monolayers to determine whether stimulation of Ca(super2+) changes in Ec by leukocytes was induced by molecules that mediate leukocyte adherence to EC. Endothelial selectins and vascular cell adhesion moelcule-1 mediate endothelial stimulation though leukocyte adhesion.

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone-ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of anglogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone-ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.