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Results for “Endothelialization”

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At least 163 records · Page 9Linked to original sources

PubMed2002

NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells.

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.

Adolescent↗

PubMed1999

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.

Amino Acid Sequence↗

PubMed2001

Triptolide inhibits vascular endothelial growth factor expression and production in endothelial cells.

AIM: To investigate the effects of triptolide on vascular endothelial growth factor (VEGF) expression and secretion by endothelial cells, and explore the mechanism of anti-proteinuric effect of triptolide on glomerulonephritis. METHODS: A human umbilical endothelium derived cell line (ECV-304) from American Type Culture Collection (ATCC) was used in this study. The effects of triptolide on VEGF mRNA expression, production, and secretion induced by 12-o-tetradecanoyl-phorbol-13-acetate (TPA) were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and enzyme linked immunosorbent assay (ELISA) respectively. The endothelial c-fos/c-jun mRNA expression were also detected by RT-PCR after treatment of triptolide. RESULTS: VEGF mRNA expression was markedly up-regulated by TPA-stimulation. In addition, the production and secretion of VEGF in endothelial cells also increased in TPA treated cells. It was founded that triptolide inhibited VEGF mRNA expression, protein production and secretion in endothelial cells induced by TPA. Interestingly, TPA-induced c-fos/c-jun mRNA expression in endothelial cells was also inhibited by triptolide. CONCLUSION: Triptolide is a potent inhibitor of VEGF expression and production in endothelial cells. The inhibitory effects of triptolide on VEGF expression and production can contribute to its anti-proteinuric effect on glomerulonephritis. Down-regulation of c-fos/c-jun expression in endothelial cells by triptolide is one of the mechanisms of the inhibitory effect of triptolide on VEGF expression.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Perturbation of endothelial junction proteins by Staphylococcus aureus alpha-toxin: inhibition of endothelial gap formation by adrenomedullin.

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell-cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus alpha-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus alpha-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, alpha-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked alpha-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after alpha-toxin application. Although no RhoA activation was noted after endothelial alpha-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.

Adrenomedullin↗

PubMed1998

Uptake of lithium carmine by sinusoidal endothelial and Kupffer cells of the rat liver: new insights into the classical vital staining and the reticulo-endothelial system.

Sinusoidal cells in the rat liver were studied in vivo and in vitro using the original vital staining with lithium carmine, which has contributed much to the development of the concept of the reticulo-endothelial system. Immunohistochemical and electron-microscopic studies revealed that the dye-incorporating cells were sinusoidal endothelial cells, Kupffer cells, and monocytes. The endothelial cells took up much more dye than did the Kupffer cells and bulged largely into the sinusoidal lumen. Electron microscopy revealed that small particles of lithium carmine were associated with coated vesicles of endothelial cells and ruffled membranes of Kupffer cells. In the endothelial cells, these particles were present in various concentrations within vacuolated structures and condensed in the lysosomes forming large aggregates of lithium carmine lumps. These lumps showed crystalline structures, within which the size of the individual particle was up to 30 nm in width and 50 nm in length. A few endothelial cells containing abundant dye underwent degeneration, and some were taken up by Kupffer cells. Liver endothelial cells isolated from lithium carmine-administered rats endocytosed fluorescence-labeled collagen. Isolated endothelial cells from normal rat liver, when cultured with lithium carmine, did not take up any dye, and their endocytosis of formaldehyde-treated albumin was inhibited dose-dependently. We conclude that in the liver, endothelial cells, but not Kupffer cells, predominantly take up lithium carmine. Furthermore, we propose the existence of a generalized cell system based on its vital staining capacity.

Animals↗

PubMed1985

Influx of [3H,14C]cholesterol-labelled lipoprotein into re-endothelialized and de-endothelialized areas of ballooned aortas in normal-fed and cholesterol-fed rabbits.

The entry of [3H]- and [3H,14C]cholesterol-labelled lipoprotein into de-endothelialized and re-endothelialized areas of balloon-injured rabbit aortas was studied in normal-fed and cholesterol-fed rabbits. Studies were carried out 11-15 weeks after the initial injury when endothelial regeneration involved approximately half of the aortic area. The entry into the aorta of 3H-labelled free and ester cholesterol in lipoprotein over a 72-h period was studied following the ingestion of a single dose of 3H-labelled cholesterol. The entry of double labelled [3H,14C]cholesterol-labelled lipoprotein was also studied over a 6-h period following the injection of plasma from donor rabbits. The accumulation of cholesterol and cholesterol ester in the aorta in both the normal- and cholesterol-fed rabbits was significantly greater for the re-endothelialized (white) areas than for the de-endothelialized (blue) areas or the sham-operated aortas. Where the rabbits were cholesterol-fed 4-10 times the amount of cholesterol accumulated in re-endothelialized intima compared to normal intima. Both entry (micrograms/day/100 mg wet weight aortic intima) and clearance (mu 1 plasma/day/cm2) of free and ester cholesterol were increased in the neointima compared with the normal intima for both normal-fed and cholesterol-fed rabbits. Hydrolysis of cholesterol ester occurred in the neointima and was greater than in the corresponding de-endothelialized area but less than for the sham-operated intima. Synthesis of cholesterol ester was minimal in all areas. Removal of labelled cholesterol and cholesterol ester from the intima during a 20-h efflux period following the initial 72-h loading period indicated that for aortas of both normal-fed and cholesterol-fed rabbits, there was greater removal for normal intima than for either re-endothelialized or de-endothelialized intima. However, no clear difference between the blue and white areas was observed. It is concluded that the accumulation of cholesterol in neointima after balloon injury is associated with a marked increase in permeability to lipoprotein of the neointima as well as to possible binding of lipoprotein to glycosaminoglycan in the artery.

Animals↗

PubMed2004

Optimizing endothelial nitric oxide activity may slow endothelial aging.

The capacity of vascular endothelium to generate bioactive nitric oxide (NO) decreases with advancing age, even in healthy subjects with a relatively benign risk factor profile; this phenomenon may reflect decreased expression of NO synthase, as well as increased production of superoxide, and evidently contributes importantly to the increased vascular risk associated with aging. Studies with cultured endothelial cells suggest that the rate of endothelial aging is determined primarily by the rate of cell turnover and the associated progressive shortening of telomeres; endothelial cells transfected with the catalytic subunit of telomerase--which preserves a youthful telomere length--do not show a reduction in NO synthase expression after numerous doublings, in contrast to the marked reduction observed in control cells. Also consistent with this view is the fact that, following balloon denudation of arteries, the regenerated endothelium makes less NO. In the vasculature of adults, the rate of endothelial cell mitosis is evidently a reflection of the rate of endothelial cell apoptosis. Numerous cell culture studies demonstrate that physiological levels of NO protect endothelial cells from apoptosis induced by a wide range of noxious stimuli--including vascular risk factors such as oxidized LDL, angiotensin II, and hyperglycemia. In the human vasculature, endothelial cells with disproportionately short telomeres are found capping atheromatous lesions and in atheroma-prone areas where blood flow is turbulent; these findings evidently reflect increased endothelial cell turnover in regions where NO bioactivity is relatively weak. It can be deduced that lifelong adherence to an "endotheliophilic lifestyle" that optimizes vascular NO production, while minimizing that of superoxide, will literally slow the rate of aging of vascular endothelium, such that, at any given advanced age, the optimal functional capacity of the vascular endothelium will be superior to that of age-matched controls. These considerations underline the desirability of actively promoting vascular health in younger and middle-aged individuals in whom risk for vascular events may still be quite low. The impact of lifelong caloric restriction on endothelial aging requires further study, preferably in primates.

Administration, Oral↗

PubMed2000

ATP-dependent astrocyte-endothelial calcium signaling following mechanical damage to a single astrocyte in astrocyte-endothelial co-cultures.

In the brain, endfeet of perivascular astrocytes make close contact with capillary endothelial cells that form the blood-brain barrier. The aim of the present work was to investigate whether and how calcium signals can be communicated from astrocytes to endothelial cells following acute mechanical cell damage. The experiments were performed on astrocyte-endothelial co-cultures prepared from primary rat brain astrocytes and an endothelial cell line (ECV304). A single astrocyte was acutely damaged by mechanical stimulation of sufficient strength with a micropipette, and the resulting cytoplasmic calcium changes were monitored using fura-2 and digital calcium imaging. Mechanical damage to a single astrocyte triggered a large intercellular calcium wave that propagated to surrounding astrocytes and also to even remotely located (several hundred micrometers) endothelial cells. Astrocyte-endothelial calcium waves induced by mechanical cell damage were largely deflected by fast superfusion, were able to cross a cell-free lane, were dose-dependently inhibited by suramin, a P2-purinoceptor blocker, and were largely reduced in size in the presence of the ATP-degrading enzyme apyrase. Our results indicate that mechanical damage to a single astrocyte can produce far reaching calcium signals that are propagated by the release of a calcium mobilizing P2-purinergic agonist and that can be communicated to endothelial cells. As endothelial cytoplasmic calcium is an important factor in the regulation of blood-brain barrier permeability and transport, mechanical cell damage-induced astrocyte-endothelial calcium signals are hypothesized to play a role in the initiation of brain edema and the stimulation of brain glucose uptake.

Adenosine Triphosphate↗

PubMed2005

Ketamine reduces nitric oxide biosynthesis in human umbilical vein endothelial cells by down-regulating endothelial nitric oxide synthase expression and intracellular calcium levels.

OBJECTIVE: Ketamine, an intravenous anesthetic agent, can modulate vascular tone. Nitric oxide (NO), constitutively produced in endothelial cells, contributes to vasoregulation. In this study, we attempted to evaluate the effects of ketamine on NO biosynthesis in human umbilical vein endothelial cells and its possible mechanism. DESIGN: Controlled laboratory study SETTINGS: Research laboratory in a universal hospital. SUBJECTS: Human umbilical vein endothelial cells prepared from human umbilical cord veins were exposed to 1, 10, 100, and 1000 microM ketamine for 1, 6, and 24 hrs. MEASUREMENTS AND MAIN RESULTS: Exposure to 1, 10, and 100 microM ketamine for 1, 6, and 24 hrs was not cytotoxic to human umbilical vein endothelial cells. However, ketamine at 1000 microM significantly caused cell apoptosis. A therapeutic concentration of ketamine (100 microM) time-dependently reduced the levels of nitrite in human umbilical vein endothelial cells. Immunoblot analysis revealed that ketamine time-dependently decreased endothelial NO synthase protein production in human umbilical vein endothelial cells. Results of an assay by reverse-transcription polymerase chain reaction showed that ketamine significantly inhibited levels of endothelial NO synthase messenger RNA. Ketamine time-dependently reduced bradykinin-enhanced intracellular calcium concentrations. Analysis by confocal microscopy further demonstrated the suppressive effects of ketamine on bradykinin-induced calcium mobilization. CONCLUSIONS: A clinically relevant concentration of ketamine can reduce NO biosynthesis. The suppressive mechanisms occur not only by pretranslational inhibition of eNOS expression but also by a posttranslational decrease in endothelial NO synthase activity due to a reduction in intracellular calcium levels.

Analgesics↗

PubMed1995

Human endothelial cells bioactivate organic nitrates to nitric oxide: implications for the reinforcement of endothelial defence mechanisms.

Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemoglobin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 0.1-100 mumol L-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2.97 +/- 0.98 pmoles NO min-1 x mg protein with 100 mumol L-1 GTN; n = 8) were similar to those elicited upon challenge of the cells with 100 nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with consequent increases in the intracellular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100 mumol L-1) nitrate bioactivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.

Biotransformation↗

PubMed1998

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells.

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.

Adult↗

PubMed2006

CRP promotes monocyte-endothelial cell adhesion via Fcgamma receptors in human aortic endothelial cells under static and shear flow conditions.

Monocyte-endothelial cell adhesion is a key early event in atherogenesis. C-reactive protein (CRP), a cardiovascular risk marker, is known to stimulate ICAM and VCAM in human aortic endothelial cells (HAEC) and induces monocyte-endothelial cell adhesion. In this study, we examined the mechanisms by which native CRP promotes monocyte-endothelial cell adhesion under static conditions and tested the effect of CRP on adhesion under shear flow. Incubation of HAEC with CRP (>25 microg/ml) upregulated NF-kappaB activity, and this resulted in a significant increase in ICAM (54% increase, P<0.001), VCAM (41% increase, P<0.01), and monocyte-endothelial cell adhesion (44% increase, P<0.02) compared with those of control. Preincubation with antibodies to CD32 and CD64 but not CD16 effectively inhibited this activation. Blocking NF-kappaB activity with inhibitors or a dominant negative inhibitory kappaB significantly decreased ICAM, VCAM upregulation, and subsequent monocyte-endothelial cell adhesion. Preincubation with antibodies to CD32 and CD64 or transient transfection with small interference RNA to CD32 attenuated CRP-induced NF-kappaB activity, ICAM, VCAM, and monocyte-endothelial cell adhesion under static conditions. Also, the Syk kinase inhibitor piceatannol and MG-132, a proteasome degradation inhibitor, produced similar attenuation in NF-kappaB activity, ICAM, VCAM, and adhesion. Furthermore, CRP-activated endothelial cells supported monocyte rolling, arrest, and transmigration in shear flow (2 dyn/cm2), and this was also inhibited by preincubation with antibodies to CD32 and CD64. Thus, in HAEC, CRP upregulates monocyte-endothelial adhesion by activation of NF-kappaB through engaging the Fcgamma receptors CD32 and CD64.

Antibodies↗

PubMedDate not supplied

Evidence that an endothelial cytosolic protein binds to the 3'-untranslated region of endothelial nitric oxide synthase mRNA.

Changes in the endothelial nitric oxide synthase (eNOS) expression could be involved in the endothelium-dependent vasorelaxing dysfunction associated with cardiovascular diseases. We have recently demonstrated the existence of endothelial cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In the present work, we have characterized the cytosolic proteins that bind to 3'-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically bound to 3'-UTR eNOS mRNA as determined by a cross-linking assay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The endothelial cytosolic protein recognized a cytidine (C)-rich region within 3'-UTR eNOS mRNA. Furthermore, tumor necrosis factor-alpha (TNF-alpha) increased the level of the 60-kD endothelial cytosolic protein. In addition, TNF-alpha reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide. Cycloheximide also prevented the binding activity of the endothelial cytosolic protein to 3'-UTR eNOS mRNA. In summary, these data suggest that a 60-kD endothelial cytosolic protein binds to 3'-UTR eNOS mRNA. TNF-alpha increased the 60-kD protein levels. Cycloheximide prevented the binding activity of the cytosolic protein to 3'-UTR eNOS mRNA related to TNF-alpha; this effect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosolic protein in the regulation of eNOS mRNA stabilization and in the endothelial dysfunction associated with cardiovascular diseases.

3' Untranslated Regions↗

PubMed2003

Endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface.

OBJECTIVE: A thick endothelial glycocalyx provides the endothelial surface with a nonadherent shield. Oxidized LDL (Ox-LDL) degrades the endothelial glycocalyx. We hypothesized that glycocalyx degradation stimulates leukocyte-endothelial cell adhesion, whereas intravascular supplementation with sulfated polysaccharides reconstitutes the endothelial glycocalyx and attenuates Ox-LDL-induced leukocyte-endothelial cell adhesion. METHODS AND RESULTS: Degradation of the endothelial glycocalyx by local microinjection of heparitinase (10 to 50 U/mL) into mouse cremaster venules dose-dependently increased the number of adherent leukocytes. Systemic administration of Ox-LDL (0.4 mg/100 g body weight) induced 10.1+/-0.9 adherent leukocytes/100 microm at 60 minutes. In the venules perfused with 500-kDa dextran sulfate (1 mg/mL), the number of adherent leukocytes at 60 minutes after Ox-LDL bolus application was not influenced (9.2+/-1.0 leukocytes/100 microm). However, the venules locally perfused with heparan sulfate (10 mg/mL) or heparin (1 mg/mL) displayed a significantly lower number of adherent leukocytes induced by Ox-LDL: 5.1+/-0.7 and 5.4+/-0.9 leukocytes/100 microm, respectively (P<0.05). Fluorescently labeled heparan sulfate and heparin, but not dextran sulfate, attached to the venule luminal surface after Ox-LDL administration. CONCLUSIONS: Endothelial glycocalyx degradation stimulates leukocyte immobilization at the endothelial surface. Circulating heparan sulfate and heparin attach to the venule wall and attenuate Ox-LDL-induced leukocyte immobilization.

Abdominal Muscles↗

PubMed2006

Endothelial cell-restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury.

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

Animals↗

PubMed1992

Endothelial cell senescence inhibits unidirectional endothelialization in vitro.

We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between cell-adhesion and noncell-adhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endothelialization.

Animals↗

PubMed1975

Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening.

A new model was developed to study endothelial regeneration and the effects of endothelial denudation in large arteries. Complete endothelial loss was produced in a sharply defined, unbranched segment of the rat common carotid artery by brief drying with a gentle stream of air along the lumen of the vessel. Platelets became attached to the denuded surface, but no polymorphonuclear or mononuclear leukocytic adherence or infiltration was observed. Regeneration occurred by the ingrowth, from each end of the denuded segment, of sheets of rapidly dividing endothelial cells. Endothelial replacement was complete by 7 to 10 days after drying. It was also noted that, by 14 days after drying, a striking myointimal thickening developed in the central region of the denuded segment. This central region was the last area to be covered with new endothelium; while still denuded, the region showed edema and signs of cellular proliferation in the innermost medial layer. The myointimal thickening consisted of smooth muscle-like cells in a fibroelastic stroma in the deeper zones, and a layer of rounded, relatively undifferentiated cells in the superficial zones. Progressive regression and condensation of the thickening was observed between 14 days and 3 months. In this model, the clear demarcation and completeness of endothelial denudation in the arterial segment allow study of the over-all process of endothelial replacement, avoiding the confusion imposed by islands of residual endothelium or contributions from branch vessels. The results suggest that the new endothelium in this situation is derived from each end of the segment. In addition, because the method of inducing denudation does not cause significant damage to the underlying media (as shown by the absence of inflammatory cell attachment or infiltration), the model offers a unique opportunity to study the relationship between endothelial loss and changes in the vessel wall. The correlation between the duration of endothelial denudation and the extent of myointimal thickening suggests that sustained insudation of certain, as yet undefined, factors from the lumen may provoke cellular proliferation in the arterial wall. Further investigation of this model should provide information relevant to the pathogenesis of the fibromuscular thickenings of human atherosclerosis.

Animals↗

PubMed1992

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.

Antigens, CD↗