Monitoring oxidative stress in vascular endothelial cells in response to fluid shear stress: from biochemical analyses to micro- and nanotechnologies.

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherosclerosis. Shear stress induces vascular oxidative stress via the activation of membrane-bound NADPH oxidases present in vascular smooth muscle cells, fibroblasts, and phagocytic mononuclear cells. Shear stress acting on the endothelial cells at arterial bifurcations or branching points regulates both NADPH oxidase and nitric oxide (NO) synthase activities. The former is considered a major source of oxygen-centered radicals (i.e., superoxide anion [O2(.-)]) that give rise to oxidative stress; the latter is a source of nitrogen-centered radicals (i.e., nitric oxide [NO]) that give rise to nitrative/nitrosative stress. In addition to conventional biochemical analyses, the emerging microelectromechanical systems (MEMS) provide spatial and temporal resolutions to investigate the mechanisms whereby the characteristics of shear stress regulate the biological activities of endothelial cells at the complicated arterial geometry. In parallel, the development of MEMS liquid chromatography (LC) provides a new venue to measure circulating oxidized low-density lipoprotein (ox-LDL) particles as a lab-on-a chip platform. Nanowire-based field effect transistors further pave the way for a high throughput approach to analyze the LDL redox state. Integration of MEMS with oxidative biology is synergistic in assessing vascular oxidative stress. The MEMS LC provides an emerging lab-on-a-chip platform for ox-LDL analysis. In this context, this chapter has integrated expertise from the fields of vascular biology and oxidative biology to address the dynamics of inflammatory responses.

Angiopoietin-2 levels in the hepatic vein as a useful predictor of tumor invasiveness and prognosis in human hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is characteristically a hypervascular tumor and its progression is known to be closely related to angiogenesis. In this study, we investigated angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) levels in the hepatic vein draining from HCC, as well as in the peripheral vein, to evaluate their relation to clinicopathological features and prognosis. METHODS: To obtain hepatic venous blood samples, a catheter was placed into the main branch of the hepatic vein draining from HCC in 21 patients. The Ang-2 and VEGF levels in both the hepatic and peripheral veins were investigated. Furthermore, Ang-2 mRNA expression in surgically resected HCC was evaluated by quantitative reverse-transcription polymerase chain reaction (RT-PCR), as well as microvessel density (MVD) by CD34 immunostaining. RESULTS: Ang-2 levels in the hepatic vein significantly correlated with Ang-2 mRNA expression in HCC, but Ang-2 levels in the peripheral vein did not correlate. Furthermore, a significant correlation was found between hepatic venous Ang-2 and MVD levels, whereas there was no significant correlation with hepatic venous VEGF levels. When hepatic venous Ang-2 levels were compared with clinicopathological features, a significant relationship was found between high Ang-2 levels and portal vein invasion. The survival for patients in the high hepatic venous Ang-2 group was significantly poorer when compared with the low group. CONCLUSION: Preoperative hepatic venous Ang-2 levels may be a good predictor for portal vein invasion and also prognosis in patients with HCC.

Microvascular network in renal carcinomas. Quantitative and tissue microarray immunohistochemical study.

The aim of the study was to investigate differences in microvessels between renal tumors. The material consisted of 97 clear cell carcinomas (CCRCC), 20 papillary carcinomas (PapRCC), 33 chromophobe carcinomas and 15 oncocytomas (RO). The endothelia were stained immunohistochemically for CD34 antigen. The vascular features were analyzed with the AnalySIS image processing system. The stains for VEGF, GLUT-1 and Ki67 were performed on tissue microarrays. The mean microvascular density (MVD) was 163.62 profiles/mm2 and microvascular area (MVA) was 3.75%. The highest values were seen in CCRCC and the lowest in PapRCC. The size and shape parameters of the individual vessels were also different between the tumors under consideration. The tumor diameter, MVD and MVA were inversely correlated, the relationship being the strongest for RO. The minimum spanning tree parameters were different between histological types, especially between CCRCC and PapRCC. The mean fractal dimension was 1.32, and similar in all cases. VEGF, Ki67 and GLUT-1 expression was the highest in CCRCC, and lowest in RO. The vascular parameters were correlated with Ki67, GLUT-1 VEGF expression, tumor grade, and inversely correlated with tumor diameter. The relationships in each tumor type were slightly different.

Evidence for an age-related attenuation of cerebral microvascular antioxidant response to oxidative stress.

Effects of aging and oxidative stress were studied in cerebral microvessels and microvessel-depleted brain from 6-, 18-, and 24-month-old C57Bl/6J mice exposed to normoxia, 24 or 48 h hyperoxia, or 24 h hyperoxia followed by 24 h normoxia. Microvessels lacked smooth muscle and consisted predominantly of endothelium. Following exposure and isolation of microvessel and parenchymal proteins, Western blot analysis was performed for detection of cytosolic thioredoxin 1 (TRx 1) and mitochondrial thioredoxin 2 (TRx 2), protein carbonyl, and mitochondrial superoxide dismutase (MnSOD). Both microvessel and parenchymal TRx 1 levels were increased by hyperoxia; however, the microvascular response was limited and delayed in comparison to that of the parenchymal fraction. Whereas TRx 2 levels in microvessels were increased in older mice, irrespective of exposure condition, hyperoxia per se had little or no apparent effect. Parenchymal cells showed no age-related increase in TRx 2 level under normoxic conditions, but showed increased levels following hyperoxia. Microvessel MnSOD was lower than that in parenchymal cells, but increased with age under normoxia, and also was correlated with the duration of hyperoxia. Although hyperoxia augmented MnSOD levels in young (6 months) and middle-aged (18 months) animals, the response was less pronounced in microvessels from senescent, 24-month-old mice. Unlike microvessels, which showed a sustained age-related increase in MnSOD level under each exposure condition, parenchymal cells from normoxic mice showed no increase, and hyperoxia-induced elevations declined with prolonged 48 h exposure. These results indicate that the microvessel endothelium is (1) subjected to a more intense oxidative environment than neurons and glia and (2) is limited by aging in its ability to respond to oxidative insult.

Impact of hemorheological and endothelial factors on microcirculation.

Previous studies showed that endothelial alterations caused by physical stress worsened the hemorheological parameters mainly in patients affected by ischemic vascular diseases: major vascular alterations have been found in patients with very high endothelial dysfunction indexes: these indexes are given by the various substances produced by the endothelium, but it is very difficult to have a value which clearly identifies the real state of the endothelial alteration. The function of the NO, an endogenous vasodilator whose synthesis is catalyzed by NOs, can be determined by the Citrulline/Arginine ratio, which represents the level of activity of the enzyme. A very good index of the endothelial dysfunction is asymmetric dimethylarginine (ADMA), a powerful endogenous inhibitor of NOs; in fact several studies have demonstrated a strong relationship between ischemic vascular disease and high levels of plasmatic ADMA. Our recent studies on heart failure and on ischemic cerebrovascular diseases evaluate endothelial dysfunctions and hemorheological parameters.

Influence of extra corporeal circulation on myocardial oxygen tension: results of an animal model.

BACKGROUND: Experimental data have shown the potential risk of cellular damage of the myocardium during extra corporeal circulation (ECC). The influence of ECC on myocardial oxygen tension however remained unclear. Therefore, the influence of ECC on the oxygen tension in a beating heart was investigated. METHODS: In a pig animal model flexible pO2 microcatheters were positioned in the midmyocardium of the left ventricle and the skeletal muscle and tissue oxygen tension during ECC were monitored and compared with data of a control group without ECC. RESULTS: ECC and unload of the heart caused a significantly higher increase of myocardial pO2 than in a non-ECC control group. CONCLUSION: Our findings show the beneficial effect of ECC on myocardial pO2. This may support the use of ECC in coronary artery bypass grafting because the potential myocardial injury due to ECC is not related to myocardial ischemia. On the contrary, myocardial pO2 was even increased during extracorporeal circulation in this study.

Microcirculatory detection of Toll-like receptor 4 in rat pancreas and intestine.

This paper was aimed to detect Toll-like receptor 4 (TLR4) microcirculatory expression and localization in rat pancreas and intestine. Acute pancreatitis (AP) was induced by twice injections of cerulein (20 mug in total) and acute necrotizing pancreatitis (ANP) was induced by intraductal injection of 5% taurocholate (1 ml/kg.bw). Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to detect and localize TLR4 in the pancreas and intestine. Results showed that RT-PCR of RNA isolated from pancreatic and intestinal tissue yielded the predicted amplicon for TLR4; IHC analysis localized TLR4 expression to the endothelium of pancreatic arteriole, venule, acinar capillary network and sinusoidal capillary of endocrine islet; TLR4 expression in intestine was principally in the microvascular endothelium and leucocytes within the mucosa lamina propria. TLR4 staining in intestine was more intense in taurocholate-induced pancreatitis (TIP) than that in cerulein-induced pancreatitis (CIP). In conclusion, TLR4 could be detected in the pancreatic and intestinal microcirculation, suggesting TLR4 involved in the microcirculatory impairment in AP; the more intense intestinal TLR4 expression in TIP suggests a potential risk for secondary infection.

Purification and identification of mouse lung microvessel endothelial cell-derived chemoattractant for lung-metastasizing murine RAW117 large-cell lymphoma cells: identification as mouse monocyte chemotactic protein 1.

We reported recently that medium conditioned with mouse lung microvessel endothelial cells possessed chemotactic activity for a highly lung-metastatic variant (L17) of the RAW117 murine large-cell lymphoma cell line but not for the poorly metastatic parental cells (P) or a liver-metastasizing variant (H10). The chemotactic factor was purified to homogeneity by a five-step procedure involving hydrophobic interaction, Cibacron blue F3GA affinity, metal affinity, anion exchange, and reversed phase chromatography, followed by preparative gel electrophoresis. The purified material appeared as a single broad band when analyzed by SDS-PAGE, with an average molecular weight of 26,000. The factor was cleaved by cyanogen bromide treatment, and a partial amino acid sequence of one of the cleaved polypeptides proved identical to mouse monocyte chemotactic protein 1 (mMCP-1/JE). The amino acid composition of the factor also indicated similarity to mMCP-1/JE. Separately purified mMCP-1/JE significantly stimulated the chemotactic migration of RAW117 cells (L17 >> H10, P). When recombinant human monocyte chemotactic protein 1 was compared to the purified endothelial cell chemotactic factor as a chemoattractant, similar migratory responses were observed in the RAW117 sublines. The chemotactic activity for L17 cells was significantly reduced from lung microvessel endothelial cell-conditioned medium after treatment with anti-mouse MCP-1 antibody. In contrast, the migration-stimulating activity of liver sinusoidal endothelial cell-conditioned medium to H10 cells was not affected by anti-mouse MCP-1. A major function of mMCP-1/JE is to recruit monocytes to inflammatory sites, and our results suggest that mMCP-1/JE also facilitates lymphoma lung invasion and metastasis.

Stimulation of arteriogenesis in skeletal muscle by microbubble destruction with ultrasound.

BACKGROUND: The application of ultrasound to microbubbles in skeletal muscle creates capillary ruptures. We tested the hypothesis that this bioeffect could be used to stimulate the growth and remodeling of new arterioles via natural repair processes, resulting in an increase in skeletal muscle nutrient blood flow. METHODS AND RESULTS: Pulsed ultrasound (1 MHz) was applied to exposed rat gracilis muscle after intravenous microbubble injection. Capillary rupturing was visually verified by the presence of red blood cells in the muscle, and animals were allowed to recover. Ultrasound-microbubble-treated and contralateral sham-treated muscles were harvested 3, 7, 14, and 28 days later. Arterioles were assessed by smooth muscle alpha-actin staining, and skeletal muscle blood flow was measured with 15- micro m fluorescent microspheres. An approximately 65% increase in arterioles per muscle fiber was noted in treated muscles compared with paired sham-treated control muscles at 7 and 14 days after treatment. This increase in arterioles occurred across all studied diameter ranges at both 7 and 14 days after treatment. Arterioles per muscle fiber in sham-treated and untreated control muscles were comparable, indicating that the surgical intervention itself had no significant effect. Hyperemia nutrient blood flow in treated muscles was increased 57% over that in paired sham-treated control muscles. CONCLUSIONS: Capillary rupturing via microbubble destruction with ultrasound enhances arterioles per muscle fiber, arteriole diameters, and maximum nutrient blood flow in skeletal muscle. This method has the potential to become a clinical tool for stimulating blood flow to organs affected by occlusive vascular disease.

Thrombomodulin deficiency in human diabetic nerve microvasculature.

Human diabetic neuropathy is multifactorial in etiology, with ischemia as a final common pathology. Although impaired vascular endothelial cell function in diabetic microvascular injury is established, the role of thrombomodulin (TM)-dependent protein C antithrombotic mechanism in the pathogenesis of neuropathy is unclear. This neuropathologic case-control study investigated whether vascular endothelial TM expression is deficient in peripheral nerve microvessels in diabetic neuropathy. Sural nerve biopsies from 7 patients with diabetic neuropathy and 10 with axonal neuropathy without vasculopathy were immunostained with anti-TM and anti-von Willebrand factor (vWF; an endothelial cell marker) antibodies. The proportion of TM-positive microvessels was expressed relative to total vWF-staining vessels, according to vessel caliber and regional distribution within the nerve. In diabetic nerves compared with reference controls, the proportion of TM-positive endoneurial microvessels was 15-fold lower (0.02 vs. 0.30 in diabetic nerves vs. controls, P < 0.004), and the proportion of small-caliber epineurial microvessels was 10-fold lower (0.04 vs. 0.43, P < 0.001). No TM expression was detected at the perineurium in diabetic or control nerves. We demonstrate a substantial reduction of vascular endothelial TM expression throughout human diabetic neuropathy. These findings suggest that an impaired native TM-dependent protein C antithrombotic mechanism may contribute to microvascular ischemia in the pathogenesis of diabetic neuropathy.

Quantitative analysis of bronchial wall vascularity in the medium and small airways of patients with asthma and COPD.

BACKGROUND: Submucosal hypervascularity is part of airway remodeling in patients with asthma; however, its existence in the small airways and its contribution to airflow limitation remain controversial. METHODS: We investigated bronchial wall vascularity and angiogenic cells between medium airways (inner diameter, 2 to 5 mm) and small airways (inner diameter, < 2 mm) in patients with asthma (n = 9) and COPD (n = 11), and in 8 control subjects. The lung specimens obtained during surgery were immunostained to detect CD31, CD34, vascular endothelial growth factor, and basic fibroblast growth factor. RESULTS: The number of vessels in both the medium and small airways in patients with asthma was significantly (p < 0.01) increased compared to those in patients with COPD and control subjects, and the percentage of vascularity was significantly (p < 0.01) increased in the medium airways in asthma patients and in the small airways in COPD patients. Patients with moderate asthma showed a greater increase in vascularity than those with mild asthma (p < 0.01), and the number of angiogenic factor-positive cells increased in asthma patients compared with control subjects. In asthmatic subjects, inverse correlations were found between FEV(1) percentage of predicted and the number of vessels (r = -0.85; p < 0.01), or the percentage of vascularity (r = -0.72; p < 0.03) in the inner area of the medium airways, but they were not found for the small airways. In COPD patients, no correlations were demonstrated. CONCLUSIONS: The number of vessels in the medium and small airways in asthma patients shows a greater increase than those in COPD patients, and the vascular area in the small airways is increased in COPD patients but not in asthma patients. Enhanced vascularity in the inner area of the medium airways, but not in the small airways, might contribute to airflow limitation in asthma patients.

Correlation of bone marrow angiogenesis and mast cells with tryptase activity in myelodysplastic syndromes.

Bone marrow samples from 30 patients with myelodysplastic syndromes (MDS) grouped according to the International Prognostic Scoring System for MDS were investigated for counts of microvessels, total metachromatic mast cells (MC) and MC expressing tryptase, an angiogenesis-inducing molecule. Counts were higher in patients with a poor prognosis. The observation of a high correlation between microvessel counts and both total metachromatic and tryptase-reactive MC in all samples suggests that angiogenesis in MDS increases with their progression and that MC may intervene in the angiogenic response in MDS through tryptase contained in their secretory granules.

Astroglia-microvessel relationship in the developing human telencephalon.

The telencephalon of 12 and 18 week-old human foetuses was examined for evidence of astroglia-microvessel relationship. Immature astroglia cells (radial glia and astroblasts) and astrocytes were immunostained using antibodies to the cytoskeletal proteins vimentin (VIM) and glial fibrillary acidic protein (GFAP). The microvessels were detected using an antibody to the blood-brain barrier (BBB)-specific glucose transporter GLUT1. Two extracellular matrix (ECM) glycoproteins, laminin (LM), an endothelial-derived molecule, and tenascin-C (TN-C), a glia-derived molecule, were also analyzed. In the two stages examined, VIM- and GFAP-positive fibers of the radial glia establish close relationships with the radial and periventricular microvessels, which are GLUT1-positive and lined by an LM-positive basal lamina-like matrix. At the 18th week, also radial glia transitional forms and immature astrocytes exhibit extensive contacts with the microvasculature. A TN-C-rich ECM is revealed around the vascular plexus of ventricular zones at the 12th week, and around the newly growing radial microvessels and the microvessel branching sites at the 18th week. The observations taken as a whole, suggest that during the telencephalon morphogenesis the immature astroglia cells play a role in the early establishment of the distribution pattern of the neural microvessels and in their growth and maturation.

Lectin analysis of common glycoproteins detected on the surface of continuous microvascular endothelium in situ and in culture: identification of sialoglycoproteins.

For many years, molecular interactions with vascular endothelium have been studied in vitro on cultured endothelial cells. Yet, it is clear that the different environmental conditions in vivo vs. in vitro may cause phenotypic drift and altered expression of cell surface molecules. In this study, we identify several endothelial surface proteins of similar apparent molecular mass by radioiodination of cultured microvascular cells and by intravascular radioiodination of rat heart endothelium in situ. The radioiodinated surface polypeptides detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (followed by autoradiography) were subjected to lectin affinity chromatography in order to provide an additional screen for identifying common surface glycoproteins and a means for partial characterization of their glycans. With a battery of 18 lectins, seven major (gp140, gp120, gp100, gp85, gp75, gp60, gp47) and 6 minor (gp330, gp300, gp180, gp160, gp150, gp42) glycoproteins were identified on the cultured cells each with a different lectin binding profile. The lectin binding profiles of many endothelial glycoproteins in situ were similar to those of their counterparts in culture. A common set of seven major glycoproteins with the same apparent molecular masses was found in situ as well as in vitro. These common glycoproteins were characterized further using both sialidase digestion and sequential lectin affinity chromatography of cell lysates. Most of the glycoproteins appear to have both complex-type N-linked and O-linked glycans except for gp60 with only O-linked glycans, gp47 with only complex N-linked sugars, and gp42 with only simple N-linked sugars. A subset of sialoglycoproteins (gp140, gp120, gp100, gp60, gp47) was identified. One of them, gp120, is podocalyxin based on immunoprecipitation with specific antiserum and another one, gp60, is a recently identified albumin binding protein on the surface of cultured microvascular endothelial cells. This study shows that gp60 is indeed present on the surface of endothelium in situ and that it is a sialoglycoprotein with typical O-linked glycans. It is apparent that the continuous type of microvascular endothelium can indeed express in culture and in situ a common set of major glycoproteins.

Isolation and cultivation of endothelial cells derived from human placenta.

Endothelial cells were isolated from human full-term placenta by perfusion with trypsin solution via the umbilical cord vein. Human placental endothelial cells (HPEC) were successfully grown and kept in culture. HPEC exhibited endothelial characteristics as judged by morphology of confluent monolayers, staining with low density lipoprotein, binding of Ulex europaeus I lectin, and immunostaining against von Willebrand factor, alpha-thrombomodulin, VE-cadherin and a series of integrins. Different growth requirements and particular morphological characteristics indicated the different vascular origin of HUVEC and HPEC.

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Human dermal endothelial cells express membrane-associated mast cell growth factor.

Mast cell growth factor (MGF), a molecule that serves as a ligand for the receptor tyrosine kinase c-kit, is important in mast cell differentiation, migration, and activation. Previous studies of paraffin-embedded human skin using antibody to murine MGF and reverse transcription-polymerase chain reaction have demonstrated MGF protein and mRNA expression in keratinocytes and isolated dermal cells. We utilized a monoclonal antibody to human MGF to further define patterns of immunoreactivity in frozen specimens of neonatal and adult skin from normal individuals and from patients with urticaria pigmentosa. In addition to keratinocytes and isolated dermal cells in normal and urticaria pigmentosa skin, MGF was detected in cells lining superficial and mid-dermal vessels. Co-expression of MGF and the vascular antigen CD31, and immunoelectron microscopy, identified MGF-positive cells as endothelial cells. Patterns of endothelial MGF expression were not influenced by mast cell degranulation and endothelial E-selectin induction in vitro. By ultrastructure, unfixed specimens demonstrated MGF expression both within the endothelial cytoplasm and in association with lumenal, but not ablumenal, surfaces. Specimens fixed with Nakane's solution had diminished endothelial cytoplasmic MGF reactivity, but lumenal expression was maintained, suggesting persistence of a membrane-associated reactivity. MGF mRNA was also detected in cultured dermal microvascular endothelial cells using reverse transcription-polymerase chain reaction. These data establish human dermal endothelial cells as sites of MGF production and expression in human skin. Mast cell precursors must home to skin via vascular channels and differentiate in the immediate perivascular space. Thus, endothelial MGF may be an important determinant of adhesion and differentiation of mast cell progenitors expressing receptors for MGF.

A factor in human plasma permits persistent expression of E-selectin by human endothelial cells.

E-selectin is an inducible endothelial cell adhesion protein that is a critical element in the binding of leukocytes to activated endothelial cells. It is induced by a variety of pro-inflammatory soluble substances including interleukin-1 (IL-1), tumor necrosis factor (TNF), or bacterial lipopolysaccharide (LPS). In vitro studies of a large vessel endothelial cells demonstrate that stimulation with TNF or IL-1 leads to a rapid, but transient, induction of E-selectin expression that disappears within 24 hours. However, in vivo studies have shown that microvascular endothelial cells persistently express E-selectin in chronic inflammatory states, particularly in the skin where it serves as a homing receptor for memory T cells. Stimulation of dermal-derived microvascular endothelial cells (HDMECs) with single doses of IL-1 alpha, TNF alpha, or LPS resulted in transient but slightly more persistent expression of E-selectin than seen after stimulation of large vessel derived umbilical vein endothelial cells (HUVECs). However, stimulation of either HDMECs or HUVECs with repetitive doses of IL-1 alpha, TNF alpha, or LPS in the presence of human serum or plasma resulted in persistent E-selectin expression in vitro. The persistent E-selectin cell surface expression was associated with persistent E-selectin mRNA expression and correlated with E-selectin-mediated HL-60 binding to endothelial cell monolayers. The effect of human plasma or serum was dose dependent, and fractionation of human plasma by gel filtration demonstrated that the E-selectin persistence activity resolved into high and low molecular peaks. These data demonstrate that human endothelial cells are capable of persistent E-selectin expression in vitro and that factors in human serum or plasma are critical in preventing cytokine refractoriness and loss of E-selectin expression. This study provides a basis to resolve the apparent discrepancies between previous in vivo and in vitro dynamics of E-selectin expression.

Polarized expression and basic fibroblast growth factor-induced down-regulation of the alpha 6 beta 4 integrin complex on human microvascular endothelial cells.

Endothelial cells rest on a basement membrane that anchors them to the vessel wall. The alpha 6 beta 4 integrin complex has been described on epithelial cells, frequently localizes to basement-membrane structures, and appears to play a role in binding epithelial cells to laminin. We have determined that human microvascular endothelial cells express the beta 4 integrin chain in vivo and that it preferentially localizes to the endothelial basement membrane. Human microvascular endothelial cells and human umbilical vein endothelial cells also express cell-surface beta 4 in vitro. In addition, the expression of beta 4 appears to be polarized to the undersurface of endothelial cell monolayers in vitro, mimicking its in vivo localization. Stimulation of microvascular endothelial cells with basic fibroblast growth factor or phorbol 12-myristate 13-acetate, agents previously shown to induce endothelial cell migration in vitro, resulted in a marked decrease in cell-surface expression of the beta 4 integrin chain, associated with a decrease in beta 4 mRNA. These data demonstrate that human endothelial cells express the beta 4 integrin chain in vivo and in vitro, the expression of this integrin chain is polarized, and its expression is regulated on microvascular endothelial cells by factors important in wound healing and vascular regeneration.

Jejunal/kidney glucose transporter isoform (Glut-5) is expressed in the human blood-brain barrier.

The recently cloned Glut-5, glucose transporter isoform, is expressed in human jejunum and kidney. Employing previously characterized polyclonal antibodies directed towards the C-terminus region of the derived human Glut-5 peptide and Western blot analysis, a 50-55 kilodalton Glut-5 protein was detected in adult human brain homogenates. The amount of Glut-5 protein in brain was 4-fold lower when compared to the levels in adult kidney. Immunohistochemical analysis using cerebral and cerebellar sections demonstrated Glut-5 immunoreactivity in only some of the Glut-1 and factor VIII-positive brain microvascular endothelial cells, the intravascular red and white blood cells being negative. This selective localization pattern was confirmed by the 5-fold enrichment of Glut-5 vs. a 20-fold enrichment of Glut-1 in an isolated human cerebral cortical microvascular preparation, when compared to whole cerebral homogenates. We conclude that Glut-5 is localized in the endothelial cells of human brain microvasculature. Unlike other fructose using tissues, where Glut-5 may subserve the role of a fructose carrier, in brain where fructose is not used as a substrate, Glut-5 may transport glucose alone. This role of Glut-5 in conjunction with the previously characterized brain endothelial Glut-1 and Glut-3 needs further elucidation.