Biomedical subjects

M E Gerritsen

Publications and source records attributed to M E Gerritsen.

At least 55 records · Page 3Linked to original sources

PubMed1995

Activation-dependent isolation and culture of murine pulmonary microvascular endothelium.

OBJECTIVE: Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study. METHODS: Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice. Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91). Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS). Positive cell populations were collected and cultured for 1-2 weeks. When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL). RESULTS: The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake. MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices. MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation. Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta. The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions. CONCLUSION: The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study. This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

Animals↗

PubMed1995

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Base Sequence↗

PubMed1995

Altered proliferation of retinal microvascular cells on glycated matrix.

PURPOSE: To investigate the effect of nonenzymatic glycosylation (glycation) of basement membranes (BM) and isolated BM proteins on the growth of retinal pericytes and retinal endothelial cells. METHODS: Type IV collagen, laminin, Engelbreth-Holm-Swarm tumor basement membrane (EHS-BM) and bovine retinal basement membrane (RBM), after incubation in the presence of reducing sugars to induce glucose-mediated modifications, or in the absence of any sugar (control), were used as a substrate to culture bovine retinal microvascular cells. Cell growth on the nonenzymatically glycosylated and the corresponding control substrates was measured daily, using an automated cell counter. RESULTS: Retinal pericytes seeded on glycated type IV collagen proliferated consistently more slowly than on control type IV collagen (P = 0.02), showing a 20% to 33% decrease throughout most of the growth curve, whereas on glycated laminin the difference from control was not significant. In contrast, proliferation increased by 16% to 25% for retinal endothelial cells on glycated laminin compared with control substrate (P = 0.025), whereas on glycated type IV collagen the growth curve was not significantly different from the curve for the control. When seeded on whole glycated EHS-BM or RBM, proliferation of pericytes decreased by 20% to 30% (P = 0.04); the endothelial cells showed no difference on glycated EHS-BM, however, the growth rate increased on glycated RBM by 25% to 30% more than it did for the control (P = 0.01). CONCLUSIONS: Nonenzymatic glycosylation of intact BM or individual BM macromolecules resulted in reduced proliferation of retinal pericytes and increased proliferation of retinal endothelial cells. These in vitro observations resemble some of the pathologic changes of the retinal microvascular cells observed in situ, when diabetic retinopathy develops.

Animals↗

PubMed1994

Induction of cyclooxygenase II in human synovial microvessel endothelial cells by interleukin-1. Inhibition by glucocorticoids.

OBJECTIVE: To characterize the effects of interleukin-1 alpha (IL-1) on prostanoid biosynthesis by human rheumatoid synovium microvessel endothelium (HSE). METHODS: HSE cells were treated with cytokines, metabolic inhibitors, and steroids under various conditions, and prostaglandin biosynthesis was determined by radioimmunoassay. Newly synthesized cyclooxygenase (COX) was quantitated by immunoprecipitation of metabolically labeled HSE cell lysates. The effects of IL-1 on levels of messenger RNA (mRNA) for COX II were also determined. RESULTS: IL-1 induced an increase in COX activity (as assessed by prostaglandin E2 release) that was dose- and time-dependent and was blocked by cycloheximide, actinomycin D, and dexamethasone. IL-1 induced a selective increase in COX II mRNA and biosynthesis of COX II protein that was blocked by dexamethasone. CONCLUSION: IL-1 treatment of HSE cells induces COX II, as demonstrated by both Northern blotting and immunoprecipitation. The induction of COX II expression provides, at least in part, a mechanism for the pronounced increase in prostanoid synthesis observed in HSE cells following incubation with IL-1. The selective up-regulation of HSE COX II by inflammatory cytokines such as IL-1 suggests that development of specific pharmaceutical inhibitors for this novel isozyme may provide significant new therapeutic advantages in the treatment of RA.

Cells, Cultured↗

PubMed1994

Acidic fibroblast factor induction of cyclooxygenase II in rabbit cardiac muscle microvessel endothelial cells: mediation by protein kinase C.

OBJECTIVE: To determine if the novel cyclooxygenase, COX II, was induced by acidic fibroblast growth factor (aFGF) in rabbit cardiac muscle microvessel (RCME) endothelial cells and to determine the role of protein kinase C (PKC) activation in the mediation of the aFGF induction of COX activity. METHODS: Cultured RCME cells were treated with aFGF (200 ng/ml). Induction of COX II activity was assessed by determination of COX activity (PGE2 production), by immunoprecipitation of metabolically labeled COX II, and by Northern analyses. The role of PKC was assessed using phorbol myristate acetate and PKC inhibitors and by determination of PKC activity in cytosol and membrane fractions of RCME cells treated with aFGF and phorbol myristate acetate. RESULTS: aFGF selectively induced COX II protein and mRNA. Protein kinase C activation was implicated in the transduction of the effects of aFGF for the following reasons: (1) phorbol myristate acetate (PMA), a direct activator of protein kinase C, was a potent inducer of COX II mRNA, COX activity and synthesis of COX II protein. (2) H-7, an inhibitor of PKC, but not the inactive control, HA-1004, blocked aFGF induction of COX II mRNA, COX II protein synthesis, and COX activity. Two additional inhibitors of PKC, calphostin C and staurosporine, also inhibited aFGF induction of COX activity. (3) Downregulation of PKC by overnight incubation with 1 microM PMA blocked subsequent induction of COX II protein synthesis by aFGF. (4) aFGF treatment of RCME cells resulted in the translocation of PKC activity from the cytosol to the membrane fraction. However, aFGF, at concentrations that elicited COX II, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. CONCLUSION: aFGF induces COX II in RCME and this response in mediated, at least in part, by protein kinase C activation. However, aFGF mediated activation of PKC activation must stimulate this kinase through a pathway of signal transduction distinct from inositol phospholipid accumulation or elevation of intracellular Ca2+.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma.

The elicitation of leukocytes from the circulation to inflamed tissue depends on the activation of both the leukocyte and endothelial cell. In this study we determined the gene expression and secretion patterns for the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cytokine- and lipopolysaccharide (LPS)-treated cultured human lung microvascular endothelial cells (HLE). HLE constitutively expressed low levels of MCP-1 and IL-8. Treatment of HLE with a variety of cytokines and LPS up-regulated both IL-8 mRNA expression and release of immunoreactive IL-8 with an order of potency tumor necrosis factor-alpha (TNF-alpha) >> IL-1 alpha > LPS, whereas interferon-gamma (IFN-gamma) had no effect on IL-8 mRNA or antigenic levels. However, IFN-gamma, in combination with high doses of IL-1 alpha, resulted in a synergistic increase in IL-8 generation. MCP-1 gene expression and secretion was induced in a dose-dependent manner after IL-1 alpha, TNF-alpha, IFN-gamma, and LPS activation of HLE. IL-1 alpha was the most potent inducer of MCP-1 generation and LPS was relatively ineffective. IFN-gamma, in combination with low doses of IL-1 alpha, resulted in a synergistic increase in MCP-1 generation by HLE. These results demonstrate that although IL-8 and MCP-1 generation by HLE occurs on cytokine treatment, the relative ability of a given cytokine to elicit IL-8 generation is not directly parallel to effects on MCP-1 generation. These data suggest that the regulation of IL-8 and MCP-1 expression exhibit significant differences in their mechanisms. Such differences in the expression of specific chemokines may explain the specific appearance of various leukocytes at sites of inflammation and injury. These data also directly demonstrate that the lung microvascular endothelium contribute to the cytokine network of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

Base Sequence↗

PubMed1994

Cytokines in vitreous humor: interleukin-6 is elevated in proliferative vitreoretinopathy.

PURPOSE: To measure the levels of interleukins (IL) 1 beta, 6, and 8, and tumor necrosis factor-alpha (TNF alpha) in the vitreous of patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), vitreous hemorrhage, and macular pucker. METHODS: Vitreous samples were collected, undiluted, from patients with PVR, PDR of varying severity, and miscellaneous lesions (vitreous hemorrhage from trauma, macular degeneration, vein occlusion, and non-PVR patients with giant tear, retinal detachment, and macular pucker). Immunoreactive levels of the cytokines, IL-1 beta, IL-6, IL-8, and TNF alpha were determined by enzyme-linked immunoadsorbent assays, and samples were analyzed for protein and hyaluronic acid content using standard assays. RESULTS: The levels of TNF alpha were below detection limits of the assay (< 3 pg/ml). In 45 of the 47 samples tested, IL-1 beta levels also were below detection limits of the assay (< 3 pg/ml). IL-6 levels ranged from < 30 to 5487 pg/ml, with the highest values observed in the PVR patients. IL-8 levels ranged from < 20 to 1900 pg/ml, and were consistently high in the miscellaneous group. Some of the PVR patients with C2 and C3 level severity also exhibited IL-8 levels exceeding 100 pg/ml. In a second study, IL-6 content of vitreous from miscellaneous and PVR patients was compared. In this study, significantly elevated levels of IL-6 were observed in the PVR patients (91.5 +/- 18 pg/ml) compared to the miscellaneous group (10.3 +/- 3.7 pg/ml) CONCLUSIONS: Elevated levels of IL-6 in the vitreous occur in PVR, implicating a role for this cytokine in the pathogenesis of this ocular disorder.

Adult↗

PubMed1993

Endothelial cell gene expression in response to injury.

Endothelial cells respond to injury with acute alterations in mediator generation and surface molecule expression. Endothelial cells may respond, depending on the stimulus, by altering the expression of various genes whose products are central to endothelial cell matrix remodeling, coagulation, and fibrinolysis, as well as interactions with polymorphonuclear leukocytes, platelets, and lymphocytes and in angiogenesis. These responses comprise endothelial activation or differentiation. The molecular events involved in endothelial perception of injury and in regulation of gene expression by cytokines, reactive oxygen intermediates, and mechanical forces are discussed. In addition, because endothelial cells from different sites exhibit heterogeneity regarding their injury responses, where possible this heterogeneity in endothelial gene expression is highlighted.

Animals↗

PubMed1993

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Adolescent↗

PubMed1993

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor.

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5-15 cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: 1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. 2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained a transferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. 3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure.

Analysis of Variance↗

PubMed1993

Isolation and characterization of human and rat cardiac microvascular endothelial cells.

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Animals↗

PubMed1993

Cellular signaling responses mediated by a novel nucleotide receptor in rabbit microvessel endothelium.

The adenine nucleotide, ATP, elicits an elevation in intracellular ionized calcium concentration ([Ca2+]i) and phospholipase C-mediated phosphatidylinositol hydrolysis and stimulates the synthesis of the prostaglandins E2 and I2 in cultured endothelial cells derived from rabbit cardiac muscle. Use of various ATP analogues indicated that these events did not fit the classical definition of P1 or P2 purinergic receptors and, furthermore, indicated that the receptor(s) mediating these activities was not specific for purines. The rank order of agonist potency on prostaglandin release, elevations in [Ca2+]i, and inositol phosphate response was UTP > or = ATP > ADP > ADP[beta]S = 2-methylthio ATP > adenosine, suggesting that these three cellular responses are coupled to the same or similar receptors. However, the sensitivity of these three cellular responses to added nucleotides was somewhat different. The half-maximum effective concentration (EC50) for ATP stimulation of prostaglandin release was 100 microM, for inositol phosphate turnover it was 25 microM, and for elevations in [Ca2+]i it was < 1 microM. Similar discrepancies in EC50 UTP values for these three cellular responses were also noted. These observations indicate that purine and pyrimidine nucleotides elicit at least three cellular responses in rabbit cardiac muscle microvessel endothelial cells, all demonstrating similar rank orders of potency. However, the differences in EC50 suggest that if these responses are mediated by a single receptor type, it exhibits divergent coupling to various cellular signaling pathways.

Adenine Nucleotides↗

PubMed1993

Cytokine activation of human macro- and microvessel-derived endothelial cells.

The effects of the inflammatory cytokines, tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1), and gamma interferon (IFN gamma) on macro- and microvessel-derived endothelial cell proteolytic, adhesion protein and prostaglandin synthetic activities were compared. TNF alpha treatment of human umbilical vein endothelial (HUVE) cells induced urokinase-type plasminogen (uPA) activity, increased HUVE uPA-dependent extracellular matrix (ECM) degradation, and accelerated matrix remodeling and endothelial differentiation into tubes or cord-like structures. All of the aforementioned effects of TNF alpha on HUVE uPA-dependent activities were abrogated by co- or pretreatment with IFN gamma. In contrast, endothelium derived from human lung (HLE) exhibited high constitutive uPA and uPA-dependent matrix degradation and rapid tube formation in Matrigel, activities all unaffected by TNF alpha or IFN gamma. Endothelium derived from human rheumatoid synovium (HSE) exhibited uPA-dependent activities intermediate between the HLE and HUVE. TNF alpha or IL-1 treatment of HUVE potently induced surface ICAM-1 expression, whereas these cytokines were relatively ineffective on HLE and HSE ICAM-1 expression. Co-incubation with IFN gamma synergistically elevated TNF alpha or IL-1 induced ICAM-1 expression in HUVE, HLE, and HSE. The major prostaglandin synthesized by HUVE was PGI2, in contrast to HLE and HSE which produced PGE2 as the major product. Although cytokine treatment increased prostanoid production in all three cell types, HLE were not responsive to IL-1, and HSE demonstrated the greatest increase in prostaglandin synthetic capacity. These studies underline important differences not only in the "constitutive" activities expressed by EC from different vascular beds, but also in the responsiveness to proinflammatory cytokines alone or in combination. These observations further emphasize the need to study the endothelial cell derived from the vascular bed of interest rather than extrapolate from results obtained with HUVE or other macrovessel-derived endothelium.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelial cells by a glucocorticoid independent mechanism.

The present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time- and dose-dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor beta, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 microM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells.

Animals↗

PubMed1992

Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung.

Primary cultures of peripheral lung lobes were grown in a highly supplemented medium. Human lung endothelial cells (HLE) were isolated from the mixed population by FACS. The cells proliferated rapidly and were serially cultivated for at least 16 passages. Both early and late passage cells were positive for the standard endothelial markers. Factor VIII related-antigen (Factor VIII R-Ag), angiotensin-converting enzyme, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake, and bound the lectin Ulex europaeus agglutinin (UEA). Prostaglandin E2 was the major cyclooxygenase product of HLE, in contrast to human umbilical vein endothelial cells (HUVE), which synthesized PGI2 in excess of PGE2. Factor VIII R-Ag exhibited a diffuse cytoplasmic as well as an extracellular fibrillar distribution in HLE, in contrast to a vesicular (Weibel-Palade body) cytoplasmic distribution in HUVE. The HUVE did demonstrate some extracellular fibrillar Factor VIII R-Ag as well. Urokinase was the predominant plasminogen activator (PA) secreted by HLE, whereas tissue PA was predominant in HUVE cultures. HLE formed tube-like structures within 2 h of plating on a Matrigel matrix whereas HUVE formed larger tube-like structures only after 1 or more days. The properties described here indicate that human lung microvessel endothelium can be isolated and continuously grown from small tissue segments and express a number of properties that differ from those of HUVE. These studies provide further support for the concept that endothelial cells from different sources can exhibit considerable heterogeneity relating to their phenotypic and biochemical properties.

Cell Separation↗

PubMed1992

Polymyxin B-induced bronchial neutrophilia does not alter airway responsiveness to methacholine in cynomolgus monkeys.

The purpose of this study was to develop a primate model of chronic bronchial neutrophilia to investigate the role of neutrophils in the pathogenesis of airway hyperresponsiveness. Ten adult male cynomolgus monkeys (Macaca fascicularis) were anaesthetized and intubated for each study. Six animals each received a total of seven inhalation treatments with polymyxin B (200 micrograms) over a 24 day period. Four control animals received an identical treatment regime with vehicle inhalations. Airway cellular composition was assessed by bronchoalveolar lavage (BAL). Airway responsiveness was assessed by methacholine cumulative dose response determinations. There were no significant changes in airway cellular composition or airway responsiveness in the control group. In contrast, Polymyxin B inhalation resulted in an influx of neutrophils (PMN) into the lungs which peaked at day 10 of the study (%PMN in BAL fluid rose from 5 +/- 2 to 51 +/- 8, P less than 0.001) and persisted out to day 24 (41 +/- 8, P less than 0.01). Increases in PMNs were associated with an increase in BAL levels of myeloperoxidase (MPO levels in BAL fluid increased from 2 +/- 3 to 180 +/- 23 OD P less than 0.05). Although airway PMN percentages and MPO concentrations were chronically elevated, airway responsiveness did not change. These results suggest that the neutrophil does not play a functional role in the onset of hyperresponsive airways in primates.

Administration, Inhalation↗

PubMed1991

Glucocorticoid-mediated alterations in fluidity of rabbit cardiac muscle microvessel endothelial cell membranes: influences on eicosanoid release.

Experiments were conducted to determine effects of the synthetic glucocorticoid, dexamethasone, on the lipid fluidity of cultured rabbit cardiac muscle microvessel endothelial cells and the possible role(s) for altered fluidity in the steroid inhibition of cellular eicosanoid production. Following a sixteen hour exposure to 10(-7) M dexamethasone, membranes prepared from treated cells exhibited a decreased fluidity compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH). Examination of the effects of temperature on the anisotropy values of DPH using Arrhenius plots revealed consistent differences in the steroid treated cells over the entire temperature range (40-5 degrees C). These dexamethasone-dependent fluidity changes were associated with increases in the cholesterol/phospholipid ratio of membrane lipids. Restoration of membrane fluidity to control values with the fluidizing agent, 2-(2-methoxyethoxy)ethyl-8-(cis- 2-n-octylcyclopropyl)octanoate (A2C), partially reversed dexamethasone induced inhibition of A23187-stimulated eicosanoid release. These observations suggest that at least part of dexamethasone's inhibitory actions on eicosanoid generation in microvessel endothelial cells are mediated by alterations in membrane composition and fluidity.

6-Ketoprostaglandin F1 alpha↗

PubMed1991

12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea.

Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood-aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗