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Biomedical subjects

U S Ryan

Publications and source records attributed to U S Ryan.

At least 19 recordsLinked to original sources
PubMed1997

Studies on the mechanisms involved in antigen-evoked pleural inflammation in rats: contribution of IgE and complement.

Immunoglobulin E (IgE) has been shown to play a critical role in the allergic late-phase reaction, which is marked by intense leukocyte infiltration and edema. In this study we assessed the allergic pleural inflammation triggered by intrapleural (i.pl.) challenge in sensitized rats. We examined pleural effluent from actively sensitized rats following anti-IgE monoclonal antibody (mAb) (MARE-1) provocation for protein exudation, neutrophil as well as eosinophil accumulation. Inflammatory changes triggered by antigen after passive sensitization with IgE mAb was also assessed for comparison. Total serum level of IgE was found to be about threefold increased 7-8 days post-active sensitization, remaining augmented for at least 30 days. Increased levels of peritoneal leukocyte-bound IgE and serum IgE with specificity to ovalbumin were also detected. Nevertheless, the anti-IgE challenge in 14-day actively sensitized was shown to be a weak stimulus of neutrophil and eosinophil accumulation, despite being able to cause intense protein extravasation. Similarly, antigen challenge of IgE-passively sensitized rats caused protein leakage that was comparable to that induced by anti-IgE mAb in actively sensitized rats but led to a much lower neutrophil/eosinophil infiltration. Also, blockade of complement with recombinant human soluble C receptor-1 (sCR1) treatment prevented actively sensitized rats from reacting to antigen with neutrophil and eosinophil recruitment without modifying protein extravasation. These data suggest that IgE and complement-mediated mechanisms probably account for the exudation and leukocyte infiltration that is characteristic of the pleural inflammatory response observed in actively sensitized rats.

Aluminum Chloride↗
PubMed1996

Manganese-based superoxide dismutase mimetics inhibit neutrophil infiltration in vivo.

In a previous study (Hardy et al. (1994) J. Biol. Chem. 269, 18535-18540), we observed that the manganese-based superoxide dismutase mimetic Mn(II)-dichloro(1,4,7,10,13-pentaazacyclopentadecane) (MnPAM) inhibited neutrophil-mediated cell injury in vitro. We have extended these studies with the low molecular weight superoxide dismutase mimic to evaluate the role of superoxide in neutrophil-mediated tissue injury in vivo. In a dose-dependent manner, MnPAM inhibited colonic tissue injury and neutrophil accumulation into the colonic tissue induced by the intracolonic instillation of dilute aqueous acetic acid in mice. Tissue injury was assessed by visual and histological analysis. Neutrophil infiltration was determined by tissue myeloperoxidase activity and confirmed by histological analysis. Two novel Mn(II) dichloro complexes of the carbon-substituted macrocycles 2-methyl-1,4,7,10,13-pentaazacyclopentadecane (MnMAM) and 2-(2-methylpropyl)-1,4,7,10,13-pentaazacyclopentadecane (MnBAM) effectively catalyzed the dismutation of superoxide with catalytic rate constants (kcat) of 3. 31 x 10(7) M-1 s-1 and 1.91 x 10(7) M-1 s-1, respectively, as determined by stopped-flow kinetic analysis at pH 8.1 and 21 degrees C. The superoxide dismutase mimetics MnMAM and MnBAM also attenuated dilute aqueous acetic acid-induced tissue injury and neutrophil infiltration into colonic tissue; however, two Mn(II) complexes that had little or no detectable SOD activity (kcat </= 0.1 x 10(7) M-1 s-1), specifically the Mn(II) dichloro complexes of 1,4,7,10,13-pentaazacyclohexadecane and 1,4,7,11,14-pentaazacycloheptadecane, failed to inhibit the colonic tissue injury or infiltration of neutrophils in mice treated intracolonically with dilute aqueous acetic acid. These results are consistent with a proinflammatory role for superoxide in the mediation of neutrophil infiltration in vivo.

Animals↗
PubMed1996

Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival.

Hyperacute xenograft rejection may be modified by the activation and depletion of complement (C) using cobra venom factor (CVF). This method of prolonging xenograft survival is toxic and associated with systemic inflammation, which may potentially contribute to the pathologic features of delayed xenograft rejection. Soluble complement receptor type 1 (sCR1) inhibits both the classical and alternative C pathways and thus limits the production of proinflammatory products such as the anaphylatoxins. Hence, we investigated the effects of various sCR1 and CVF regimens, and combinations thereof, in the discordant guinea pig-to-Lewis rat cardiac xenograft model. Mean graft survival time (MST) was significantly prolonged with repetitive dosing (MST=22 hr) or continuous infusion of sCR1 (MST=32 hr) as compared with unmodified controls (MST=15 min). However, sCR1 did not prevent intragraft deposition of C3 or neutrophil infiltration and resulted in only partial inhibition of C-mediated hemolytic activity in vitro. Grafts in rats treated with a single dose of CVF (MST=67 hr) or repetitive doses of CVF (MST=69 hr) survived significantly longer than those treated with sCR1 alone, and lacked C3 deposition or neutrophil accumulation. Sera from these animals were completely depleted of C-mediated hemolytic activity. Animals treated with a single dose of CVF, or sCRI plus a single dose of CVF (MST=64 hr), had similar xenograft survival times. However, immunohistologic studies showed that addition of sCR1 to a single dose of CVF resulted in decreased macrophage activation and reduced levels of cytokines (tumor necrosis factor-alpha and interleukin-1beta) within xenografts as compared with that in recipients treated with CVF alone. Such decreased macrophage activation may result from the binding of C4b by sCR1, since combination therapy was associated with decreased intragraft C4b as compared with either therapy alone. High doses of sCR1 were well tolerated by rats and significantly prolonged discordant xenograft survival (MST=32 hr), although not to the same extent as CVF. The modification of the intragraft immune responses seen with CVF/sCR1 combination therapy may augment further therapeutic manipulations to achieve discordant xenograft survival without the attendant toxicity associated with repeated CVF administration.

Animals↗
PubMed1996

Towards endothelial-cell-directed cancer immunotherapy: in vitro expression of human recombinant cytokine genes by human and mouse primary endothelial cells.

Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. In order to determine whether primary endothelial cells can express cytokine transgenes efficiently, we constructed two retroviral vectors containing a cDNA encoding either recombinant human interleukin-1 alpha (rhIL-1 alpha) or recombinant human interleukin-2 (rhIL-2), called LNCIL-1 alpha and LNCIL-2 respectively, and studied the expression of the two cytokines in vitro in non-immortalized endothelial cells. Human umbilical vein endothelial cells (HUVEC) transduced with LNCIL-1 alpha or LNCIL-2 secreted 1.8-33 ng/10(6) cells/24 h and 40-246.7 ng/10(6) cells/24 h of biological active rhIL-1 alpha and rhIL-2 respectively. Mouse microvascular endothelial cells (MMEC) transduced with LNCIL-1 alpha and LNCIL-2 secreted 1.5 ng/10(6) cells/24 h and 5.8-24.7 ng/10(6) of biologically active rhIL-1 alpha and rhIL-2 proteins respectively. Cocultivation of HUVEC/IL-2 and MMEC/IL-2 with normal human bone marrow cells generated potent cytotoxic activity against K562, Daudi and other cell targets in a 51Cr-release assay. While IL-2 transgene-expressing HUVEC and MMEC retained their normal morphology, rhIL-1 alpha transgene expression inhibited the growth and altered the morphology of both HUVEC and MMEC in culture. The cytokine-gene-transduced endothelial cells retained other endothelial cell features, including uptake of acetylated low-density lipoprotein (Ac-LDL) and expression of von Willebrand factor, and were euploid as shown by flow cytometry. These results demonstrate that endothelial cells, by sustaining the production of biologically active rhIL-2 at levels that are sufficient for the activation of potent cytotoxic lymphocyte activity, may be useful agents for cancer gene therapy.

Animals↗
PubMed1995

Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intimal hyperplasia.

OBJECTIVE: To determine if halofuginone hydrobromide, a specific type I collagen inhibitor, could prevent intimal hyperplasia at a vascular anastomosis. DESIGN: Intimal hyperplasia is characterized by smooth muscle cell proliferation and extracellular matrix accumulation. Halofuginone was used to block collagen production and smooth muscle cell proliferation in cell cultures and in a rabbit model of an end-to-end anastomosis of the right common carotid artery. Animals were fed a nontoxic dose of halofuginone. Eighteen rabbits were fed the inhibitor in a randomized blinded fashion and were examined after 4 weeks by harvesting the arteries after perfusion fixation at physiologic pressures. RESULTS: Halofuginone inhibited smooth muscle cell proliferation in vitro and had no effect on cell viability. Morphometric quantification verified that halofuginone treatment significantly attenuated anastomotic intimal thickness. CONCLUSION: Oral administration of halofuginone inhibits intimal hyperplasia at vascular anastomoses. Intimal hyperplasia inhibition by halofuginone may be a therapeutic option for preventing arterial stenosis in vascular surgery.

Anastomosis, Surgical↗
PubMed1995

Potentiation of endogenous nitric oxide with superoxide dismutase inhibits platelet-mediated thrombosis in injured and stenotic arteries.

OBJECTIVES: We tested the hypothesis that dismutation of superoxide anion increases endogenous levels of nitric oxide, resulting in inhibition of cyclic variations in blood flow in arteries that are injured and stenotic. BACKGROUND: Platelet adhesion and aggregation leading to cyclic flow variations might result, in part, from generation of superoxide anion that can deplete endogenously produced nitric oxide. METHODS: Spontaneous cyclic flow variations, monitored with a proximal Doppler probe, were induced in the carotid artery of anesthetized rabbits by clamping the vessel with forceps and placing a high grade stenosis at the site of injury. Bovine copper/zinc superoxide dismutase (12 mg/kg body weight, n = 5), a synthetic low molecular weight mimetic (12 mg/kg, n = 8) or buffer vehicle (n = 8) was administered intravenously as divided boluses over 45 min, and the frequency of cyclic flow variations was monitored for 4 h. RESULTS: Cyclic flow variations remained stable for 4 h in vehicle-treated animals (15 +/- 1 [mean +/- SEM]/30 min at baseline and 16 +/- 1/30 min after 4 h, n = 8) but exhibited a marked and persistent reduction in animals given copper/zinc superoxide dismutase (from 14 +/- 1/30 min at baseline to 4 +/- 1/30 min after 4 h) or the mimetic (from 15 +/- 1/30 min at baseline to 3 +/- 1/30 min after 4 h, p < 0.005). They were restored in three of four mimetic-treated animals during infusion of NG-monomethyl- L-arginine (100 mg/kg), an inhibitor of nitric oxide production. In addition, levels of cyclic guanosine 5'-monophosphate in platelets were elevated after administration of the mimetic (from 2.4 +/- 0.5 fmol/10(6) platelets at baseline to 4.9 +/- 0.6 fmol/10(6) platelets 45 min after the mimetic, p < 0.03, n = 6), whereas mean arterial blood pressure was decreased and flow velocity in the carotid artery was increased consistent with mediation of the effect on cyclic flow variations by increased endogenous nitric oxide. CONCLUSIONS: Dismutation of superoxide anion appears to attenuate platelet thrombus formation at a site of vessel injury by potentiation of endogenously produced nitric oxide. This approach may have utility to inhibit platelet-rich thrombosis in injured and stenotic arteries where production of superoxide anion is increased.

Animals↗
PubMed1995

Molecular regulation of lung endothelial glutamine synthetase expression.

BACKGROUND: The lungs play a crucial role in maintaining amino acid homeostasis by exporting glutamine. Lung glutamine release is increased markedly in patients with sepsis, and in rat models injection of endotoxin causes up-regulation of glutamine synthetase (GS), the principal enzyme of glutamine synthesis. To investigate the molecular regulation of this response in the lung microvasculature we studied the effects of several hormones and cytokines that mediate the septic response on the expression of GS in rat microvascular pulmonary endothelial cells (MPECs). METHODS: MPECs were grown to confluence and incubated with the synthetic glucocorticoid dexamethasone, prostaglandins, cytokines, or activated complement C5a. Cellular lysates were prepared and total cellular RNA was extracted, hybridized with a GS complementary DNA derived probe, and normalized to reduced glyceraldehyde-phosphate dehydrogenase. GS protein content was determined by Western blotting with a GS antibody. RESULTS: Of the compounds tested, only dexamethasone caused a marked increase (tenfold or greater) of GS messenger RNA and protein levels in MPECs. Dexamethasone-induced accumulation of GS messenger RNA was rapid, dose-dependent, and maximal after 4 hours of exposure. GS protein levels were maximal after 8 hours and remained elevated for at least 48 hours. The dose of dexamethasone sufficient to induce 50% of maximal GS messenger RNA and protein level increase was approximately 10 nmol/L. The dexamethasone-induce increase of GS messenger RNA level was completely blocked by the glucocorticoid receptor antagonist RU38486 and by the transcriptional inhibitor actinomycin D but was not inhibited by the translational inhibitor cycloheximide. CONCLUSIONS: Glucocorticoids augment GS expression in rat lung microvascular endothelial cells in a manner consistent with a direct transcriptional response via glucocorticoid receptors. Other septic response mediators had minimal effect on GS expression. Induction of GS expression by adrenocorticoids is likely to contribute to the marked ability of the lungs to augment glutamine production during septic states.

Animals↗
PubMed1995

Complement inhibitory therapeutics and xenotransplantation.

Once complement-mediated HAR has been inhibited, the full spectrum of cellular and antibody-mediated inflammatory and immune responses characteristic of acute and chronic rejection will need to be counter-manded. But the fact remains that if xenotransplantation is to become a clinical reality, a clinically relevant means of inhibiting complement activation will be required. Soluble complement receptor type 1 provides such a therapeutic option and an option where the dosing regimen is under the control of the physician and can be adjusted in response to the needs of the patient.

Animals↗
PubMed1994

Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro.

In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.

Antioxidants↗
PubMed1994

Inhibition of neointimal hyperplasia by blocking alpha V beta 3 integrin with a small peptide antagonist GpenGRGDSPCA.

PURPOSE: Neointimal hyperplasia is a leading cause of restenosis after vascular procedures. Recent findings showed that smooth muscle cell (SMC) migration from the media into the neointima is a critical step in the development of the hemodynamically compromising neointimal lesion. Moreover, integrins are believed to play a role in SMC motility. Therefore we studied the role of one ubiquitous integrin, alpha V beta 3, in SMC migration. METHODS: Transwell assay was used to study in vitro migration of human and rabbit SMCs after stimulation with platelet-derived growth factor (PDGF). A neutralizing monoclonal antibody to alpha V beta 3, LM609, and a specific arginine-glycine-aspartic acid (RGD) antagonist, GpenGRGDSPCA, were used in the migration assay to inhibit alpha V beta 3-mediated SMC migration. In addition, GpenGRGDSPCA was administered locally to rabbit carotid artery after balloon angioplasty to determine the effect of blocking alpha V beta 3 on neointimal hyperplasia. RESULTS: We showed that PDGF-induced human SMC migration is mediated by the alpha V beta 3 integrin by use of LM609 to inhibit migration and that SMC migration is RGD dependent by use of GpenGRGDSPCA to inhibit migration. We have also inhibited rabbit SMC migration with GpenGRGDSPCA to demonstrate the cross-species preservation of the RGD peptide sequence in SMC mortality. Finally, when we administered GpenGRGDSPCA locally to rabbit carotid artery after balloon angioplasty, there was a statistically significant reduction in neointimal lesion formation compared with arteries administered an inactive peptide or saline solution. CONCLUSIONS: We have demonstrated the important role of the alpha V beta 3 integrin in SMC migration in vitro and in neointimal hyperplasia in vivo.

Amino Acid Sequence↗
PubMed1994

Vascular permeability factor accelerates endothelial regrowth following balloon angioplasty.

Many failures of vascular reconstructions are due to thrombosis and restenosis and are often attributed to inadequate endothelial regeneration at the site of endothelial denudation. Vascular permeability factor (VPF) is a naturally occurring growth factor responsible for vessel permeability and microvascular angiogenesis. Here, we show that VPF stimulated rabbit endothelial cell proliferation in vitro at concentrations 100 ng/ml. However, VPF had no effect on smooth muscle cell proliferation at these concentrations up to 500 ng/ml. When VPF was administered for 4 weeks (120 micrograms, twice weekly, i.v.) following balloon angioplasty-induced endothelial denudation of rabbit carotid artery, there was a significant increase in the in vivo regeneration of endothelium compared to control (57.5 +/- 6.7% vs. 38.3 +/- 1.9%, P < 0.01). Moreover, 8 weeks of VPF administration resulted in 88.1 +/- 3.1% re-endothelialization compared to control (44.7 +/- 3.8%). Hence, VPF appears to be a specific mitogen for endothelial regeneration.

Angioplasty, Balloon↗
PubMed1993

Evaluation of activity of putative superoxide dismutase mimics. Direct analysis by stopped-flow kinetics.

By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.

Cyclic N-Oxides↗
PubMed1993

Inhibition of lung inflammatory reactions in rats by an anti-human IL-8 antibody.

IL-8 belongs to the family of chemotactic cytokines and may play an important role in the inflammatory response. In the current studies, a murine mAb (DM/C7) to human rIL-8 was found to have protective effects in inflammatory lung injury in rats. DM/C7 was nonreactive with the rat cytokine-induced neutrophil chemoattractant peptide. In vivo, DM/C7 blocked the glycogen-induced accumulation of neutrophils in rats and was highly protective against lung and dermal vascular injury after deposition of IgG immune complexes. The latter model of injury has recently been shown to be E-selectin dependent. The protective effects of DM/C7 correlated with reduced tissue accumulation of neutrophils, as measured by myeloperoxidase content. DM/C7 reacted with an epitope expressed by TNF-alpha-stimulated rat pulmonary artery endothelial cells and with the pulmonary vascular endothelium after intrapulmonary deposition of IgG immune complexes. In the model of IgG immune complex-induced lung injury, the protective effects of DM/C7 were abolished by prior absorption of the antibody with human rIL-8. Polyclonal antibody to cytokine-induced neutrophil chemoattractant peptide failed to protect against IgG immune complex-induced vascular injury even though this antibody blocked the in vitro chemotactic activity of cytokine-induced neutrophil chemoattractant. In the model of rapidly developing lung injury due to systemic activation of C after infusion of cobra venom factor, DM/C7 was not protective. As well, in the neutrophil-independent model of IgA immune complex-induced lung injury, treatment with DM/C7 was not protective. These data indicate that in inflammatory lung injury that is linked to E-selectin-dependent recruitment of neutrophils in rats, antibody to human IL-8 also blocks recruitment of neutrophils and thereby affords protection against lung injury. The data suggest the presence of an IL-8-like product in this model of lung injury.

Animals↗
PubMed1993

The diffusional transport of water and small solutes in isolated endothelial cells and erythrocytes.

The diffusional permeability coefficients, PD, for tritiated water (3HHO) 14C-antipyrine (AP) and 14C-iodoantipyrine (IAP) in isolated calf pulmonary artery endothelial cells and dog erythrocytes are measured with the linear diffusion technique at 11.5, 15, 20 and 37 degrees C. The PD values for both cell populations follow the sequence 3HHO > IAP > AP at each of the temperatures. PD for water is higher in the erythrocyte compared to the endothelial cells. The differences in PD for AP and IAP in the erythrocytes and endothelial cells are not dramatic and are similar to the differences seen in comparing permeation of the same solute through bilayers of different composition. A comparison of the values of PD calculated for the endothelial cells with those for isolated capillaries and the structured endothelium in whole lungs validates the use of the isolated cells as models for the endothelial cells in situ. Incubation of the endothelial cells with cis-vaccenic acid or cholesterol produces a reduction in PD for water and antipyrine. These data are analyzed in terms of Stokesian and non-Stokesian diffusion. The interpretation which best accommodates the data is that the phospholipid area of the membrane, rather than the hydrocarbon core, provides the greatest resistance to permeation for these solutes.

Alkanes↗
PubMed1993

Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells.

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet-derived growth factor (PDGF)-mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin-binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an alpha v-containing integrin receptor, but not alpha v beta 1 or alpha v beta 3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50-100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation.

Animals↗
PubMed1993

Intestinal ischemia-reperfusion injury causes pulmonary endothelial cell ATP depletion.

Intestinal ischemia-reperfusion is a common clinical event associated with both clinical and experimental distant organ injury. In particular, the pulmonary microvasculature appears to be susceptible to injury resulting from systemic inflammatory mediator activation. This study was designed to evaluate the hypothesis that noncellular humoral factors associated with intestinal ischemia-reperfusion result in pulmonary endothelial cell adenosine triphosphate (ATP) depletion. Male Sprague-Dawley rats had intestinal ischemia induced by microvascular clip occlusion of the superior mesenteric artery (SMA) for 120 minutes. Reperfusion resulted from superior mesenteric artery clip removal. After reperfusion for 0, 15, or 30 minutes, plasma samples were obtained from the portal vein. Monolayers of cultured rat pulmonary artery endothelial cells then were incubated with the plasma samples. Adenosine triphosphate levels were determined using a luciferin-luciferase assay. A 51Cr-release assay using labeled endothelial cells was performed under identical conditions to assess cytotoxicity. Potential mechanisms of ATP depletion were evaluated by analysis of cellular energy charge and assessment of microfilament architecture. Endothelial cell ATP levels decreased from 2.23 +/- 0.16 x 10(-11) moles/microgram DNA in sham preparations to 1.23 +/- 0.09 x 10(-11) moles/microgram DNA (p < 0.001) after 4 hours in plasma from animals undergoing 120 minutes of intestinal ischemia. For plasma obtained after 15 minutes of reperfusion, the decrease in cellular ATP concentration persisted (1.23 +/- 0.27 x 10(-11) moles/microgram DNA, p < 0.001 vs. sham). After 30 minutes' reperfusion, cellular ATP levels increased only slightly after the 4-hour incubation (1.39 +/- 0.26 x 10(-11) moles/microgram DNA, p < 0.005 vs. sham). No significant cytotoxic injury occurred in any group when compared with controls. Cellular energy charge was unchanged, and microfilament architecture was preserved. These data confirm the hypothesis that humoral factors, independent of the neutrophil, result in endothelial cell ATP depletion without metabolic inhibition or cell death. Depletion of energy stores by noncellular humoral factors may represent an early event that predisposes the cell to more severe injury by other mediators of the endogenous inflammatory response.

Actin Cytoskeleton↗
PubMed1993

Regulation of endothelin-1 expression in normal and transfected endothelial cells.

Calcium phosphate precipitation and retrovirus-mediated infection methods were used to stably infect bovine pulmonary artery endothelial cells (BPAECs) with mammalian expression vectors bearing human prepro-ET-1 cDNA. The calcium phosphate precipitation method afforded a stably transfected cell line that expressed approximately four times higher ET-1 than untransfected BPAEC by radioimmunoassay and at the mRNA level. The retrovirus-mediated transfection method yielded stably infected clones that secreted eightfold to 10-fold higher ET-1 than the nontransfected BPAECs; one clone continued to produce 10-fold higher levels after continuous assay for 1 year. Both transfected and nontransfected cells showed an increase (approximately twofold) in ET-1 production in response to thrombin (10 U/ml). Downregulation of ET-1 production was exhibited by both transfected and nontransfected cells in response to nitric oxide (NO) donors: sodium nitroprusside (NOPr), S-nitroso-N-acetoxy penicillamine (SNAP), and acetoxime. The potentiation of NO by superoxide dismutase (SOD) also downregulated ET-1 production. These studies show that an exogenous gene introduced into a cell type that normally expresses that gene product can be regulated by agonists and antagonists in a manner similar to the normal gene regulatory mechanisms for that cell type. This is of potential importance in gene therapy experiments, where mechanisms for regulation of expression remain elusive.

Animals↗