Human Dermal Microvascular Endothelial Cells Express Inducible Nitric Oxide Synthase In Vitro
1999; Elsevier BV; Volume: 112; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.1999.00505.x
ISSN1523-1747
AutoresGeorg Hoffmann, Josef Rieder, Michaela Smolny, Markus J. Seibel, Wolfgang Schobersberger, Christine Fürhapter, Peter Fritsch, Norbert Sepp,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoStimulation of inducible nitric oxide synthase with subsequent release of nitric oxide in large amounts may play a critical part either in host defense reactions or in the pathophysiology of the inflammatory response syndrome leading to septic shock. The aim of the present study was to investigate whether human dermal microvascular endothelial cells exhibit the typical characteristics of an inducible nitric oxide synthase expressing cell. A strong effect on inducible nitric oxide synthase gene expression could be detected when the cells were coincubated with the proinflammatory cytokines interferon-γ and tumor necrosis factor-α with inducible nitric oxide synthase cDNA concentrations averaging 11.7 ± 0.6 amol per μg total RNA at 24 h, and 25.0 ± 1.4 amol per μg total RNA at 48 h, respectively. Intracellular staining with an antibody recognizing inducible nitric oxide synthase protein and subsequent analysis by flow cytometry revealed a 4-fold increase of inducible nitric oxide synthase protein in human dermal microvascular endothelial cells treated with interferon-γ/tumor necrosis factor-α. This was accompanied by a significant elevation in nitrite/nitrate concentrations in the cell-free culture supernatants. Our results indicate that human dermal microvascular endothelial cells are provided with an inducible nitric oxide synthase system and can be regarded as an appropriate cell model for investigating inducible nitric oxide synthase gene expression and nitric oxide properties in microvascular endothelial cells. Stimulation of inducible nitric oxide synthase with subsequent release of nitric oxide in large amounts may play a critical part either in host defense reactions or in the pathophysiology of the inflammatory response syndrome leading to septic shock. The aim of the present study was to investigate whether human dermal microvascular endothelial cells exhibit the typical characteristics of an inducible nitric oxide synthase expressing cell. A strong effect on inducible nitric oxide synthase gene expression could be detected when the cells were coincubated with the proinflammatory cytokines interferon-γ and tumor necrosis factor-α with inducible nitric oxide synthase cDNA concentrations averaging 11.7 ± 0.6 amol per μg total RNA at 24 h, and 25.0 ± 1.4 amol per μg total RNA at 48 h, respectively. Intracellular staining with an antibody recognizing inducible nitric oxide synthase protein and subsequent analysis by flow cytometry revealed a 4-fold increase of inducible nitric oxide synthase protein in human dermal microvascular endothelial cells treated with interferon-γ/tumor necrosis factor-α. This was accompanied by a significant elevation in nitrite/nitrate concentrations in the cell-free culture supernatants. Our results indicate that human dermal microvascular endothelial cells are provided with an inducible nitric oxide synthase system and can be regarded as an appropriate cell model for investigating inducible nitric oxide synthase gene expression and nitric oxide properties in microvascular endothelial cells. endothelial cells human dermal microvascular endothelial cells inducible nitric oxide synthase nitric oxide Endothelial cells (EC) are situated at the vital interface between the circulating blood and the surrounding tissue. EC contribute to a number of physiologic functions, e.g., maintenance of blood pressure, regulation of cardiac muscle contractile flow, and setting of an adequate microvascular perfusion rate (Brutsaert and Andries, 1992Brutsaert D.L. Andries L.J. The endocardial endothelium.Am J Physiol. 1992; 263: H985-H1002PubMed Google Scholar;Ungureanu-Longrois et al., 1995aUngureanu-Longrois D. Ballignand J.L. Okada I. et al.Contractile responsiveness of ventricular myocytes is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture.Circ Res. 1995; 77: 486-493Crossref PubMed Scopus (72) Google Scholar). In addition, they can be dramatically affected by alterations in their mechanical, chemical, and humoral environment, thus promoting capillary leakages, cellular dysfunctions, and ultimately organ failures (Gibbs et al., 1992Gibbs L.S. Lai L. Malik A.B. Tumor necrosis factor enhances the neutrophil-dependent increase in endothelial permeability.J Cell Physiol. 1992; 145: 496-500Crossref Scopus (54) Google Scholar;Martin and Silverman, 1992Martin M.A. Silverman H.J. Gram-negative sepsis and the adult respiratory distress syndrome.Clin Infect Dis. 1992; 14: 1213-1228Crossref PubMed Scopus (101) Google Scholar). There are important phenotypic differences between EC derived from different tissues or organs (Gerritsen, 1987Gerritsen M. Functional heterogeneity of vascular endothelial cells.Biochem Pharmacol. 1987; 36: 2701-2711Crossref PubMed Scopus (213) Google Scholar). Among others, heterogenic behavior of EC derived from large conduit vessels and microvascular EC with regard to their response to inflammatory events and oxidative stress has been reported (Geiger et al., 1997Geiger M. Stone A. Mason S.N. Oldham K.T. Guice K.S. Differential nitric oxide production by microvascular and macrovascular endothelial cells.Am J Physiol. 1997; 273: L275-L281PubMed Google Scholar). Nitric oxide (NO) produced in large amounts following stimulation of inducible NO synthase (iNOS) gene expression seems to be an important messenger molecule under conditions associated with increased activity of the cellular immune system. Expression of the iNOS enzyme is stimulated by a variety of proinflammatory mediators, e.g., interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and lipopolysaccharide in a number of cell lines (Bandaletova et al., 1993Bandaletova T. Brouet I. Bartsch H. Sugimura T. Esumi H. Ohshima H. Immunhistochemical localization of an inducible form of nitric oxide synthase in various organs of rats treated with Propionibacterium acnes and lipopolysaccharide.APMIS. 1993; 101: 330-336Crossref PubMed Scopus (75) Google Scholar;Geller et al., 1993Geller D.A. Nussler A.K. Di Silvio M. et al.Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.Proc Natl Acad Sci USA. 1993; 90: 522-526Crossref PubMed Scopus (623) Google Scholar;Hoffmann et al., 1995Hoffmann G. Grote J. Friedrich F. Mutz N. Schobersberger W. The pulmonary epithelial cell line L2 as a new model for an inducible nitric oxide synthase expressing distal airway epithelial cell.Biochem Biophys Res Commun. 1995; 217: 575-583Crossref PubMed Scopus (23) Google Scholar). Interestingly, iNOS gene expression induced by proinflammatory cytokines or lipopolysaccharide has not been documented in large vessel EC derived from human tissues (MacNaul and Hutchinson, 1993MacNaul K.L. Hutchinson N.I. Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions.Biochem Biophys Res Commun. 1993; 196: 1330-1334Crossref PubMed Scopus (196) Google Scholar;Schoedon et al., 1993Schoedon G. Schneemann M. Blau N. Edgell C.J.S. Schaffner A. Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelial-cell derived relaxing factor.Biochem Biophys Res Commun. 1993; 196: 1343-1348Crossref PubMed Scopus (50) Google Scholar;Werner-Felmeyer et al., 1993Werner-Felmeyer G. Werner E.R. Fuchs D. et al.Pteridine biosynthesis in human endothelial cells.J Biol Chem. 1993; 268: 1842-1846PubMed Google Scholar), whereas recent studies provide evidence that microvascular EC derived from rat cardiac tissue (Ungureanu-Longrois et al., 1995aUngureanu-Longrois D. Ballignand J.L. Okada I. et al.Contractile responsiveness of ventricular myocytes is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture.Circ Res. 1995; 77: 486-493Crossref PubMed Scopus (72) Google Scholar), rat lungs (Geiger et al., 1997Geiger M. Stone A. Mason S.N. Oldham K.T. Guice K.S. Differential nitric oxide production by microvascular and macrovascular endothelial cells.Am J Physiol. 1997; 273: L275-L281PubMed Google Scholar), and bovine retina (Chakravarthy et al., 1995Chakravarthy U. Stitt A.W. McNally J. Bailie J.R. Hoey E.M. Duprex P. Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.Curr Eye Res. 1995; 14: 285-294Crossref PubMed Scopus (102) Google Scholar) can be stimulated to express the iNOS gene and release NO. Nevertheless, it should be noted that there are marked differences between human cells and cells derived from other species concerning their NO-producing capacity (Weinberg et al., 1995Weinberg J.B. Misukonis M.A. Shami P.J. et al.Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.Blood. 1995; 86: 1184-1195Crossref PubMed Google Scholar). Therefore, the aim of the present study was to investigate whether human microvascular EC express the gene for iNOS upon stimulation with the proinflammatory cytokines IFN-γ and TNF-α. Human dermal microvascular endothelial cells (HDMEC) were isolated from human foreskins from different donors as described previously (Sepp et al., 1995Sepp N.T. Lynn A.C. Romani N. Lian-Jie L. Wright Caughman S. Lawley T.J. Swerlick R.A. Polarized expression and basic fibroblast growth factor-induced down-regulation of the α6β4 integrin complex on human microvascular endothelial cells.J Invest Dermatol. 1995; 104: 266-270Abstract Full Text PDF PubMed Scopus (24) Google Scholar) with the following modifications: instead of trypsinization we incubated the foreskins overnight in 1.2 units dispase II per ml Puck’s salt solution at 4°C, separated the epidermis from the dermis and used only the dermal layer for the HDMEC isolation. Cells were cultured in endothelial cell basal medium supplemented with 10% normal human serum, 5 ng epidermal growth factor per ml, 2 mM L-glutamine, 2 mM L-arginine, 100 μg streptomycin per ml, 100 U penicillin per ml, 250 ng amphotericin B per ml. In the first two passages of culturing 5 × 10–5 M dibutyryl cyclic adenosine monophosphate was added. The resulting cell cultures were consistently 100% pure as assessed by morphologic and immunhistochemical criteria. The cells were kept in culture through sequential passages, and passages 3–6 were used in the present experiments. At the end of the 6 h, 24 h, 48 h, and 72 h experiments, cells were washed with sterile phosphate-buffered saline and lyzed with 4 M guanidine isothiocyanate containing 0.1 M 2-mercaptoethanol. Total RNA was isolated by acid phenol–chloroform extraction according to the method ofChomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-162Crossref PubMed Scopus (62241) Google Scholar, redissolved in water, and the concentration was determined photometrically at a wavelength of 260 nm. One micrograms of total RNA was reverse-transcribed into first-strand cDNA using oligo(dT)15 as primer for reverse transcriptase. Reverse transcriptase-generated cDNA encoding for human iNOS was amplified using PCR as described recently (Schobersberger et al., 1995Schobersberger W. Hoffmann G. Grote J. Wachter H. Fuchs D. Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells.FEBS Lett. 1995; 377: 461-464Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase served as control, RNA with no glyceraldehyde-3-phosphate dehydrogenase band was excluded from further investigations. For quantitation of iNOS cDNA, a competitive PCR was performed using a DNA fragment derived from the viral oncogene V-erb B to which the human iNOS primer template sequences have been added (Competitive DNA MIMIC, Clontech, Heidelberg, Germany), as described bySiebert et al., 1992Siebert P.D. Larrick J.W. Competitive P.C.R. Nature. 1992; 359: 557-558Crossref PubMed Scopus (642) Google Scholar). Densitometric analysis of the scanned photographs was done by using the Imaging densitometer GS 670 (BioRad, Munich, Germany) with regard to the different amount of ethidium-bromide intercalation according to the length of the fragments. Results are expressed as amol iNOS-cDNA per μg total RNA taking into consideration that 1 μl of the 25 μl reverse transcriptase reaction yield was used per quantitative PCR. Synthesis of the stable NO metabolites nitrite and nitrate was determined in the cell-free culture supernatants following incubation of cells for 6 h, 24 h, 48 h, and 72 h in L-arginine enriched medium without Phenol Red as described previously (Schobersberger et al., 1995Schobersberger W. Hoffmann G. Grote J. Wachter H. Fuchs D. Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells.FEBS Lett. 1995; 377: 461-464Abstract Full Text PDF PubMed Scopus (82) Google Scholar). HDMEC were grown in the appropriate medium and stimulated with IFN-γ (100 U per ml)/TNF-α (500 U per ml) for 24 h. Cells were washed with phosphate-buffered saline, permeabilized with 2% saponine for 10 min at room temperature and then fixed with 4% paraformaldehyde. After washing with phosphate-buffered saline, cells were incubated with an antibody recognizing iNOS protein (1:500 dilution) and vimentin (1:5 dilution; as control for intracellular staining and permeabilization of the cell). Anti-rabbit fluoroscein isothiocyanate-conjugated and anti-mouse antibodies were used for visualization. Mean channel fluorescence was determined for each data point using a minimum of 10,000 events. Endothelial cell basal media, and epidermal growth factor were purchased from Clonetics (Santa Anna, CA). Recombinant rat IFN-γ and TNF-α were from IC Chemicals (Ismaning, Germany). Amphotericin B, dibutyryl-cAMP, β-NADPH, penicillin, phenol–chloroform–isoamylalcohol mixture, and streptomycin were from Sigma (Deisenhofen, Germany). Normal human serum and glutamine were from Irvine (Santa Anna, CA). dNTP-mix, Oligo-(dT)15, primer sets, Taq polymerase, trypsin-ethylenediamine tetraacetic acid, and M-MLRV superscript reverse transcriptase were purchased from Gibco Life Technologies (Eggenstein, Germany). Sulfanilamide was from Serva (Heidelberg, Germany). Guanidine isothiocyanate and rotiphorese gel 40 were from Roth (Karlsruhe, Germany); dispase II and nitrate reductase were from Boehringer (Mannheim, Germany). Antibody, recognizing iNOS protein, was purchased from Calbiochem–Novabiochem (San Diego, CA). Rabbit and mouse fluoroscein isothiocyanate conjugated antibodies for second step staining as well as vimentin antibody were obtained from Dako (Glostrup, Denmark). Results are expressed as mean ± SEM. To test for significance of differences between the mean value of a control versus the mean value of treated cells, the Student’s t test was used; p < 0.05 were considered to be significant. Figure 1 shows a representative result of the qualitative analyses of iNOS mRNA expression detected as iNOS cDNA (fragment length 487 bp) following 6 h, 24 h, 48 h, and 72 h incubations, respectively. No iNOS gene expression could be found under control conditions (C), and in cells treated with IFN-γ (100 U per ml) alone. In contrast, incubations of HDMEC with TNF-α (500 U per ml) resulted in a detectable stimulation of iNOS gene expression following either 6 h, 24 h, 48 h, or 72 h incubations, respectively. As derived from band intensities, co-treatment of cells with IFN-γ (100 U per ml) plus TNF-α (500 U per ml) strongly enhanced the single effect of TNF-α on iNOS mRNA synthesis during the given incubation periods. Figure 2 summarizes the results of the quantitative analyses of iNOS cDNA concentrations following incubations of HDMEC with either TNF-α (500 U per ml) or IFN-γ (100 U per ml) plus TNF-α (500 U per ml) for 6 h, 24 h, 48 h, and 72 h, respectively. Single application of TNF-α resulted in iNOS cDNA concentrations of 1.3 ± 0.1 amol per μg total RNA (6 h, n = 5), 6.7 ± 0.8 amol per μg total RNA (24 h, n = 5), 13.3 ± 0.9 amol per μg total RNA (48 h, n = 5), and 17.5 ±1.2 amol per μg total RNA (72 h, n = 5), respectively. Co-incubation of cells with IFN-γ plus TNF-α resulted in a pronounced stimulation of iNOS gene expression with iNOS cDNA concentrations averaging 4.7 ±0.2 amol per μg total RNA after 6 h, 11.7 ± 0.6 amol per μg total RNA after 24 h, 25.0 ± 1.4 amol per μg total RNA after 48 h, and 33.0 ±1.7 amol per μg total RNA after 72 h incubation (n = 5 for each time point). Results of NO-metabolite concentrations (nitrate plus nitrite) in cell-free culture supernatants are presented in Figure 3 (n = 8 for each incubation). As compared with controls, no significant increase in NO release was found in cells treated with IFN-γ (100 U per ml) for 6 h, 24 h, 48 h, and 72 h. Time-dependent increases in NO-production were detectable in HDMEC stimulated with TNF-α for any given incubation period. The results were as follows: 16.8 ±0.4 nmol nitrite per 105 cells following 6 h incubation, 18.9 ± 0.6 nmol nitrite per 105 cells following 48 h incubations, 48.8 ± 0.8 nitrite per 105 cells following 48 h incubations as well as 60.6 ±0.7 nmol nitrite per 105 cells following 72 h incubations (n = 8 for each experiment; p < 0.05 as compared with controls incubated for the same time intervals). A significantly stronger effect on nitrite/nitrate concentrations in the supernatants was observed when cells were co-incubated with IFN-γ (100 U per ml) plus TNF-α (500 U per ml) with values averaging 21.5 ±1.2 nmol nitrite per 105 cells after 6 h incubations, 26.4 ± 0.7 nitrite per 105 cells after 24 h incubations, 81.9 ±0.9 nmol nitrite per 105 cells after 48 h incubations, and 107.1 ±2.0 nmol nitrite per 105 cells after 72 h incubations (n = 8; p < 0.05 as compared with the single incubation protocols with TNF-α). Examination of intracellular iNOS protein by FACS analysis demonstrated a 4-fold increase of iNOS protein after 24 h incubation with IFN-γ (100 U per ml) plus TNF-α (500 U per ml; Figure 4). Median values increased from 111 to 437 (n = 3). In contrast, control antibody recognizing intracellular vimentin filaments did not show any difference comparing untreated and stimulated cells (median values: 1255 and 1486, untreated versus stimulated cells). The results of this study show that human dermal microvascular endothelial cells derived from neonatal foreskins express the gene for iNOS upon stimulation with distinct cytokines. This iNOS gene expression is accompanied by a 4-fold elevation of intracellular iNOS protein, as detected by FACS analysis, and an increase in the nitrate/nitrite concentrations in the cell-free culture supernatants, thus indicating that HDMEC were activated to produce NO. The human microvascular EC line used here may serve as a model for studying iNOS gene expression. iNOS mRNA expression has previously been shown in rat and bovine microvascular EC derived from different tissues (Chakravarthy et al., 1995Chakravarthy U. Stitt A.W. McNally J. Bailie J.R. Hoey E.M. Duprex P. Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.Curr Eye Res. 1995; 14: 285-294Crossref PubMed Scopus (102) Google Scholar;Ungureanu-Langrois et al., 1995bUngureanu-Langrois D. Ballignand J.L. Kelly R.A. Smith T.W. Myocardial contractile dysfunction in the systemic inflammatory distress syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes.J Mol Cell Cardiol. 1995; 27: 155-167Abstract Full Text PDF PubMed Scopus (181) Google Scholar;Geiger et al., 1997Geiger M. Stone A. Mason S.N. Oldham K.T. Guice K.S. Differential nitric oxide production by microvascular and macrovascular endothelial cells.Am J Physiol. 1997; 273: L275-L281PubMed Google Scholar). This behavior seems to be in contrast to large vessel endothelial cells, where iNOS gene expression due to proinflammatory cytokines and/or lipopolysaccharide is not a common, species-independent event. High output NO synthase has been demonstrated thus far in brain murine EC (Gross et al., 1991Gross S.S. Jaffe E.A. Levi R. Kilbourne R.G. Cytokine activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages.Biochem Biophys Res Commun. 1991; 178: 823-829Crossref PubMed Scopus (364) Google Scholar), porcine aortic EC (Radomski et al., 1990Radomski M.W. Palmer R.M.J. Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells.Proc Natl Acad Sci USA. 1990; 87: 10043-10047Crossref PubMed Scopus (1035) Google Scholar), and bovine aortic EC (Lamas et al., 1991Lamas S. Michel T. Brenner B.M. Marsden P.A. EDRF synthesis by endothelial cells: evidence for a pathway induced by TNF-α.Am J Physiol. 1991; 261: C634-C641PubMed Google Scholar). In contrast, NO synthesis could not be detected in substantial amounts in human aortic endothelial cells (MacNaul and Hutchinson, 1993MacNaul K.L. Hutchinson N.I. Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions.Biochem Biophys Res Commun. 1993; 196: 1330-1334Crossref PubMed Scopus (196) Google Scholar), in the human vascular endothelium-derived hybrid cell line EA.hy 926 (Schoedon et al., 1993Schoedon G. Schneemann M. Blau N. Edgell C.J.S. Schaffner A. Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelial-cell derived relaxing factor.Biochem Biophys Res Commun. 1993; 196: 1343-1348Crossref PubMed Scopus (50) Google Scholar), and in human umbilical vein endothelial cells (Werner-Felmeyer et al., 1993Werner-Felmeyer G. Werner E.R. Fuchs D. et al.Pteridine biosynthesis in human endothelial cells.J Biol Chem. 1993; 268: 1842-1846PubMed Google Scholar) following incubation of these cells with different proinflammatory agonists. In general, contrary to animal cells induction of iNOS in human cell types and tissues has been difficult to characterize (Weinberg et al., 1995Weinberg J.B. Misukonis M.A. Shami P.J. et al.Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.Blood. 1995; 86: 1184-1195Crossref PubMed Google Scholar). One possible explanation for these observations might be that human cells, e.g., macrophages, have particularly low activities of the tetrahydrobiopterin (BH4) generating pathway (Fuchs et al., 1993Fuchs D. Weiss G. Wachter H. Neopterin, biochemistry and clinical use as a marker for cellular immune reactions.Int Arch Allergy Immunol. 1993; 101: 1-6Crossref PubMed Scopus (205) Google Scholar). Being an essential cofactor for iNOS, BH4 limitation may reduce the potential of human cells to produce large amounts of NO. There is, however, in vivo evidence for the importance of iNOS in physiologic and pathophysiologic situations, as interleukin-2 chemotherapy is associated with an activation of the L-arginine-NO pathway leading to a septic shock-like state (Hibbs et al., 1992Hibbs J.J. Westenfelder C. Tainter R. et al.Evidence for cytokine-induced nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.J Clin Invest. 1992; 89: 867-877Crossref PubMed Scopus (446) Google Scholar). Moreover, patients with sepsis regularly have high concentrations of nitrite in plasma, indicating a pronounced release of NO (Ochoa et al., 1991Ochoa J.B. Udekwu A.O. Billiar T.R. Curren F.B. Cerra R.L. Simmons H. Peizman A.B. Nitrogen oxide levels in patients after trauma and during sepsis.Ann Surg. 1991; 214: 612-626Crossref Scopus (553) Google Scholar). It is likely that iNOS induction during sepsis is the final common pathway for vasodilation in septic shock. Microvascular endothelial cells may represent a source for high-output NO. An inappropriate stimulation of the iNOS enzyme may lead to excessive release of NO, which is supposed to promote cellular dysfunctions, capillary leakages, and multiple organ failure as observed in the systemic inflammatory response syndrome (Ungureanu-Langrois et al., 1995bUngureanu-Langrois D. Ballignand J.L. Kelly R.A. Smith T.W. Myocardial contractile dysfunction in the systemic inflammatory distress syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes.J Mol Cell Cardiol. 1995; 27: 155-167Abstract Full Text PDF PubMed Scopus (181) Google Scholar). Enhanced NO formation may not only play a part in the development of septic shock but is suggested to be involved in the pathogenesis of inflammatory vascular diseases, e.g., temporal arteritis and systemic lupus erythematosus. Recently,Wagner et al., 1996Wagner A.D. Björnsson J. Goronzy J.J. Weyand C.M. Potential role of TGF-β1 and nitric oxide in human vasculitis.Arthritis Rheum. 1996; 39 (Abstract): S80Google Scholar reported that iNOS is expressed in vascular lesions associated with temporal arteritis. Although the site of NO production in temporal arteritis were macrophages accumulated in the intimal layer of the inflamed artery, a potential source of NO in systemic lupus erythematosus were activated endothelial cells via upregulated iNOS (Belmont et al., 1997Belmont H.M. Levartovsky D. Goel A. et al.Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus.Arthritis Rheum. 1997; 40: 1810-1816Crossref PubMed Scopus (146) Google Scholar). In addition, vascular NO formation in active systemic lupus erythematosus was reflected by elevations of serum nitrite. Increased iNOS activity, however, in response to a stimulation of the cellular immune system might be beneficial due to its microbicidal and tumoricidal potential (Hibbs et al., 1988Hibbs J.B. Taintor R.R. Vavrin Z. Tachlin E.M. Nitric oxide: a cytotoxic activated macrophage effector molecule.Biochem Biophys Res Commun. 1988; 157: 87-94Crossref PubMed Scopus (1765) Google Scholar;Stuehr and Nathan, 1989Stuehr D.J. Nathan C.F. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells.J Exp Med. 1989; : 1543-1555Crossref PubMed Scopus (1556) Google Scholar). Thus, HDMEC may contribute to host defense reactions following an adequate stimulation of their iNOS system by proinflammatory substances. Moreover, the expression of endothelial surface adhesion molecules like P-selectin and monocyte chemoattractant protein-1 is suppressed by NO synthesis and/or application of exogenous NO donors (Gauthier et al., 1994Gauthier T.W. Davenpeck K.L. Lefer A.M. Nitric oxide attenuates leukocyte and endothelial interaction via P-selectin in splanchnic ischemia-reperfusion.Am J Physiol. 1994; 267: G562-G568PubMed Google Scholar;Zeiher et al., 1995Zeiher A.M. Fissthaler B. Schray-Utz B. Busse R. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells.Circ Res. 1995; 76: 980-986Crossref PubMed Scopus (371) Google Scholar). These data suggest that NO inhibits adhesion of leukocytes on EC and subsequent migration of monocytes through the endothelial barrier, thus exhibiting anti-atherosclerotic mechanisms. Owing to the decreased blood flow velocity, this might be of special importance in the microvasculature. In summary, the ability of HDMEC to produce NO in considerable amounts might be of clinical importance in situations associated with increased activity of the inflammatory cytokine cascade. Moreover, due to their localization in the vascular bed, NO derived from these cells may contribute to the regulation of microcirculatory blood flow under physiologic as well as pathophysiologic conditions. G.H. is supported by a grant from the BONFOR-Kommission (160/11).
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