Treatment of no-reflow and impaired flow with the nitric oxide donor nitroprusside following percutaneous coronary interventions: initial human clinical experience.

OBJECTIVES: The objective of this study was to test the hypothesis that the intracoronary administration of a direct donor of nitric oxide is a safe and effective method to treat impaired blood flow (no-reflow phenomenon) that occurs during percutaneous transluminal coronary interventions (PTCI). BACKGROUND: The absence of blood flow or decreased blood flow in a coronary artery following PTCI despite the presence of a patent epicardial vessel or graft is designated "no-reflow" or "impaired flow." This alteration in blood flow is a serious complication of percutaneous revascularization strategies that results in an increased incidence of morbidity, myocardial infarction and mortality. METHODS: Nineteen consecutive patients undergoing standard percutaneous revascularization procedures complicated by either no-reflow or impaired flow that received intracoronary nitroprusside treatment were studied. One patient had two procedures performed on two separate grafts on two successive days. Interventions were performed on either saphenous vein grafts or native vessels and utilized angioplasty, stent deployment or rotational atherectomy strategies. Following interventions that were associated with impaired flow, varying total doses (of nitroprusside 50 to 1,000 microg) were administered into the coronary artery or saphenous vein graft. The angiographic archives before and after intracoronary administration of nitroprusside were analyzed for TIMI grade flow and a frame count method was used to quantitate blood flow velocity. RESULTS: Following a PTCI that resulted in either no-reflow or impaired flow, nitroprusside (median dose 200 microg) was found to lead to a highly significant and rapid improvement in both angiographic flow (p < 0.01 compared with pretreatment angiogram) and blood flow velocity (p < 0.01 compared with pretreatment angiogram). No significant hypotension or other adverse clinical events were associated with nitroprusside administration. CONCLUSIONS: The direct nitric oxide donor nitroprusside is an effective, safe treatment of impaired blood flow and no-reflow associated with PTCI. The use of nitroprusside to treat syndromes secondary to microvascular dysfunction may provide a novel therapeutic strategy for treating no-reflow or impaired blood flow following percutaneous interventions.

12-lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation.

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.

NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium.

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso-N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 microM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-D-penicillamine or the NO synthase (NOS) inhibitor 1 mM N(G)-nitro-L-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 microg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 microM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF(1alpha) release. LPS-stimulated COX-2 gene expression was decreased by approximately 50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF(1alpha) release. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.

Induction of angiotensin II subtype 2 receptor-mediated blood pressure regulation in synthetic diet-fed rats.

OBJECTIVE: Chronic feeding of a purified synthetic diet induces renin-angiotensin system-dependent moderate high blood pressure in normal Sprague-Dawley rats. The present study was designed to characterize the angiotensin II (Ang II) receptor type 2 (AT2)-specific mechanism of blood pressure regulation in these rats. METHODS: The effect of the AT2 receptor antagonist PD123319 (PD) on blood pressure was examined in vivo in synthetic diet-fed rats. Ang II-dependent contraction of aortic rings prepared from the synthetic diet-fed rats was also investigated. RESULTS: After 8 weeks of feeding the synthetic diet, the mean arterial pressure (MAP) was significantly elevated above levels measured in control rats (117 +/- 2 versus 102 +/- 3 mmHg, P < 0.05). Intravenous administration of PD to conscious hypertensive rats elicited an immediate dose-dependent increase in MAP that was sustained for approximately 7.4 min with 3 mg/kg PD. The angiotensin converting enzyme inhibitor captopril, but not the Ang II type 1 receptor blocker losartan, significantly attenuated the effect of PD on blood pressure. PD did not increase the plasma level of catecholamines. The PD-dependent blood pressure increase was not observed in normotensive control rats. Aortic ring assays revealed that functional activation of the AT2 receptor occurs only in the hypertensive rats, and this AT2 response is abolished by indomethacin (5 micromol/l) but not by Nomega-nitro-L-arginine methyl ester (100 Fmol/l). CONCLUSION: These results clearly demonstrate that AT2 receptor-mediated blood pressure regulation is functional in this experimental model of hypertension. Furthermore, cyclooxygenase metabolites might be the key mediators for the AT2 receptor-mediated blood pressure-lowering action.

Endothelial cell regulation of nitric oxide production during hypoxia in coronary microvessels and epicardial arteries.

Nitric oxide (NO) synthesized by endothelial cell nitric oxide synthase (eNOS) elicits vasodilation of resistance-sized coronary microvessels. Since coronary blood flow increases during hypoxia, we tested the hypotheses that: (1) hypoxia results in increased blood flow through increased NO production mediated by the upregulation of both eNOS mRNA and protein and (2) the regulation of NO production in response to hypoxia differs in microvascular endothelial cells and nonresistance, epicardial endothelial cells. Monocultures of vascular endothelium from resistance (approximately 100 micro) and nonresistance epicardial arteries were established and characterized. Nitric oxide was quantitated using a chemiluminescence method. Hypoxia (pO(2) = 10 mmHg) significantly increased NO production in both cell lines, with less NO produced in microvascular endothelium. Western blots demonstrated that hypoxia caused a time-dependent increase in eNOS protein in both lines, with an average 2.5-fold increase in nonresistance, epicardial endothelial cells compared to an average 1.7-fold increase in protein from microvascular endothelium. Total mRNA recovery increased 2.4 +/- 0.6-fold within 30 min of hypoxia in nonresistance, epicardial endothelial cells with no increase in microvascular endothelial cells. Although hypoxia increased NO production in both populations of endothelial cells, the increase in NO production and eNOS protein in microvascular endothelium was less compared to nonresistance, epicardial endothelial cells. Furthermore, there was no significant upregulation of total mRNA for eNOS in microvascular endothelium. The data indicate that increased NO production in microvascular endothelium during hypoxia may be through translational or posttranslational modifications of the enzyme, whereas transcriptional upregulation may account for the increased NO production in nonresistance, epicardial endothelial cells. Oxygen-sensitive response mechanisms that modulate NO production may be different in endothelium from different coronary artery vascular beds.

The direct release of nitric oxide by gypenosides derived from the herb Gynostemma pentaphyllum.

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Gypenosides are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum and are reported to be effective in the treatment of cardiovascular diseases; however, the mechanism underlying the therapeutic effect is not known. We tested the hypothesis that gypenosides extracted from G. pentaphyllum elicit vasorelaxation through the direct release of endothelium-derived nitric oxide. Nitric oxide production in bovine aortic endothelial cells grown under standard tissue culture conditions was quantitated using a chemiluminescence method. Arterial vasomotion was assayed using isolated porcine coronary artery rings under standard isometric recording conditions. The extract of G. pentaphyllum at 0.1-100 microg/ml elicited concentration-dependent vasorelaxation of porcine coronary rings that was antagonized by the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. Indomethacin had no significant effect on G. pentaphyllum-induced relaxation. The G. pentaphyllum extract elicited a concentration-dependent increase in nitric oxide production from cultured bovine aortic endothelial cells. At the concentrations utilized, there was no morphological evidence for cellular toxicity. These results demonstrate that extracts of G. pentaphyllum directly stimulate nitric oxide release, but not prostanoid production. Nitric oxide production in response to gypenosides may be one mechanism whereby this herbal medicine elicits its therapeutic effects.

Enhanced prostanoid-mediated vasorelaxation in pulmonary arteries isolated during experimental endotoxemia.

Endotoxemia secondary to gram-negative sepsis has been shown to inhibit endothelium-dependent vasomotion in numerous vascular beds, including guinea pig aortae and coronary arteries. We tested the hypothesis that in vivo endotoxin impairs endothelium-dependent nitric oxide-mediated relaxation responses of pulmonary arteries isolated from guinea pigs given intraperitoneal injections of Escherichia coli endotoxin lipopolysaccharide (LPS) or saline (control) 16 h before sacrifice. Pulmonary rings from the main artery and primary branches were isolated and studied in vitro using conventional isometric techniques. Interestingly, endotoxemia resulted in enhanced pulmonary artery relaxation in response to the endothelium-dependent receptor agonists acetylcholine (10(-10) -10(-5) M) and adenosine diphosphate (ADP; 10(-9) -10(-5) M), as compared with control responses (p < .05). Nitric oxide synthase inhibitors N-monomethyl-L-arginine (300 microM) and N-nitro-L-arginine methyl ester (100 microm) reduced acetylcholine- and adenosine diphosphate-mediated relaxation in both groups (p < .05); however, vasodilation responses in arteries from LPS animals remained enhanced relative to those of control arteries. In contrast to nitric oxide synthase inhibitors, the cyclooxygenase inhibitor indomethacin markedly inhibited acetylcholine- and adenosine diphosphate-mediated relaxation responses of pulmonary arteries isolated from LPS-treated animals (p < .05) but not control arteries; indomethacin effectively reversed LPS-induced enhanced vasodilation of pulmonary arteries. Relaxation responses to the receptor-independent calcium ionophore (A23187) and to the direct smooth muscle vasodilator sodium nitroprusside (+ N-nitro-L-arginine methyl ester) were not significantly altered by LPS treatment (p > .05). These data suggest that in pulmonary arteries, unlike aortae and coronary arteries isolated from the same model, in vivo LPS enhances agonist-mediated endothelium-dependent vasodilation responses to acetylcholine and adenosine diphosphate. Underlying mechanisms appear to involve increased dependency upon vasodilator prostanoids and decreased dependency on nitric oxide synthesis/release for LPS-induced alterations in pulmonary relaxation responses.

Ethanol increases endothelial nitric oxide production through modulation of nitric oxide synthase expression.

Moderate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO.

Lipopolysaccharides and cytokines downregulate the angiotensin II type 2 receptor in rat cardiac fibroblasts.

The present study examines the effect of lipopolysaccharides and proinflammatory cytokines on the expression of the second isoform of the angiotensin II receptor (AT(2)), which may have a role in lowering collagen deposition in cardiac tissue. Cardiac fibroblasts express high levels of both angiotensin II type 1 (AT(1)) and type 2 receptors. Incubation with lipopolysaccharides for 24 h dose- and time-dependently decreased angiotensin II AT(2) receptor expression with no apparent difference in the affinity. Actinomycin D, cycloheximide, N(omega)-nitro-L-arginine methyl ester and the protein tyrosine kinase inhibitor herbimycin A, but not the protein kinase C inhibitors bisindolylmaleimide and calphostin C, abolished the inhibitory action of lipopolysaccharides. The cytokines interleukin-1beta and tissue necrosis factor-alpha mimicked the effect of lipopolysaccharides. All three compounds induced inducible nitric oxide synthase (iNOS). The nitric oxide donor sodium nitroprusside and the cGMP analog 8-bromoguanosine cyclic monophosphate downregulated angiotensin II AT (2) receptor expression. The findings are consistent with the pathway in which lipopolysaccharides or cytokines induce iNOS. The data suggest that lipopolysaccharide- or cytokine-dependent induction of iNOS and resultant production of nitric oxide leads to the production of cGMP, which in turn downregulates expression of the angiotensin II AT (2) receptor in cardiac fibroblasts.

The upregulation of endothelin and its receptors in porcine coronary arteries in a double balloon injury model of restenosis.

Endothelin, a potent vasoconstrictor, mitogen, and stimulant of collagen synthesis, is reported to be increased after vascular injury. We tested the hypothesis that tissue endothelin levels and its receptor expression are increased following double balloon injury in a porcine coronary artery model of restenosis. Male miniature swine maintained on a hyperlipidemic diet underwent oversized balloon injury to both the proximal right coronary artery and left circumflex coronary artery. Two weeks following the initial injury, the arteries were repeat injured at the same site and subsequently harvested four weeks later. Proximal balloon injured (BI) and distal non-balloon-injured (NBI) segments from the same artery were collected. Tissue endothelin-1 (ET-1) levels were measured by ELISA. Endothelin receptors were assayed by radioligand binding using 125I-ET-1 and also immunolabeling. Tissue endothelin levels were 4-5 fold greater in BI arteries as compared to NBI. There was a significant increase in tissue ET-1 levels and endothelin receptor binding following double balloon injury relative to NBI control arteries. Western blots showed an increased expression of ET(A) receptor protein in injured vessels compared to non-injured arteries. Immunohistochemistry using an ET(A) receptor specific antibody confirmed increased receptor density following balloon injury. Thus, in an in vivo double balloon injury model for coronary artery restenosis, the response to vascular injury is increased tissue ET-1 content and upregulation of ET(A) receptor density associated with increased receptor protein.

Development of endothelium-dependent relaxation in canine coronary collateral arteries.

BACKGROUND: Little information exists regarding development of vasomotor control mechanisms during coronary collateral artery maturation. Therefore, we studied endothelium-dependent relaxation of canine collateral arteries isolated 2, 4, and 9 months after placement of an ameroid occluder around the proximal left circumflex coronary artery. RESULTS: Collateral arteries isolated after 2 months exhibited markedly reduced endothelium-dependent relaxation in response to acetylcholine (ACh; 10(-10) to 10(-4) mol/L) and bradykinin (BK; 10(-11) to 10(-6) mol/L) compared with relaxation of noncollateral coronary arteries (P<0.01). In contrast, endothelium-independent relaxation of collateral arteries to nitroprusside was only slightly reduced compared with relaxation of noncollateral arteries (P<0.05). Endothelium-dependent relaxation of collateral arteries isolated after 4 and 9 months was increased significantly, to the extent that relaxation to ACh and BK was not significantly different between collateral and noncollateral arteries at these periods. Inhibition of nitric oxide synthesis with NT-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L) markedly inhibited ACh-induced relaxation in all noncollateral arteries and in collateral arteries isolated after 9 months. However, neither L-NAME nor indomethacin (5 micromol/L) alone inhibited ACh-mediated relaxation of collateral arteries isolated after 4 months. ACh-induced relaxation of these collateral arteries was only inhibited when arteries were preconstricted with 30 mmol/L K+ and pretreated with L-NAME and indomethacin (ie, when synthesis/effects of nitric oxide, prostaglandins, and endothelium-derived hyperpolarizing factor were inhibited). CONCLUSIONS: Development of endothelium-dependent relaxation in canine coronary collateral arteries is not complete after 2 months. After 4 months, endothelium-dependent relaxation of collateral arteries is similar to relaxation of noncollateral arteries, but the relaxation exhibits decreased dependence on synthesis of nitric oxide and increased involvement of prostaglandins and endothelium-derived hyperpolarizing factor(s). After 9 months of development, collateral arteries exhibit normal nitric oxide-dependent relaxation, similar to noncollateral arteries.

Vascular endothelial cell regulation of extracellular matrix collagen: role of nitric oxide.

Endothelium-derived products have been implicated in the regulation of vascular wall structure through their effects on extracellular matrix metabolism. The purpose of this study was to further understand the paracrine mechanisms underlying endothelial cell regulation of extracellular matrix metabolism by testing the hypothesis that endothelium-derived nitric oxide decreases the concentration of soluble collagens derived from vascular smooth muscle cells (VSMCs). Porcine coronary endothelium and VSMCs were grown under a coculture configuration to assess the paracrine effects of nitric oxide produced by the endothelium on VSMC collagen types I and III. Endogenous endothelial cell nitric oxide production was blocked with N(omega)-nitro-L-arginine methyl ester. Collagen type I and type III were quantitatively measured using an enzyme-linked immunosorbent assay method. The endothelium elicited a time-dependent increase in the concentration of soluble VSMC-derived collagen type I; in contrast, collagen type III was decreased. After inhibition of nitric oxide production, there was a marked increase in both collagen types I and III concentration. These results demonstrated that endothelium-derived nitric oxide differentially alters collagen subtypes produced by VSMCs. The data support the hypothesis that nitric oxide functions via a paracrine mechanism to decrease VSMC collagen types I and III concentration, a finding consistent with an integral role for the endothelium in modulating extracellular matrix metabolism.

Nitric oxide modulates basal and endothelin-induced coronary artery vascular smooth muscle cell proliferation and collagen levels.

The mechanisms governing the pathological accumulation of collagen in the extracellular matrix following angioplasty are complex, but may involve interactions between endothelium-derived paracrine agents and vascular cellular components. We tested the hypothesis that nitric oxide (NO) directly decreases collagen levels and decreases endothelin (ET-1)-stimulated increases in levels of specific collagen subtypes in coronary vascular smooth muscle cells (VSMC). Cultured VSMC were incubated for 48 h with the NO donor CAS 754 (10(-4) M), ET-1 (10(-8) M), or ET-1 plus CAS 754. In some experiments, angiotensin II (Ang II; 10(-8) M) was utilized in place of ET-1. Soluble collagen types I and III were quantitated with an ELISA method, and cell counts were performed. CAS 754 significantly inhibited cell proliferation (-17+/-2% v control), basal total protein synthesis (-65+/-7% v control), and basal collagen type I levels (-39+/-6% v control), but not collagen type III levels. ET-1 and Ang II both significantly stimulated cell proliferation (26+/-5% v control), total protein synthesis (169+/-6% v control), and collagen type I levels (200+/-11% v control). Ang II, but not ET-1, significantly increased collagen type III levels. Co-incubations of ET-1 and CAS 754 resulted in a significant decrease in cell proliferation, protein synthesis, and collagen levels (-23+/-2% v control, 90+/-5% v control, and 63+/-3% v control, respectively) compared to ET-1 alone. In contrast, co-incubation of Ang II and CAS 754 had no significant effect on cell proliferation, protein synthesis, and collagen levels seen with Ang II alone. These results demonstrate that NO inhibits basal collagen levels and cell division. Additionally, NO alters ET-1 stimulation of VSMC proliferation, protein synthesis, and production of extracellular matrix components. Thus, an imbalance in key endothelium-derived compounds could significantly impact upon extracellular matrix deposition following mechanical revascularization.

Chronic endotoxemia and endothelium-dependent vasodilation in coronary arteries.

Endotoxin acutely decreases the production of nitric oxide, leading to abnormal regulation of coronary vascular tone: however, the effects of chronic endotoxemia on vasomotion are unknown. We therefore tested the hypothesis that chronic, low-level endotoxemia inhibits endothelium-dependent vasodilation. Rabbits were continuously infused with a subclinical dose of Escherichia coli endotoxin (.6 microgram/24 h, intraperitoneal) or saline for 5 wk. Endotoxin at this concentration elicited no significant sustained pyretic or hemodynamic responses. Both endothelium-dependent and independent vasomotor responses were determined in coronary arteries (250-500 mu). Vasorelaxation in response to acetylcholine, but no adenosine diphosphate (ADP), was significantly enhanced in endotoxin-challenged animals (EC50 = 62.6 +/- 11.1 nM, control vs. 33.97 +/- 5.7 nM, endotoxin-challenged; p < .05). Vasoconstriction to PGF2 alpha, but not KCl, was significantly decreased in endotoxin-challenged animals. These results indicate that endothelium-dependent and independent vasomotor responses are altered during chronic endotoxemia and are due, in part, to alterations in signal-transduction mechanisms specific for certain types of receptors.

Differences in nitric oxide production in porcine resistance arteries and epicardial conduit coronary arteries.

The purpose of this study was to test the hypothesis that endothelial cells from resistance arteries and epicardial conduit coronary arteries differ in their expression of nitric oxide synthase (NOS) and calcium metabolism, and that these differences contribute to the mechanism underlying disparate physiological vasodilator responses observed between the two populations of vessels. The functional vasodilator responses of isolated resistance arteries and epicardial conduit coronary arteries were compared in vitro using both the receptor-independent agonist A23187 ionophore to increase intracellular calcium and the receptor-dependent agonist bradykinin. Constitutive NOS (cNOS) activity in monocultures of endothelial cells derived from resistance arteries and conduit arteries was assayed using a fibroblast-reporter cell method. Intracellular calcium concentration was assessed using fura-2 microfluorometry. Nitric oxide production was determined using a chemiluminescence technique, while cNOS protein was quantitated by Western blot analysis. A23187 was a less potent vasodilator of resistance arteries studied in vitro, compared to epicardial conduit arteries (EC50 = 1.6 microM, resistance artery vs. EC50 = 0.03 microM, conduit artery); however, bradykinin was more potent in resistance arteries (EC50 = 0.3 nM, resistance artery vs. EC50 = 2 nM, conduit artery). In pure monocultures of endothelium, nitric oxide production measured by chemiluminescence both basally and in response to A23187 was significantly less in resistance arteries (6.1 +/- 0.5, basal vs. 10.80 +/- 0.55, stimulated nmol/microgram protein), compared to conduit arteries (7.7 +/- 0.5, basal vs. 17.00 +/- 1.52, stimulated nmol/microgram protein; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). cNOS enzyme activity assessed by cGMP production in reporter cell fibroblasts was also lower in resistance arteries compared to conduit arteries (0.17 +/- 0.03 vs. 0.33 +/- 0.05 fmol cGMP/microgram protein, respectively; P < 0.05 resistance artery endothelium vs. conduit artery endothelium). Conduit arteries expressed 2.1 x more cNOS protein than resistance arteries, as assessed by Western blotting of cellular homogenates. No significant differences were found with microfluorimetry in either basal or ionophore-stimulated intracellular calcium concentrations. The results signified that porcine resistance arteries expressed less NOS and produced less nitric oxide than epicardial conduit arteries both basally and in response to an increase in intracellular calcium. This difference was reflected functionally as a decreased vasodilatory response to increased intracellular calcium in resistance arteries that could not be explained on the basis of differences in the metabolism of intracellular calcium. In contrast, the functional vasodilator response of intact vessels to a receptor-mediated agonist was enhanced in resistance arteries compared to conduit arteries, suggesting an important role of signal transduction mechanisms in specific physiological responses. Thus, the ability of the endothelium to regulate on a regional basis the expression of NOS and integrate receptor-mediated responses with these differences may provide a mechanism for diverse vasomotor responses in different populations of vessels.

Restenosis is associated with decreased coronary artery nitric oxide synthase.

The purpose of the present study was to test the hypothesis that restenosis is associated with decreased constitutive nitric oxide synthase activity. Male miniswine with moderately elevated serum cholesterol levels underwent cardiac catheterization and oversized balloon injury to the right and left circumflex coronary arteries, followed 2 weeks later by repeat injury on the same coronary segments. After 4 weeks, the coronary arteries were either immediately frozen in liquid nitrogen or pressure-perfusion fixed and prepared for histologic examination. Constitutive nitric oxide synthase activity was quantified using a fibroblast reporter cell method, while constitutive nitric oxide synthase protein was compared between balloon-injured and non-balloon-injured arteries using Western blot analysis. Immunohistochemical studies were performed using a specific antibody against constitutive nitric oxide synthase protein. Following balloon injury, there was decreased constitutive nitric oxide synthase activity in balloon-injured coronary arteries, compared to distal non-balloon-injured segments from the same artery. Histological examination demonstrated an intact endothelium. Specific antibody staining revealed that there was less constitutive nitric oxide synthase protein reactivity by immunohistochemical analysis. Western analysis confirmed less constitutive nitric oxide synthase protein. The data are consistent with the hypothesis that restenosis is associated with decreased endothelial cell nitric oxide production. The data suggest this is secondary to a decreased amount of constitutive nitric oxide synthase enzyme in the endothelium. A deficiency in constitutive nitric oxide synthase enzyme may contribute to the impaired second messenger and paracrine functions of the endothelium observed during restenosis following balloon injury, including abnormal vasomotion, extracellular matrix formation, and platelet aggregation.

Effects of oxygen tension on flow-induced vasodilation in porcine coronary resistance arterioles.

Vascular tone has been shown to be importantly influenced by flow-induced release of endothelium-derived vasodilators. The purpose of the present study was to test the hypothesis that in porcine coronary resistance-size arterioles, flow-induced vasodilation is sensitive to oxygen tension. Arterioles (55-150 mu m) were studied in vitro under conditions of constant intraluminal pressure to dynamically measure arteriolar diameter in response to changes in flow or, alternatively, in response to bradykinin under three conditions: hyperoxia (pO(2) 400 mm Hg), normoxia (pO(2) 160 mm Hg), and hypoxia (p0(2) 40 mm Hg). Under conditions of constant pressure and no flow, hypoxia alone resulted in vasodilation that was blocked by the nitric oxide synthase inhibitor omega-nitro-L-arginine methyl ester (L-NAME). Hypoxia did not alter the vasodilator response to bradykinin when compared to the vasodilator response to bradykinin during normoxia. During hyperoxia, flow-induced vasodilation was significantly reduced by either indomethacin, or L-NAME. Indomethacin and L-NAME combined completely abolished flow-induced vasodilation under conditions of hyperoxia. Under conditions of normoxia and hypoxia, indomethacin or L-NAME alone only partially blocked flow-induced vasodilation. No further inhibition was observed when indomethacin and L-NAME were combined. Glybenclamide failed to alter flow-induced vasodilation either alone or in combination with indomethacin and L-NAME. The results suggest that the mechanisms responsible for flow-induced vasodilation in coronary arterioles are complex and are different depending upon the oxygen tension. During hyperoxia, vasodilation is due to the combined actions of prostanoids and nitric oxide, while under conditions of normoxia and hypoxia, flow-induced vasodilation is the result of not only prostanoids and nitric oxide, but of another as of yet unidentified oxygen-sensitive endogenous vasodilator.

Extracellular matrix collagen synthesis and degradation following coronary balloon angioplasty.

Percutaneous transluminal coronary angioplasty is associated with intimal hyperplasia and extracellular matrix deposition of collagen, leading to restenosis in a significant number of cases. The purpose of the present study was to determine the effects of balloon angioplasty on extracellular matrix collagen content and collagenase activity in a porcine coronary artery restenosis model 6 weeks following balloon injury. We tested the hypothesis that in balloon-injured arteries the neointimal extracellular matrix was characterized by increased collagen content and decreased metalloproteinase activity relative to non-injured arteries. Male miniswine maintained on a high cholesterol diet underwent cardiac catheterization and double balloon injury to the right and left circumflex coronary arteries. The coronary arteries were either pressure-perfusion-fixed and prepared for histological examination, or dissected free of adventitia for further collagen and matrix metalloproteinase studies. Collagen synthesis in balloon-injured coronary arteries was compared to non-injured arteries using Northern blot analysis and histochemical stains. Comparative studies on differences between balloon-injured and non-balloon-injured arterial matrix metalloproteinase activity were done using zymography. Balloon angioplasty arterial injury resulted in a significant increase in type I collagen mRNA expression, with increased collagen deposition in the extracellular matrix. In contrast, matrix metalloproteinase activity was markedly decreased. The results suggest that the increased neointimal extracellular matrix observed late in the injury response may be due to not only increased collagen synthesis, but also reduced degradation. The failure to achieve a balance between the synthesis and degradation of extracellular matrix collagen could serve as an important mechanism responsible for restenosis.

The effects of endothelin-1 on collagen type I and type III synthesis in cultured porcine coronary artery vascular smooth muscle cells.

Restenosis is the single most important factor limiting a favorable long-term outcome following mechanical revascularization. The vascular endothelium, through the release of key regulatory compounds, may regulate vascular structure by exerting fundamental control over collagen synthesis following injury to the vessel wall. We tested the hypothesis that endothelin (ET-1), an endothelium-derived peptide previously shown to be increased in pathological states, differentially stimulates porcine coronary vascular smooth muscle cell collagen types I and III synthesis. Monocultures of porcine coronary vascular smooth muscle were exposed to varying concentrations of endothelin over a 24-96-h time period. The medium was assayed for soluble collagen types I and III using a sensitive and specific ELISA method. Experiments were also done with the ET-1 antagonists PD 145065 and BQ123. Cell counts and viability were serially monitored. Experiments were also conducted with angiotensin II (A-II). A-II and ET-1 stimulated cell proliferation. ET-1 maximally stimulated collagen type I synthesis at 48 h at an optimal concentration of 10(-8) M, with no significant stimulation of collagen type III synthesis. The ETA specific antagonist BQ123 significantly inhibited the stimulatory effects of ET-1. A-II also stimulated collagen type I synthesis above basal levels, but was less efficacious than endothelin (95 +/- 5%, A-II, v 189 +/- 14% ET-1). In contrast to ET-1, A-II stimulated collagen type III synthesis (31 +/- 6% above basal, compared to -4 +/- 5% for ET-1). Results are also reported using smooth muscle cells from porcine aorta. The data demonstrate that ET-1 and A-II stimulate collagen synthesis by coronary artery vascular smooth muscle, and that they exert a differential effect over the two types of collagen that are present in the intima following balloon injury. Thus, the over expression of key regulatory compounds by endothelium following balloon injury could enhance collagen deposition and, consequently, play an integral role in intimal hyperplasia and restenosis.

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells.

OBJECTIVE: Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. METHODS: Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. RESULTS: Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. CONCLUSIONS: These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.