PR39, a peptide regulator of angiogenesis.

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1alpha protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1alpha degradation may underlie the mechanism of inflammation-induced angiogenesis.

Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis.

Coronary occlusive disease is the leading cause of death in industrial nations and affects one in four adults. Although heart attacks are caused by occlusion of a coronary artery, some patients have occlusions without infarction because they have sufficient collateral vessels providing an alternate pathway for blood supply. Vascular endothelial growth factor (VEGF) is an angiogenic peptide that can stimulate collateral vessel development in the ischaemic myocardium. We used magnetic resonance imaging (MRI) and image processing to identify and quantify non-invasively the benefits related to VEGF infusion on collateral development in the heart. This was accomplished as a placebo-controlled study in the porcine model of chronic ischaemia that most closely mimics the human pathophysiology of progressive coronary occlusion. Image series converted to a space-time map demonstrated that with treatment the ischaemic zone was smaller and the contrast arrival delay was less, which resulted in better ejection fraction and regional wall thickening. These findings demonstrate in a manner applicable to humans, that VEGF improves collateral blood supply, resulting in improved cardiac global and regional function after and in spite of coronary artery occlusion.

Basic fibroblast growth factor improves myocardial function in chronically ischemic porcine hearts.

The effect of basic fibroblast growth factor (bFGF) administration on regional myocardial function and blood flow in chronically ischemic hearts was studied in 26 pigs instrumented with proximal circumflex coronary artery (LCX) ameroid constrictors. In 13 animals bFGF was administered extraluminally to the proximal left anterior descending (LAD) and LCX arteries with heparin-alginate beads and 13 other animal served as controls. bFGF-treated pigs showed a fourfold reduction in left ventricular infarct size compared to untreated controls (infarct size: 1.2 +/- 0.4% vs. 5.1 +/- 1.3% of LV mass, mean +/- SEM, P < 0.05). Percent fractional shortening (% FS) in the LCX area at rest was reduced compared with the LAD region in both bFGF and control pigs. However, there was better recovery in the LCX area after rapid pacing in bFGF-treated pigs (% FSLCX/% FSLAD, 22.9 +/- 7.3%-->30.5 +/- 8.5%, P < 0.05 vs. prepacing) than in controls (16.0 +/- 7.8%-->14.3 +/- 7.0%, P = NS). Furthermore, LV end-diastolic pressure rise with rapid pacing was less in bFGF-treated than control pigs (pre-pacing; pacing; post-pacing, 10 +/- 1; 17 +/- 3; 11 +/- 1* mmHg vs 10 +/- 1; 24 +/- 4; 15 +/- 1 mmHg, *P < 0.05 vs. control). Coronary blood flow in the LCX territory (normalized for LAD flow) was also better during pacing in bFGF-treated pigs than in controls. Thus, periadventitial administration of bFGF in a gradual coronary occlusion model in pigs results in improvement of coronary flow and reduction in infarct size in the compromised territory as well as in prevention of pacing-induced hemodynamic deterioration.

Biochemical and structural evidence for pig myocardium adherens junction disruption by cardiopulmonary bypass.

BACKGROUND: Given that cardiopulmonary bypass (CPB) is associated with edema and heart dysfunction and that adherens junctions may regulate vascular permeability barrier integrity and cardiomyocyte function, we investigated adherens junction protein steady-state levels in a pig model of CPB. METHODS AND RESULTS: Pigs were subjected to normothermic CPB for 90 minutes, followed by post-CPB perfusion for 90 minutes. Atrial and ventricular myocardium tissue samples were harvested before institution of bypass (basal levels) and at the end of post-CPB perfusion. Adherens junctions were analyzed by either total lysate or cadherin immunoprecipitates that were immunoblotted for pan-cadherin, VE-cadherin, beta-catenin, and gamma-catenin. Adherens junction solubility was addressed with Triton X-100 extraction. Frozen tissue sections were labeled with the same antibodies, and adherens junctions were visualized by confocal microscopy. Immunoblotting of total lysates revealed an increase in smaller-molecular-weight fragments of VE-cadherin, beta-catenin, and gamma -catenin after post-CPB perfusion, indicating partial protein degradation. Smaller-molecular-weight fragments recognized by VE-cadherin and beta-catenin antibodies were also obtained from VE-cadherin immunoprecipitation, indicating degradation of endothelial cell adherens junctions. A prominent increase in adherens junction complex solubility was observed in post-CPB perfusion samples. Confocal microscopy of hearts obtained before CPB showed a continuous, homogeneous pattern of cell-cell labeling that contrasted with an irregular, discontinuous, punctuate, or zigzag pattern observed in post-CPB perfusion samples, corroborating biochemical data. CONCLUSIONS: These results indicate that CPB is associated with signs of degradation of endothelial and cardiomyocytes adherens junctions, pointing to a molecular mechanism leading to increased vascular permeability and cardiomyocyte dysfunction.

Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery: results of a phase I randomized, double-blind, placebo-controlled trial.

BACKGROUND: Angiogenesis is a promising treatment strategy for patients who are not candidates for standard revascularization, because it promotes the growth of new blood vessels in ischemic myocardium. METHODS AND RESULTS: We conducted a randomized, double-blind, placebo-controlled study of basic fibroblast growth factor (bFGF; 10 or 100 microg versus placebo) delivered via sustained-release heparin-alginate microcapsules implanted in ischemic and viable but ungraftable myocardial territories in patients undergoing CABG. Twenty-four patients were randomized to 10 microg of bFGF (n=8), 100 microg of bFGF (n=8), or placebo (n=8), in addition to undergoing CABG. There were 2 operative deaths and 3 Q-wave myocardial infarctions. There were no treatment-related adverse events, and there was no rise in serum bFGF levels. Clinical follow-up was available for all patients (16.0+/-6.8 months). Three control patients had recurrent angina, 2 of whom required repeat revascularization. One patient in the 10-microg bFGF group had angina, whereas all patients in the 100-microg bFGF group remained angina-free. Stress nuclear perfusion imaging at baseline and 3 months after CABG showed a trend toward worsening of the defect size in the placebo group (20.7+/-3.7% to 23.8+/-5.7%, P=0.06), no significant change in the 10-microg bFGF group, and significant improvement in the 100-microg bFGF group (19.2+/-5.0% to 9.1+/-5.9%, P=0.01). Magnetic resonance assessment of the target ischemic zone in a subset of patients showed a trend toward a reduction in the target ischemic area in the 100-microg bFGF group (10.7+/-3.9% to 3. 7+/-6.3%, P=0.06). CONCLUSIONS: This study of bFGF in patients undergoing CABG demonstrates the safety and feasibility of this mode of therapy in patients with viable myocardium that cannot be adequately revascularized.

Clinical trials in coronary angiogenesis: issues, problems, consensus: An expert panel summary.

The rapid development of angiogenic growth factor therapy for patients with advanced ischemic heart disease over the last 5 years offers hope of a new treatment strategy based on generation of new blood supply in the diseased heart. However, as the field of therapeutic coronary angiogenesis is maturing from basic and preclinical investigations to clinical trials, many new and presently unresolved issues are coming into focus. These include in-depth understanding of the biology of angiogenesis, selection of appropriate patient populations for clinical trials, choice of therapeutic end points and means of their assessment, choice of therapeutic strategy (gene versus protein delivery), route of administration, and the side effect profile. The present article presents a summary statement of a panel of experts actively working in the field, convened by the Angiogenesis Foundation and the Angiogenesis Research Center during the 72nd meeting of the American Heart Association to define and achieve a consensus on the challenges facing development of therapeutic angiogenesis for coronary disease.

Efficacy of intracoronary or intravenous VEGF165 in a pig model of chronic myocardial ischemia.

OBJECTIVES: We sought to optimize vascular endothelial growth factor (VEGF) treatment for therapeutic angiogenesis in myocardial ischemia, we explored the efficacy of five different regimens. BACKGROUND: Although VEGF165 is one of the most potent pro-angiogenic growth factors, VEGF165 treatment for myocardial ischemia has been hampered by low efficacy and dose-limiting hypotension after systemic or intracoronary delivery. METHODS: This study evaluated the effect of intravenous or intracoronary rhVEGF165 in the presence or absence of nitric oxide (NO) synthase inhibition in a porcine model of chronic myocardial ischemia. Forty-two Yorkshire pigs with chronically occluded left circumflex coronary arteries were randomly assigned to receive 10 microg/kg of VEGF165: 1) rapid (40 min) intravenous VEGF165 0.25 microg/kg/min, 2) slow (200 min) intravenous VEGF165 0.05 microg/kg/min, 3) rapid intracoronary VEGF165 0.25 microg/kg/min, 4) rapid intracoronary VEGF165 0.25 microg/kg/min + nitro-L-arginine methyl ester hydrochloride (L-NAME) or 5) rapid vehicle infusion. RESULTS: Intracoronary and intravenous VEGF165 induced hypotension. Intracoronary VEGF-induced hypotension was blocked by L-NAME. Coronary angiography three weeks after treatment showed improvement in collateral index in both intracoronary groups but not the intravenous VEGF165 groups. Likewise, myocardial blood flow and microvascular function in the ischemic territory improved in both intracoronary groups but not in the intravenous groups. Global and regional myocardial function showed no significant improvements in any groups. CONCLUSIONS: Intracoronary infusion of VEGF165 significantly improves blood flow to the ischemic myocardium. Concomitant administration of L-NAME inhibits VEGF-induced hypotension while most likely preserving VEGF-induced angiogenesis. Intravenous infusion of VEGF165 was not effective in augmenting either myocardial flow or function in this model.

Responses of porcine epicardial venules to neurohumoral substances.

OBJECTIVE: The coronary microvenous system may be determinant of ventricular distensibility and of resistance to coronary flow under conditions of arteriolar dilatation. Because responses of venules have not been well defined, in vitro responses of porcine venules and arterioles to neurohumoral substances were examined. METHODS: Porcine subepicardial venules (125-250 microns in diameter) and arterioles (100-200 microns in diameter) were studied in a pressurised no flow state with video microscopical imaging and electronic dimension analysis. After precontraction of vessels with the thromboxane A2 analogue U46619, agents were applied extraluminally. RESULTS: Responses of precontracted venules to sodium nitroprusside, adenosine, and adenosine 5' diphosphate (ADP) were comparable with those of similarly sized arterioles. By contrast, precontracted venules responded minimally to noradrenaline, whereas arterioles were dilated potently by this agent. Precontracted arterioles were dilated minimally by serotonin and were contracted slightly by acetylcholine, whereas venules dilated in response to either serotonin or acetylcholine. Serotonin and acetylcholine both produced greater contraction in non-precontracted arterioles than in non-precontracted venules. Responses of venules to most substances were minimally affected by changes in oxygen tension except when this was very low. Relaxation of venules by ADP, serotonin, and acetylcholine were all inhibited to varying degrees by indomethacin, methylene blue, and NG-methyl-L-arginine (L-NMMA). Both methylene blue and L-NMMA produced minimal but significant contraction of non-precontracted venules, indicating a small amount of basal release of endothelium derived relaxing factor. CONCLUSION: Responses of coronary venules to acetylcholine, serotonin, and noradrenaline are opposite or of a significantly different magnitude when compared with the respective arteriolar responses. Relaxation of venules to ADP, serotonin, and acetylcholine is likely mediated at least in part by the release of endothelium derived relaxing factor and prostaglandins.

Therapeutic angiogenesis in cardiology using protein formulations.

Therapeutic angiogenesis in cardiovascular disease aims at improving myocardial function by increasing blood flow to ischemic myocardium that is not amenable to traditional forms of revascularization. Preclinical data have provided proof of the concept that angiogenic growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelium growth factor (VEGF) may indeed improve myocardial flow and function when administered in ways that ensure prolonged tissue exposure to these short-lived molecules. Although other cytokines have been shown to enhance angiogenesis in vivo, FGF-2 and VEGF have been most widely studied and may serve as prototype proangiogenic drugs. Currently, several delivery techniques that are clinically applicable are being studied with respect to tissue distribution and retention as well as angiogenic efficacy of FGF-2 and VEGF. Although tissue distribution and retention of FGF-2 after intramyocardial injection compares favorably with other routes of administration, efficacy studies are not yet conclusive. At the same time, different protein- and gene-based formulations are being investigated. Arguments for and against protein and gene therapy are presented, showing that protein-based therapy seems to have advantages over gene therapy at the present time, although continuous efforts should be made to increase the tissue exposure time after a single administration of protein. While delivery systems and growth factor formulations are being improved, double-blind, placebo-controlled trials designed with existing animal data in mind, are needed to firmly establish the utility of therapeutic angiogenesis in cardiovascular disease.

Myogenic and agonist induced responses of coronary venules after cold hyperkalaemic cardioplegia.

OBJECTIVE: The aim was to examine agonist induced and myogenic venular responses after crystalloid cardioplegia in conditions simulating cardiopulmonary bypass. METHODS: Hearts of pigs were arrested with cold hyperkalaemic ([K+] = 25 mM) crystalloid cardioplegic solution for 1 h under conditions of cardiopulmonary bypass. In another group, hearts were arrested and then reperfused with warm blood for 1 h while being separated from cardiopulmonary bypass. In a third group, animals were supported on cardiopulmonary bypass for 75 min without diversion of coronary blood flow. Hearts from non-instrumented animals served as controls. Coronary venules (91-197 microns in internal diameter) were studied in vitro in a pressurised no flow state using video microscopy. Agonist induced responses were assessed in vessels precontracted with the thromboxane A2 analogue U46619. RESULTS: Endothelium dependent relaxations to adenosine diphosphate (ADP, P = 0.11 v control), or serotonin (P = 0.67), and endothelium independent relaxations to the beta adrenergic cyclic AMP mediated agonist isoprenaline (P = 0.20), adenosine (P = 0.98), or the KATP channel opener pinacidil (P = 0.40) were not significantly altered after cold cardioplegia alone. After cardioplegia followed by 1 h of warm blood reperfusion, venular responses to ADP (P = 0.003 v control), isoprenaline (P = 0.013), adenosine (P = < 0.001), and pinacidil (P = 0.005) were reduced compared to the respective control responses, while the response to serotonin (P = 0.97) remained unchanged. Endothelium independent cyclic GMP mediated relaxation to sodium nitroprusside was similar in all groups (P > 0.90). Myogenic reactivity was assessed after incremental increases in the intraluminal pressure from 2-40 mm Hg. As intraluminal pressure was increased, the diameter of control venules increased and reached a plateau. Following cardioplegia, the pressure-diameter relationship of venules was shifted upward (P = 0.04 v control) suggesting impaired myogenic tone. After reperfusion, myogenic tone partially recovered. Extracorporeal circulation without diversion of coronary perfusion did not significantly affect venular responses. CONCLUSIONS: Ischaemic cardioplegia using a cold hyperkalaemic solution under conditions of cardiopulmonary bypass does not significantly alter agonist induced venular responses, whereas myogenic contraction is slightly reduced. After 1 h of reperfusion, agonist induced relaxations of coronary venules are significantly impaired, whereas myogenic contraction recovers. These findings may have implications for the control of myocardial perfusion and diastolic properties of the heart after ischaemic cardioplegia under conditions of extracorporeal circulation.

Mechanisms causing coronary microvascular dysfunction following crystalloid cardioplegia and reperfusion.

OBJECTIVE: The aim was to examine the mechanisms of coronary microvascular dysfunction during cardiopulmonary bypass and ischaemic arrest using a crystalloid cardioplegic solution. METHODS: Porcine hearts were arrested with cold hyperkalaemic (K+ = 25 mmol.litre-1) cardioplegic solution for 1 h during cardiopulmonary bypass and then reperfused for 1 h. Selected hearts were arrested but not reperfused. Coronary vessels of non-instrumented pigs were used as controls. In vitro vascular responses of subepicardial and subendocardial arterioles were examined in a pressurised (40 mm Hg) no flow state with video microscopy. RESULTS: Following 1 h of ischaemic cardioplegia, endothelium dependent relaxations of epicardial arterioles to the receptor mediated agent ADP and the non-receptor-mediated agent calcium ionophore A23187 were moderately reduced, and the contractile responses to KCl or the thromboxane A2 analogue U46619 were reduced compared to responses of vessels from control animals. After 1 h of reperfusion, U46619 caused contraction greater than control values, while contraction to KCl and endothelium dependent relaxations to ADP or A23187 were further reduced. Responses of endocardial microvessels to serotonin were slightly more affected by cardioplegia and reperfusion than were epicardial vessels, while the effect on responses of epicardial and endocardial vessels to bradykinin or A23187 were similar. Endothelium independent relaxation to sodium nitroprusside was not altered in any of the experimental groups. The addition of manganese superoxide dismutase to the cardioplegic solution markedly preserved endothelium dependent responses to ADP and A23187 and contractile response to U46619, compared to the responses of vessels from the plain crystalloid cardioplegia group, but had no effect on relaxation to sodium nitroprusside or on contraction to KCl. Five hours of normokalaemic hypothermia (5-10 degrees C) in Krebs buffer had minimal effect on vasodilator responses. Electron microscopy revealed preserved endothelial and smooth muscle cell structure, and focal mononuclear leucocyte-endothelium adherence following cardioplegic arrest and reperfusion. CONCLUSIONS: Ischaemic cardioplegia-reperfusion induced endothelium dependent and direct smooth muscle microvascular dysfunction is at least partially mediated by prolonged exposure of vessels to hyperkalaemia and to the generation of oxygen derived free radicals. Leucocytes probably mediate injury during reperfusion, while hypothermia has minimal effect on recovery of vasomotor function.

VEGF administration in chronic myocardial ischemia in pigs.

OBJECTIVE: Previous investigations have shown the effectiveness of sustained intra- or extravascular administration of vascular endothelial growth factor (VEGF) in chronic myocardial ischemia in improvement of left ventricular function. The present investigations were undertaken in order to evaluate efficacy of a single bolus or local intracoronary delivery. METHODS: Yorkshire pigs underwent placement of a left circumflex artery ameroid occluder. Three weeks later the animals were randomized to treatment with VEGF (20 micrograms) accomplished by local intracoronary delivery system (InfusaSleeve, n = 10), intracoronary bolus infusion (n = 7) or by epicardial implantation of an osmotic delivery system (n = 7). An additional group of animals received intracoronary administration of saline and served as a control (n = 9). Three weeks after initiation of therapy, the animals were evaluated with regard to myocardial perfusion and global as well as regional ventricular function. RESULTS: All three VEGF treatment groups but not the control animals demonstrated a significant increase in the left-to-left (but not right-to-left) collateral index, myocardial blood flow (pre-therapy LCX vs. LAD (average of all groups): 0.76 +/- 0.35 vs. 0.96 +/- 0.38 ml*min-1*g-1, p = 0.03; post-therapy: LCX vs. LAD: 1.16 +/- 0.39 vs. 1.15 +/- 0.28 ml*min-1*g-1, p = NS) and coronary vasodilatory reserve 3 weeks after growth factor administration. The observed increase in VEGF-induced perfusion correlated with improvement in regional ventricular function in all VEGF-treated groups (pre-therapy vs. post-therapy: i.c. VEGF 20 +/- 5.1 vs. 33 +/- 4.8; local VEGF 16 +/- 2.8 vs. 33.6; pump VEGF 17 +/- 3.8 vs. 34 +/- 4.9 p < 0.05 for all) but not control animals (21 +/- 3.3 vs. 27 +/- 5.8, p = NS). CONCLUSION: Single intracoronary delivery (intravascular bolus or local delivery) of VEGF is effective in stimulating physiologically significant angiogenesis in porcine model of chronic myocardial ischemia.

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.

COS cells expression cloning of tyrosine-phosphorylated proteins by immunocytochemistry.

Tyrosine phosphorylation is an important post-translational modification of proteins, essential in many aspects of the cell economy, particularly in signal transduction pathways. Despite the importance of protein tyrosine phosphorylation, the approaches available for molecular cloning remain limited. We have developed a COS cell-based eukaryotic expression cloning procedure for phosphotyrosine-containing proteins by immunocytochemistry of cell monolayers. The approach takes advantage of the low basal levels of tyrosine phosphorylated, robust transient expression, availability of specific antibodies against tyrosine-phosphorylated residues, and rescue of episomal DNA after immunocytochemistry. The technique is validated by cloning the rat proto-oncogene c-fgr in its tyrosine-phosphorylated form out of a rat kidney cDNA library containing over 10(6) primary recombinants. This technique set the grounds for expression cloning of tyrosine-phosphorylated proteins in eukaryotic cells, and it is anticipated that further modifications and refinements will allow the identification of protein tyrosine phosphatase substrates.

Normal and pathophysiologic considerations of endothelial regulation of vascular tone and their relevance to nitrate therapy.

During the past decade, it has become clear that the vascular endothelium critically influences vascular permeability, controls vessel growth, modulates hemostasis, and regulates vasomotion. This latter role of the endothelium is mediated by the liberation of a number of potent vasoactive compounds, including endothelium-derived relaxing factors, one of which is either nitric oxide or a compound that releases nitric oxide, vasoactive prostaglandins, hyperpolarizing factors, and a number of constricting factors. This role of the endothelium is dramatically altered by several diseases, including atherosclerosis, hypertension, and diabetes. Abnormalities of endothelial regulation of vascular tone may contribute to a number of clinical syndromes, including variant angina, unstable angina, syndrome X, and perhaps many others. In this review, several aspects of the endothelium-derived relaxing factor will be considered, including recent concepts regarding its synthesis, its chemical identity, and alterations in atherosclerosis. Finally, its action in the coronary microcirculation as contrasted to that of nitroglycerin will be considered.

Angiogenesis in cardiovascular disease: current status and therapeutic potential.

Therapeutic angiogenesis, in the form of growth factor protein administration or gene therapy, has emerged as a new method of treatment for patients with severe, inoperable coronary artery disease. Improved myocardial perfusion and function after the administration of angiogenic growth factors has been demonstrated in animal models of chronic myocardial ischaemia. Recently, preliminary clinical trials using growth factor proteins or genes encoding these angiogenic factors have demonstrated clinical and other objective evidence of relevant angiogenesis. Thus, therapeutic angiogenesis has the potential to extend treatment options to patients who are not optimal candidates for conventional methods of myocardial revascularisation.

Mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis.

This study was performed to assess the effects of subacute sepsis in rats on the in vitro reactivity of arterioles (internal diameter, 100-150 microm) to alpha1- and alpha2-adrenergic stimulation and to angiotensin II. Male Sprague-Dawley rats were rendered septic by intraperitoneal implantation of a gelatin capsule containing sterile rat feces and 1 x 10(6) viable colony forming units of Escherichia coli. Control rats underwent sham laparotomy and implantation of a gelatin capsule containing only sterile feces. In vitro reactivity of arterioles from mesentery and skeletal muscle were studied 48 h later in a pressurized (50 mmHg) no flow state using videomicroscopy. Subacute sepsis decreased the contractile response of nonprecontracted microvessels from both anatomical sites to phenylephrine (both p < .01 versus control) and blunted the relaxation response to staurosporine (both p < .01), an inhibitor of protein kinase C. The small contraction to angiotensin II of mesenteric vessels was inhibited by sepsis (p < .05) but was unaltered in the skeletal muscle microcirculation. In the precontracted mesenteric microvessels from septic rats, endothelium-dependent relaxation to clonidine and to adenosine 5'-diphosphate were decreased (both p < .01 versus control), whereas in skeletal muscle microvessels, clonidine and adenosine 5'-diphosphate elicited constriction (both p < .01). Relaxation to the endothelium independent vasodilators sodium nitroprusside and pinacidil was preserved across all vessels. In conclusion, mesenteric and skeletal muscle microvascular responses to angiotensin II and alpha1- and alpha2-adrenergic stimulation are altered in subacute sepsis. This may in part lead to systemic hypotension and altered organ perfusion during states of chronic sepsis.

Escherichia coli endotoxemia alters coronary and pulmonary arteriolar responses to platelet products.

In order to examine the effects of Escherichia coli endotoxemia on coronary and pulmonary microvascular responses to serotonin (5-HT) and ADP, arterioles (80-190 micros diameter) were isolated from pigs 3 h after administration of E. coli endotoxin (150 micrograms/kg, intravenously over 1 h, n = 8) or Ringer's lactate (control, n = 8). Arterioles were studied in vitro in a pressurized, partially contracted, no-flow state with video-microscopy. Precontracted (30-50% of baseline diameter) control coronary arterioles dilated in responses to either 5-HT (24 +/- 2%) or ADP (89 +/- 2%). These relaxations were partially inhibited by indomethacin, but were markedly reduced with nitric oxide synthase inhibition. After 3 h of endotoxemia, 5-HT caused contraction of coronary arterioles which was inhibited with indomethacin. In the presence of indomethacin, coronary vessels from endotoxic pigs relaxed slightly, but significantly, more to 5-HT than did control vessels exposed to indomethacin. In contrast, the relaxation response to ADP was unchanged following endotoxemia. Precontracted (15-30% of baseline diameter) pulmonary arterioles dilated in response to 5-HT (13 +/- 1%) or ADP (67 +/- 3%). Following 3 h of endotoxemia, the pulmonary arteriolar relaxation induced by 5-HT was reduced, whereas the response to ADP was not altered. In both coronary and pulmonary arterioles, relaxation induced by the endothelium-independent vasodilator, sodium nitroprusside, was unaffected by endotoxemia. Thus, coronary and pulmonary microvascular relaxation response to ADP are minimally affected by 3 h of endotoxemia, but relaxation responses to 5-HT are significantly reduced or converted to contractile responses.

Altered effects of vasopressin on the coronary circulation after ischemia.

Ischemia and reperfusion alter the reactivity of large coronary arteries, but the effect of ischemia and reperfusion on the coronary microcirculation has been less well defined. Elevated circulating levels of vasopressin are associated with cardiopulmonary bypass and numerous other clinical states in which vascular ischemia and reperfusion may occur. We examined the effects of ischemia with and without reperfusion on the responses to vasopressin of both large coronary arteries and coronary arterial microvessels. Studies were performed on vessels from control dogs (n = 8), dogs undergoing 1 hour of ischemia only (n = 8), and dogs undergoing 1 hour of ischemia followed with 1 hour of reperfusion (n = 9). Rings of proximal obtuse marginal coronary arteries distal to the site of circumflex coronary artery occlusion were studied in isolated organ chambers. Coronary microvessels (110 to 220 microns in diameter) were studied in a pressurized (20 mm Hg), no-flow state with a microvessel imaging apparatus and electronic dimension analyzer. Microvessels were preconstricted with the thromboxane A2 analog U46619. Responses of large vessel rings were studied in the nonpreconstricted state and after preconstriction with prostaglandin F2 alpha. Large vessel response to vasopressin was minimal and not altered by ischemia with or without reperfusion. In contrast, ischemia markedly affected the coronary microvascular response to vasopressin (10 to 1000 microU/ml). Control coronary microvessels constricted minimally to vasopressin (4% +/- 2% of the baseline diameter), while microvessels after either ischemia alone or ischemia followed by reperfusion constricted 22% +/- 5% and 21% +/- 3%, respectively (p less than 0.05 versus control for both). Hemoglobin, which inactivates the endothelium-derived relaxing factor, augmented microvascular constrictions to vasopressin in all groups to a similar extent. Relaxations to the endothelium-independent agent nitroglycerin were not altered by ischemia. Constrictions of the coronary microcirculation to vasopressin in conditions such as cardiopulmonary bypass or myocardial ischemia, in which circulating levels of vasopressin are increased, may predispose to persistent myocardial ischemia in the perioperative setting.

Preconditioning improves cardioplegia-related coronary microvascular smooth muscle hypercontractility: role of KATP channels.

OBJECTIVES: The effect of preconditioning before hyperkalemic cardioplegia on the coronary smooth muscle remains to be elucidated. We tested the hypothesis that hypoxic preconditioning could protect coronary smooth muscle against subsequent hyperkalemic cardioplegia-induced coronary vasospasm and that this preconditioning effect could be mediated by K(ATP) channels. METHODS: Rat coronary arterioles (endothelium-denuded) were studied in a pressurized, no-flow, normothermic state. Simultaneous monitoring of luminal diameter and intracellular calcium concentration of vascular smooth muscle loaded with fura-2 was made with microscopic image analysis. All vessels were subjected to 60 minutes of hypoxic hyperkalemic cardioplegia (K(+) = 25.0 mmol/L) and were then reperfused. Six groups were studied: (1) controls, no precardioplegic intervention; (2) preconditioning, achieved with 10 minutes of hypoxia (PO2 < 30 mm Hg) and 10 minutes of reoxygenation; (3) preconditioning plus glibenclamide (10 micromol/L), achieved with 10 minutes of preconditioning in the presence of K(ATP) channel blocker glibenclamide; (4) pretreatment with K(ATP) channel opener pinacidil (100 micromol/L); (5) pretreatment with pinacidil (100 micromol/L) plus glibenclamide (10 micromol/L); and (6) pretreatment with glibenclamide (10 micromol/L) alone. RESULTS: Hypoxic preconditioning significantly (P <.01) reduced hyperkalemic cardioplegia-induced intracellular calcium concentration accumulation and prevented the hypercontractility during and after hyperkalemic cardioplegia compared with control vessels. Pinacidil provided effective microvascular protection similar to hypoxic preconditioning. These vasoprotective effects of preconditioning were significantly antagonized in glibenclamide-treated vessels. CONCLUSIONS: Hypoxic preconditioning can prevent coronary microvascular hypercontractility during and after subsequent cardioplegia by a K(ATP ) channel mechanism that regulates intracellular calcium concentration of the vascular smooth muscle.