Virginia M. Miller

Enhanced Endothelin-Mediated Coronary Vasoconstriction and Attenuated Basal Nitric Oxide Activity in Experimental Hypercholesterolemia

BACKGROUND Experimental hypercholesterolemia is associated with coronary vasomotor dysfunction. This study was designed to test the hypothesis that experimental hypercholesterolemia is characterized by altered coronary vasomotor responses to endothelin and inhibition of the endogenous NO pathway. METHODS AND RESULTS Endothelin-1 (ET-1) at 5 ng x kg(-1) x min(-1) or N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase (NOS), at 50 microg x kg(-1) x min(-1) was infused into the left anterior descending coronary artery in pigs before and after 10 weeks of cholesterol diet. There was a significant increase in serum cholesterol. At 10 weeks, ET-1 resulted in an accentuated decrease in coronary blood flow (CBF) and coronary artery diameter (CAD) compared with baseline (-88+/-6% versus -45+/-9%, P<.05, and -77+/-14% versus -18+/-8%, P<.05, respectively) and an increase in coronary vascular resistance (CVR) (242+/-18% versus 110+/-17%, P<.05); ET receptor density and binding affinity in epicardial coronary arteries were unchanged. The effect of L-NMMA on CBF, CAD, and CVR was attenuated at 10 weeks (-7+/-8% versus -48+/-4%, -2+/-3% versus -17+/-5%, and 16+/-10% versus 125+/-32%; each P<.05). Immunohistochemistry staining for constitutive NOS revealed a decrease in immunoreactivity in the coronary arteries of hypercholesterolemic pigs. CONCLUSIONS The present study demonstrates an enhanced coronary vasoconstrictive response to pathophysiological doses of endothelin and an attenuated response to the inhibition of endogenous NO activity, suggesting an alteration in coronary vascular reactivity in experimental hypercholesterolemia.

Effects of thrombolysis and venous thrombectomy on valvular competence, thrombogenicity, venous wall morphology, and function

PURPOSE The experiments were designed to compare the effects of thrombolytic therapy (TL) and balloon-catheter thrombectomy (TX) on valvular competence, thrombogenicity, venous wall morphology, and function after acute deep venous thrombosis (DVT) in canine veins. METHODS The femoral veins of male mongrel dogs were ligated proximally and distally for 48 hours to induce DVT. The thrombosed veins were treated with either TL (n = 5) or TX (n = 9), or no treatment was rendered (n = 6). Sham-operated dogs were used as controls. TL was performed with catheter-directed infusion of urokinase at 4000 U/min for 90 minutes. Three hours after the treatment, the valvular competence was determined with duplex scanning, thrombogenicity determined with deposition of radio-labeled platelet and fibrin, and function determined with response to contractile and relaxing agonists in organ chambers. The structural integrity of the endothelial layer was assessed by means of scanning electron microscopy. RESULTS The removal or lysis of the thrombus was successful in all cases. The valvular competence did not differ among the groups. The platelet deposition was the highest after TX (P <.05), and the fibrin deposition was not significantly different among the groups. In the organ chamber experiments, relaxations to adenosine diphosphate and nitric oxide were reduced after TX (P <.05). The contractions to serotonin were enhanced after TX. Scanning electron microscopy results showed a comparable (51% to 75%) endothelial loss with either treatment. CONCLUSIONS After experimental acute DVT, the TL and the TX at 3 hours had similar effects on the valvular competence and the endothelial morphology. However, the TL reduced thrombogenicity, which is consistent with the preserved endothelial responses to platelet products. These data suggest that TL may preserve vein function after DVT and may reduce the long-term potential for recurrent DVT and post-thrombotic syndrome.

Chronic changes in blood flow alter endothelium-dependent responses in autogenous vein grafts in dogs.

PURPOSE Experiments were designed to determine the effects of blood flow on endothelium-dependent relaxations in canine vein grafts. METHODS Blood flow through reversed femoral vein grafts was either increased by a distal arteriovenous fistula (increased flow), unmanipulated (normal flow), or reduced by a proximal adjustable clamp (reduced flow). Six weeks after implantation, blood flow through the graft was measured. Rings cut from grafts were suspended for the measurement of isometric force in organ chambers to determine endothelial function. RESULTS Blood flow was significantly greater in grafts with a distal fistula compared to grafts with normal or decreased flow. Endothelium-dependent relaxations to acetylcholine were absent in all grafts. Endothelium-dependent relaxations to adenosine diphosphate, thrombin, and the calcium ionophore A23187 were less in grafts with reduced flow compared with grafts with increased flow. Relaxations to these agents in grafts with increased flow were reduced by an analog of L-arginine. Neointimal hyperplasia was increased in grafts with reduced flow. CONCLUSIONS These data demonstrate that chronic diminution of blood flow decreases receptor-mediated release of endothelium-derived relaxing factors and increases neointimal hyperplasia in canine vein grafts. The production of endothelium-derived relaxing factors, one of which is nitric oxide, may influence the development of myointimal hyperplasia in vein grafts.

Cryopreserved venous allografts: Effects of immunosuppression and antiplatelet therapy on patency and function

PURPOSE Experiments were designed to determine the function of the endothelium and smooth muscle in cryopreserved and fresh canine saphenous vein allografts. METHODS Reversed interposition saphenous vein grafts were implanted into the femoral arteries of two groups of dogs: group 1 received one freshly harvested saphenous vein allograft and group 2 received one cryopreserved saphenous vein allograft. The contralateral femoral artery in each group was replaced with a freshly harvested autogenous saphenous vein. Animals received oral cyclosporine alone (n = 3) or in combination with aspirin (325 mg/day; n = 20). RESULTS Four weeks after surgery, none of the cryopreserved grafts was patent in the group treated with cyclosporine alone. All grafts were patent in animals treated with cyclosporine and aspirin. At 4 weeks, blood flow through cryopreserved allografts was significantly greater than in freshly harvested allografts. Platelet deposition was comparable in cryopreserved and freshly harvested allografts immediately after implantation and at 4 weeks. Rings cut from the grafts were suspended for the measurement of isometric force in organ chambers. alpha-Adrenergic agonists and endothelin caused concentration-dependent contractions in fresh autografts and allografts. After submaximal contraction with prostaglandin F2 alpha in these same grafts, calcium ionophore and thrombin caused relaxations in rings with endothelium, and nitric oxide caused relaxations in rings without endothelium. Cryopreserved allografts did not respond to any of the agonists tested. Histologic examination of the tissue indicated smooth muscle cells in cryopreserved grafts; evidence of rejection was observed in all allografts. CONCLUSION These results indicate that cryopreserved allografts remain patent with antiplatelet and immunosuppressive therapy in spite of loss of functional smooth muscle.

Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts

Cryopreserved veins used as arterial grafts may be affected by both rejection and the cryopreservation process. Experiments were designed to study changes in endothelial and smooth muscle function after cryopreservation but independent of rejection. One saphenous vein from each of eight dogs was cryopreserved for subsequent use as autografts. After 3 weeks one cryopreserved and one freshly harvested autogenous saphenous vein were implanted as bilateral femoral arterial interposition grafts. Platelet deposition was studied in vivo with indium 111-labeled platelets. At 4 weeks the autografts were removed, and the functional characteristics of the grafts were studied in organ chambers; and the ability of nerve terminals to uptake transmitter was studied with 3H-norepinephrine. Neither patency rates, blood flows, nor platelet deposition were significantly different between freshly harvested and cryopreserved grafts. Uptake of 3H-norepinephrine was significantly reduced in both grafts as compared to unoperated veins. The smooth muscle of the cryopreserved and fresh grafts contracted comparably to alpha-adrenergic agonists and endothelin. In cryopreserved grafts, the maximal tensions that developed to KCl, prostaglandin F2 alpha, and endothelin were greater when the endothelium was present compared to that developed by the smooth muscle alone. Calcium ionophore A23187 caused relaxations only in rings with endothelium; these were not significantly different between graft types. However, relaxations of the smooth muscle to nitric oxide were decreased in the cryopreserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Urokinase treatment preserves endothelial and smooth muscle function in experimental acute arterial thrombosis

PURPOSE Pharmacologic lysis or balloon thrombectomy are options to treat acute arterial thrombosis; however, little is known about their effects on functional changes in the arterial wall. The aim of this study was to determine function of the endothelium and smooth muscle in canine arteries revascularized after acute thrombosis with balloon thrombectomy or lytic therapy. METHODS Acute thrombosis was obtained by bilateral proximal and distal ligation of 8-cm. segments of the femoral arteries in dogs. After 24 hours, the ties were removed and the arteries randomized to treatment groups: group 1, balloon thrombectomy (# 4 Fogarty balloon catheter at 60 grams linear shear x 1 pass, n = 7); group 2, untreated, tie removal only (n = 6); group 3, regional intra-arterial urokinase infusion (4000 U/min x 90 min, n = 6); group 4, regional intra-arterial carrier infusion (0.43 ml/min x 90 min, n = 6); group 5, unoperated normal vessels (n = 5). After treatment, the arteries were removed and endothelial and smooth muscle responses examined in organ chambers. Endothelial loss was graded with light microscopy of vessel rings from each animal by an observer blinded to the treatment group. Findings were confirmed with scanning electron microscopy. RESULTS Treatment with urokinase did not alter endothelium-dependent relaxations or smooth muscle contractions compared with carrier infusion or untreated alone. Balloon catheter thrombectomy significantly reduced endothelium-dependent relaxations compared with all other groups in response to acetylcholine, bradykinin, and thrombin (p < 0.001). Contractions of smooth muscle in response to potassium chloride (60 mol/L) and phenylephrine (1 x 10-6 mol/L) were also reduced (p < 0.05). Rings from balloon thrombectomized arteries contracted in response to calcium ionophore A23187 (p < 0.001); these contractions were endothelium dependent and not reduced by indomethacin or blockade of endothelin A and B receptors. No significant differences in percentage of endothelial coverage between groups were assessed by light and electron microscopy. CONCLUSION Thrombolysis with urokinase caused no or minimal abnormalities in endothelial and smooth muscle function. Endothelium present after balloon thrombectomy produces contractile factors. Although the duration and recovery of these abnormalities in function are unknown, these findings support preferential use of urokinase over balloon thrombectomy when possible in acute arterial thrombosis or embolism.

Inferior mesenteric venous sampling to detect colonic ischemia: A comparison withlaser Doppler flowmetry and photoplethysmography

PURPOSE No single method has been identified that accurately and reliably detects patients with impending bowel infarction during aortic reconstruction. Serial sampling of blood gas from the inferior mesenteric vein (IMV) for detecting colonic ischemia was compared with two previously described techniques: laser Doppler flowmetry (LDF) and photoplethysmography. METHODS Nine dogs underwent induced partial colonic ischemia followed by complete ischemia. Serial IMV blood gas measurements were obtained at four intervals: baseline, partial ischemia, complete ischemia, and reperfusion. Simultaneous direct colon wall LDF and PPG measurements also were obtained. RESULTS Changes in pH, Po2, O2 saturation, and Pco2 demonstrated progressive acidosis, hypoxemia, and hypercapnia in association with progressive arterial occlusion and a reversal of these trends toward baseline after restoration of flow. The absence of a pulsatile photoplethysmography tracing and oxygen saturation less than 90% were predictive of altered perfusion but could not differentiate partial from complete ischemia. Although the differences in mean LDF values were statistically different during ischemia and reperfusion, there was considerable variability between each measurement. CONCLUSIONS Analysis of blood gas from the IMV and pulse oximetry are useful techniques for detecting colonic ischemia, but only the former can distinguish partial from complete ischemia. The variability in colonic measurements with LDF limits its usefulness for detecting levels of colonic perfusion.

Effects of dietary l-arginine on the reactivity of canine coronary arteries

Experiments were designed to determine the effects of supplemental dietary l-argi-nine on the endothelial and smooth muscle function of canine coronary arteries. One group of dogs was fed the standard laboratory chow while another group was supplemented with 250 mg/kg per day l-arginine. All dogs had undergone bilateral reversed interposition saphenous vein grafting and received 325 mg/day oral aspirin. After 5 weeks of arginine feeding, left circumflex coronary arteries were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. Concentration-response curves were obtained to l-arginine, UK-14,304 (a2-adrenergic agonist) and A23187 (calcium ionophore) in the absence and presence of NG-monomethyl-l-arginine (l-NMMA) and tetraethylammonium (TEA) alone or in combination. Serum concentrations of l-arginine increased by about 20% following 2 weeks of arginine feeding and remained elevated throughout the study. In rings with and without endothelium contracted with prostaglandin F2 a, l-arginine caused concentration-dependent contractions in rings from control animals but no significant change in tension in rings from arginine-fed animals. Contractions to l-arginine in control animals were reduced by either l-NMMA or TEA. Endothelium-dependent relaxations to the a2-adrenergic agonist were decreased with arginine feeding while relaxations to the calcium ionophore and the endothelium-derived factor nitric oxide were similar among groups. Relaxations to UK-14,304 were reduced by l-NMMA in both groups but by TEA only in rings from control animals. These results suggest that dietary supplementation with l-arginine modifies reactivity of endothelium and smooth muscle by at least two mechanisms: one associated with activation of potassium channels and the other with receptor-coupled release of nitric oxide.

Production of endothelium-derived factors from sodded expanded polytetrafluoroethylene grafts

PURPOSE Experiments were designed to determine whether endothelium isolated from adipose tissue and sodded onto expanded polytetrafluoroethylene grafts release endothelium-derived vasoactive factors. METHODS Thin-walled expanded polytetrafluoroethylene grafts (6 mm internal diameter, 6 cm length, 30 microm pore size), one sodded with autogenous endothelial cells, the other unsodded, were implanted bilaterally in carotid arteries of 30 male mongrel dogs. Dogs were treated with 325 mg aspirin daily. After 6 weeks grafts were excised and perfused in a bioassay system. Effluent from the grafts stimulated with either acetylcholine, thrombin, adenosine 5-diphosphate, or the calcium ionophore A23187 was superfused over rings of canine femoral arteries without endothelium contracted with phenylephrine. Effluent from the grafts was analyzed by radioimmunoassay for thromboxane B2, 6-keto-prostaglandin F1alpha, endothelin-1, and C-type natriuretic peptide. RESULTS Ninety percent of the sodded grafts and 87% of the unsodded grafts were patent after 6 weeks. Bioassay rings superfused with effluent from sodded grafts stimulated with acetylcholine relaxed significantly more than rings superfused with effluent from similarly stimulated unsodded grafts. Biochemical analysis of the effluent showed an increase in 6-keto prostaglandin F1alpha and C-type natriuretic peptide and a decrease in endothelin-1 and thromboxane B2 release from the sodded compared with the unsodded grafts. Scanning electron microscopy showed a continuous layer of endothelial cells lining only the sodded grafts. Staining for alpha-actin and heavy-chain myosin showed a differentiated layer of smooth muscle below the endothelial layer on the sodded grafts. Finally, there was positive staining for C-type natriuretic peptide and endothelin-1 in the endothelium of the sodded grafts. CONCLUSIONS These results indicate that endothelial cells of sodded expanded polytetrafluoroethylene grafts produce endothelium-derived vasoactive factors. In addition, receptor-coupled synthesis/release of these factors is retained in sodded endothelial cells.