L-arginine attenuates reduction in endothelium-dependent vasodilation in ischemic skeletal muscle

Session 8

0800-1130

Tuesday, September 12, 1995

Research ii Room B - 2 8.1 Thromboxane Az Receptor Density Increases During Chronic Exposure to Throboxane A2 Receptor Antagonism After Porcine Carotid Bypass T.E. BROTHERS, J.G. ROBISON, B.M. ELLIOTT, J.M. BOGGS and P. V. HALUSHKA, Charleston, South Carolina, USA PURPOSE: To determine whether thromboxane A 2 (TXAz) receptor density is altered by TXA2 receptor antagonism or aspirin in pigs undergoing carotid bypass. METHODS: Prior to prosthetic graft implantation, animals were randomized to daily treatment with the TXA2 receptor antagonist BMS180291 (3 mg/kg) (Group I, n = 6), aspirin (15 mg/kg) (Group II, n = 12) or no drug (Group Ill, n = 12). Treatment with BMS180291 completely inhibited ex vivo platelet aggregation induced by the TXA2 mimetic U46619. Platelet and arterial wall receptor density were measured by e q u i l i b r i u m b i n d i n g u s i n g 12SI-BOP. P l a s m a 1dehydrothomboxane B2 (I1-TXB2) was determined by radioimmunoassay. RESULTS: At six weeks, the platelet receptor density had increased significantly (P < 0.05) from baseline in Groups 1 (3.3 _+ 0.6 vs 1.8 ± 0.3 pmol/mg) and II (2.6 ± 0.6 vs 1.7 ± 0.2 pmol/mg), but not Group !I1 (1.3 ± 0.3 vs 1.2 _+ 0.2 pmol/mg). Aortic membrane TXA, receptor density was significantly (P < 0.05) greater in Groups 1 (150 +__50 fmol/mg) and II (68 ± 10 fmol/mg) compared to Group Ill (39 ± 6 fmol/mg). Plasma 11- TXB2 levels were 82% (P < 0.05'~ higher in Group 1 than in Group ill at six weeks. Neither p a t e n c y nor ]iminal n a r r o w i n g were i m p r o v e d by BMS180291. CONCLUSION: Chronic postoperative exposure to a TXA, receptor or aspirin is associated with increased platelet anct aortic receptor density. The increased density may counteract the desired benefits of these therapeutic agents. 8.2

Three (16.5%) of the devices were patent, two VLS and one VCS. The remaining 15 (83.5%) were occluded by thrombus and collapsed. Distal migration occurred in five devices. All arteries and veins with two exceptions demonstrated varying degrees of 1H. We believe failure was due to the inability of the stems used to resist ER. The 1H seen, we believe, is due to the forced dilatation required to seat the devices. Based on the observations of the patent devices, we suggest that arteries can be relined with modifications of this technique, using a more resistant stent that can be placed without forceful dilatation. This will require further study. 8.3

L-arginine Attenuates Reduction in EndotheliumDependent Vasodilation in Ischemic Skeletal Muscle E. LORENSEN, R.M. POLLINA, M. GENNARO and E. ASCER, New York, New York, USA ] h e immediate postischemic reduction in endotheliumdependent vasodilation suggests that a decrease in nitric oxide (NO) synthesis may contribute to skeletal muscle reperfusion injury. To clarify whether this is NO mediated, we studied the effectsof administering L-arginine (an NO precursor) on endothelium-dependent vasodilation in a canine gracilis model of skeletal muscle reperfusion injury. Adult mongrel dogs underwent bilateral isolated gracilis muscle preparation subjected to 5 h of normothermic ischemia and 48 h of reperfuslon. The experimental group (n = 6) was administered intravenous L-arginine (L-arg) 15 minutes prior to reperfusion (q00 mg/kg) and for l h of repefusion at 10 mg/kg/min. The control group (n = 6) received vehicle only. Gracilis arterial blood flow was recorded via an uhrasonic flow probe. Endothelium-dependent vasodilation of the gracilis artery to acetylcholine (ACh, 0.2 mcg) was determined by measuring percent change in blood flow from baseline pre-ischemia, and at one hour and 48 h of reperfusion. Muscles were weighed at 48 h. Results (expressed as mean ± SEM) are as follows:

Arterial Intimal Replacement: Animal Research A. BYER, G. USSIA and G. GALLETFI, Hackensack, New Jersey, USA and Bologna, Italy This animal model was used to test the theory that a stent lined (VLS) or covered (VCS) with autogenous vein could reline an artery. If tenable, this technique would prevent angioplastic failure due to intimal hyperplasia (IH) and elastic recoil (ER) in atherosclerotic stenosed arteries. Nine 50 kg pigs had Gianturco-Roubin VLS and VCS implanted by mounting on a balloon angioplasty catheter via transverse arteriotomy. VLS were implanted in three left iliac and six left carotid arteries. VCS were placed in the contralateral arteries, Operative angiography was performed to insure proper placement. Duplex imaging of carotid artery placements were performed at 10 to 14 days. All animals were sacrificed at 30 to 33 days and specimens examined grossly and microscopically.

CARDIOVASCULAR SURGERY SEPTEMBER 1995

(,roup

(;racilis a. blood flow(ml/min) Pre-ischemia 48 b

% Weight gain

Control (6) l.-arg (6)

4.5 ± 1.3 4.9 +_ 1.7

50.8 ±_ 13.9 93.1 ± 26.0*

Group

Increase in Blood Flow to ACh Pre-ischernia 1h

48 h

Control (6) l-arg (6)

602 ± 189 440 ± 207

43.1 ± 31.1 94.1 ± 33.2*

12.9 ± 4.9 t 17.9 ± 2.8 t*

13.9 ± 5.9 31.3 ± 14.8"

t p < 0.01 vs pre-ischemia, * P < 0.05 vs Control These data demonstrate that a postischemic reduction in

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22nd World Congress of the International Society for Cardiovascular Surgery endothelium-dependent vasodilation persists through 48 h of reperfusion. This effect was significantly attenuated by the administration of L-arg. The increased edema with L-arg may be secondary to augmentation of the increase in blood flow during reperfusion. This would suggest that a decrease in NO synthesis accounts for the postischemic loss of endotheliumdependent vasodilation but secondary to severe endothelial injury can only be partially reversed by L-arg.

8.4 Cigarette Smoke Alters Aortic Endothelium-Dependent Vasorelaxation (Nitric Oxide) H.A. GELABERT, A.S. SANTIBANEZ-GALLERANI, L.J. IGNARRO, G.M. BUGA and W.S. MOORE, Los Angeles, California, USA PURPOSE: Investigation of chronic in-vivoeffect of cigarette smoke endothelial-derived vasorelaxation (nitric oxide). METHODS: Twenty-four Sprague-Dawley rats were randomly assigned to three groups (I, II, III). Control subjects (I) were not exposed to cigarette smoke. Experimental subjects were exposed to smoke collected around the combusted cigarette tip (sidestream, II) or smoke from the aspirated end of the cigarette (mainstream, IIl). Research cigaretes (2RI) were used in a Griffith-type smoking machine. Subjects were exposed to smoke for a period of four weeks. Serum was assayed for carboxybemoglobin (HbCO and nitrate (NO3) levels. Thoracic aortic rings were assayed for endotheliabdependent vasodilation. Statistical analysis included ANOVA and Student's T-test. RESULTS: Blood HbCO levels between control (0.4 _+ 0.06%), sidestream smoke (8.5 _+ 3.2) and mainstream (33.5 _ 6.7 %) subjects differed significantly (P < 0.01). Chemiluminescense assays of serum NO3 revealed a significant increase in direct smoke exposure (llI) vs. control subjects (I) (59.86 + 3.18 vs. 30.06 _+ 0.7, P < 0.01). Arterial ring bath assays: % Relaxation induced by increasing doses of ACh and by TNG 10 8 M 10 ~ M 10 6 M 10 s M 10 - 4 M TNG 1 11 II1

0% 2% 8%

9%* 10% 26%*

25% t 16% 36°/,,t

26%* 18% 40%*

24% 20% 40%

48% 40°/,, 50%

* (P < 0.01 for 1 vs II ), ~ (P < 0.05) CONCLUSION: Exposure to mainstream cigarette smoke results in an increase in circulating HbCO, NO 3 levels, and in ACh-induced vasorelaxation of aortas. Sidestream smoke exposure data is inconclusive.

8.5 In Vivo Gene T r a n s f e r of Vascular Natriuretic Peptide

System Into Vascular Wall T. IKEDA, K. MA TSUA, H. ITOH, Y. K O MA TSU, Y. OGAWA, K. NAKAO and T. BAN, Kyoto, Japan C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is a potent vasodilator and inhibits smooth muscle cell growth in vitro. We have demonstrated

38

that CNP is produced by vascular endothelial cells and its production is markedly augmented by TGFq3. We have also reported the gene expression of CNP and its selective receptor, natriuretic peptide B receptor (NPR-B), in in vivo blood vessels, suggesting that CNP is a novel "endothelium-derived relaxing peptide'. Moreover, we have revealed that gene expression of NPR-B was augmented in a balloon-injured carotid artery compared to that in an intact artery in rat model. This evidence strongly indicates that CNP plays important roles in vascular remodeling. In the present study, to examine the possible significance of the vascular natriuretic peptide system in gene therapy, we overexpressed genes encoding CNP and NPR-B in rabbit neck vessels. First, we transfected E. coli Lac Z gene encoding ~-galactosidase and examined the localization of [3-galactosidase expression by X-gal stain, revealing that positive staining was observed mainly in medial cells. Plasmid vectors expressing either CNP or NPR-B genes under the control of SV40 promoter were then prepared. Each plasmid was conjugated with cationic liposome, then transferred to the vascular wall either by intraluminal injection or by application with pluronic gel to the exterior vascular surface from which the adventitia was removed. The carotid artery and external jugular vein of five Japanese White rabbits were transfected with each method. Animals were sacrificed 3 to 7 days after transfection and the expression of transfected genes was examined by reverse transcription-polymerase chain reaction (RT@CR). From Southern blot analysis of PCR products, specific bands of the expected size were detected in all vessels transfected. These signals were far higher than endogenous signals found in sham-operated vessels. From these results, the localized overexpression of the vascular natriuretic system in the vascular wall was feasible and may be applicable to therapeutic use in future. We also propose that our technique of gene transfer, using pluronic gel, may be useful for intraoperative gene therapy in the surgical field.

8.6 T h e Role of the Macrophage on Cellular Proliferation in

Re-Injury Lesions in the Balloon Angioplastied Atherosclerotic Rabbit Carotid Artery C.R. LA TTIMER, V. WILSON, S. HUGHES and K.G. BURNAND, London, United Kingdom INTRODUCTION: Macrophages are considered central to the process of atherosclerosis and have been identified in human and rabbit re-stenosis tissue and within the lesions of intimal hyperplasia. Their role in re-stenosis has not been determined. In our animal model of re-stenosis and after simple balloon injury we have used a cholesterol diet to substantially increase the macrophage population so as to determine whether the presence of macrophages has any influence on intima/media ratio and proliferation index profiles. METHOD: The right common carotid artery of 24 N Z W rabbits fed on a diet of 1% cholesterol were made 'atheromatous' by balloon catheter inflation and withdrawal. A further 24 rabbits were injured without cholesterol feeding. After two weeks, 12 carotid arteries from each group were re-injured by balloon angioplasty. The arteries were harvested at 3 days, 7 days, 2 weeks and 8 weeks, snap frozen and subjected to immuno-histochemical analysis using the monoclonal anti-

CARDIOVASCULAR SURGERY SEPTEMBER 1995