Lizzie Barber

Local effects of nitric oxide supplementation and suppression in the development of intimal hyperplasia in experimental vein grafts

OBJECTIVES The universal response of vein grafts after insertion into the arterial circulation is the development of intimal hyperplasia; smooth muscle cell proliferation and connective tissue deposition, which may be modulated in part by dysfunctional endothelial nitric oxide (NO) metabolism. This study examines the effects of single dose, local application by pluronic gel of a NO donor, S-nitroso-N-acetylpenicillamine (SNAP) and an NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) on the formation of intimal hyperplasia. MATERIALS Forty New Zealand white rabbits underwent jugular vein interposition grafting of the common carotid artery. DESIGN Ten animals were controls, 10 animals had the outer surface of the vein graft coated with 30% pluronic gel (2.5 ml), and 10 each were immersed for 15 min prior to insertion in Ringer lactate containing 10(-3) M of SNAP or L-NAME and then had their vein grafts coated with 2.5 ml of gel containing either SNAP (10(-3) M) or L-NAME (10(-3) M), which allows for sustained delivery for up to 6 h. On the 28th post operative day, the animals were sacrificed and vein grafts were harvested for morphology by electron microscopy (SEM and TEM) and dimensional analysis by videomorphometry. RESULTS All vein grafts developed intimal hyperplasia. On SEM the vein grafts had a confluent layer of endothelial cells with multiple layers of smooth muscle cells representing intimal hyperplasia in TEM. There were no demonstrable morphological differences between the four groups. Local treatment with SNAP produced a significant 36% decrease in mean intimal thickness (72 +/- 4 microns vs. 45 +/- 4 microns; mean +/- S.E.M.; p < 0.01) without a change in medial thickness compared to gel-only treated groups (58 +/- 6 microns vs. 61 +/- 7 microns; p = ns). Inhibition of NO synthase by L-NAME had no effect on the development of intimal hyperplasia (72 +/- 4 microns vs. 79 +/- 10 microns; p = ns); medial thickness was also unchanged. CONCLUSION These data confirm the protective effect of NO in vascular injury and suggest that NO synthase activity is either absent or reduced to such a level that further inhibition in this short time course is not relevant to the pathophysiology of vein graft intimal hyperplasia.

Localized versus systemic angiotensin II receptor inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor L158,809

BACKGROUND This study examines the effect of the angiotensin II receptor (type 1) antagonist (L158,809) on the formation of vein graft intimal hyperplasia in vivo, by both localized and systemic administration. METHODS Forty New Zealand White rabbits underwent carotid interposition bypass grafting with the external jugular vein and were killed on postoperative day 28. To determine the effect of L158,809 on the development of intimal hyperplasia, 10 animals received long-term oral therapy with L158,809 (10 mg/kg/day, begun 5 days before operation and continued until harvest), 10 animals underwent coating of the grafts with a pluronic gel containing L158,809 (10(-5) mol/L), and 20 animals were controls (10 with and 10 without pluronic gel). These grafts were harvested for either histologic analysis (n = 6 per group) or in vitro isometric tension studies to angiotensin II (n = 4 per group). RESULTS Long-term oral treatment with L158,809 produced a 43% decrease in intimal thickness from 76 +/- 6 microns (mean +/- SEM) in the control animals to 43 +/- 7 microns in the treated vein grafts (p = 0.002). There was also a significant decrease (44%) in the medial thicknesses between the control (75 +/- 4 microns) and L158,809-treated (42 +/- 6 microns) vein grafts (p = 0.007). The contractile responses to angiotensin II were abolished in the vein grafts by long-term L158,809 therapy. Local application by gel of L158,809 produced a significant decrease (33%) in the intimal thickness (48 +/- 3 microns) but no change in medical thicknesses (76 +/- 6 microns) compared with control grafts. The contractile responses to angiotensin II were unchanged in the vein grafts by local L158,809 therapy. CONCLUSIONS This study shows that a local single application of L158,809 will reduce the intimal response but not the medial response in vein grafts, whereas long-term treatment will reduce intimal hyperplasia and the medial response in experimental vein grafts. Therefore angiotensin II acting through AT1 receptors mediates a significant part of the intimal hyperplastic response in vein grafts that appears to involve two phases: an acute intimal response requiring short-term therapy and a long-term medial response that requires prolonged therapy.

Control of the structural and functional consequences of vein graft intimal hyperplasia with a 21-aminosteroid—U74389G

Following angioplasty and vein bypass grafting, there is endothelial cell injury, infiltration of leukocytes and smooth muscle cell (SMC) proliferation leading to intimal hyperplasia which may result in stenosis and can lead to eventual occlusion. This study examines the effect of the 21-aminosteroid U74389G (Upjohn Company), on the formation of vein graft intimal hyperplasia in vivo and on SMC DNA synthesis and proliferation in vitro. Twenty New Zealand White rabbits had a right carotid interposition bypass graft using the ipsilateral external jugular vein. Ten animals received chronic oral therapy with U74389G (25 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 control animals received vehicle only. All animals were sacrificed on the 28th postoperative day. Vein grafts were harvested either for histology/videomorphometry (n = 6 per group) or for in vitro isometric tension studies (n = 4; four 5 mm rings per graft). The incorporation of [3H]thymidine into the cellular DNA of serum-stimulated rabbit aortic SMC (passage 6th to 12th) was assessed in the presence of increasing concentrations of U74389G (10(-9) to 10(-4) M). The effect of U74389G on in vitro cell proliferation was also assessed. Treatment with U74389G produced a 44% decrease in overall mean intimal thickness from 82 +/- 1 microM (mean +/- S.E.M.) in the controls to 57 +/- 10 microM in the U74389G treated vein grafts (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of angiotensin II receptor mediated responses and inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor, L158,809

OBJECTIVES This study characterises pharmacologically the angiotensin II receptor in experimental vein grafts and examines the effect of the angiotensin II receptor (type 1) antagonist (L158,809) on the formation of vein graft intimal hyperplasia in vivo, as well as the in vitro physiological response to angiotensin II of vein grafts after chronic oral L158,809 treatment. MATERIALS Thirty New Zealand White rabbits had a right carotid interposition bypass graft using the external jugular vein and were killed on the 28th postoperative day. DESIGN To characterise the angiotensin II receptors, concentration response curves to angiotensin II were obtained in vitro in the presence or absence of L158,809. To determine the effect of L158,809 on the development of intimal hyperplasia, 10 animals received chronic oral therapy with L158,809 (10 mg/kg/day; begun 5 days before surgery and continued until harvest) and 10 animals received vehicle only as controls. These grafts were harvested either for histology (n = 6 per group) or for in vitro isometric tension studies to angiotensin II. RESULTS The monophasic contractile response to angiotensin II in the untreated vein grafts could be inhibited in a concentration dependent manner by L158,809 with first order kinetics. Chronic oral treatment with L158,809 produced a 48% decrease in intimal thickness from 82 +/- 1 micron (mean +/- S.E.M.) in the controls to 43 +/- 7 microns in the treated vein grafts (p = 0.002). There was also a significant decrease (45%) in the medial thickness between the control (76 +/- 6 microns) and L158,809 treated (42 +/- 6 microns) vein grafts (p = 0.007). The responses to angiotensin II were abolished in the vein grafts by chronic L158,809 therapy. CONCLUSIONS This study suggests that vein graft angiotensin II responses are mediated through a type 1 receptor and that chronic inhibition with L158,809, significantly reduces intimal hyperplasia and medial hypertrophy in experimental vein grafts and concomitantly abolishes the in vitro responses to angiotensin II. Therefore, angiotensin II acting through AT1 receptors mediates a significant part of the intimal hyperplastic response in vein grafts.

Diabetes mellitus and experimental vein graft structure and function

PURPOSE Diabetes mellitus is a known risk factor for accelerated atherosclerosis and for postangioplasty restenosis. METHODS This study examines the effect of chronic, uncontrolled, alloxan-induced diabetes on the structure and vasomotor function of vein bypass grafts in 20 male New Zealand white rabbits with diabetes and in 10 controls. After 8 weeks of diabetes, a common carotid vein bypass graft was performed. Four weeks after operation, vein grafts and contralateral jugular veins were harvested. RESULTS Diabetes induced a twofold increase in the vein graft intimal thickness compared with control. There was no change in medial thickness. Electron microscopy of the vein grafts in diabetes revealed intercellular gaps in the endothelium lining and abnormal endothelial cell junctions compared with controls. Diabetes significantly increased the maximal contractions generated in vein grafts to all contractile agonists tested without any change in sensitivity. CONCLUSIONS This study demonstrates that diabetes alters endothelial cell structure and increases the development of intimal hyperplasia with increased maximal contractility in vein grafts and therefore suggests that the vein grafts in diabetes are more susceptible to early stenosis.

Ramipril and Experimental Vein Graft Intimal Hyperplasia

Angiotensin-converting enzyme (ACE) inhibitors have been shown to reduce the intimal proliferation in animal models of arterial angioplasty and vein bypass grafting. This study examines the effect of high-dose ramipril, an ACE inhibitor that does not contain a sulfhydryl group, on the development of intimal hyperplasia in experimental vein bypass grafts. Twenty New Zealand White rabbits underwent common carotid interposition bypass grafting. Twelve were treated with ramipril (2mg/kg/day; po) five days prior to surgery and thereafter until harvest. The remaining 8 animals were used as controls. Vein grafts were harvested at twenty-eight days by pressure fixation (80 mmHg). The grafts were sectioned into proximal, middle, and distal thirds, and the thickness of the intima and the media and the area of the lumen from each segment were determined by videomorphometry. The effect of ramipril on the [H3]thymidine incorporation into DNA of serum-stimulated smooth muscle cells (culture passage 6 to 12) was also assessed. There was a 50% mortality rate in the rabbits that received ramipril, and this was assumed to be related to the high dose of the drug. Ramipril treatment reduced mean vein graft intimal area by 34% (P > 0.05), but this was accompanied by an increase of 73% in the mean medial area of the vein grafts as compared with controls. These changes resulted in a decrease in the mean intimal ratio (intima/[intima + media]) by 39% in the ramipril group as compared with controls. Ramipril did not inhibit [H3]thymidine incor poration into DNA of serum-stimulated smooth muscle cells. This study shows that treatment with the nonsulfhydryl ACE inhibitor ramipril, even at a high dose, produces only a marginal reduction in the formation of intimal hyperplasia in experimental vein grafts.

The influence of the combined presence of diabetes mellitus and hypercholesterolaemia on the function and morphology of experimental vein grafts

OBJECTIVES Diabetes and hypercholesterolaemia are known risk factors for the development of atherosclerosis and are considered to influence the development of vein graft intimal hyperplasia. This study examines the combined effect of diabetes for 12 weeks (alloxan-induced) and hypercholesterolaemia for 8 weeks (1% cholesterol diet) on the formation of intimal hyperplasia and the vasomotor function of vein grafts. MATERIALS AND DESIGN: Thirty-two New Zealand White rabbits underwent a carotid vein bypass graft. Eight were controls, eight were diabetic, eight were hypercholesterolaemic and eight had both diabetes and hypercholesterolaemia. All vein grafts were harvested at 4 weeks postoperatively for morphology (n = 4) or contractility studies (n = 4). RESULTS Compared to controls, both diabetes and hypercholesterolaemia increased intimal thickness by 20% and 63% respectively; medial thicknesses of these vein grafts were unchanged compared to control. In contrast, diabetes with hypercholesterolaemia dramatically increased intimal and medial thickness (1.8 fold and 1.6 fold respectively, compared to control). Smooth muscle cell contractility was enhanced in both the diabetic and hypercholesterolaemic groups. The presence of diabetes with hypercholesterolaemia did not further alter the enhanced smooth muscle cell contractile responses. CONCLUSIONS This study suggests that the combination of both the atherogenic risk factors, hypercholesterolaemia and diabetes, significantly augments the formation of intimal hyperplasia in experimental vein grafts.