Clowes Aw

Intimal smooth muscle (SMC) proliferation was examined in the rat left carotid in regions lacking endothelium for prolonged periods of time. Arteries of animals injected with tritiated thymidine and Evans blue were examined at intervals between 0 and 12 weeks. The endothelial layer was regenerated from the ends of the denuded segment but failed to cover the central third of the artery by 12 weeks. Autoradiography on samples from this central region (stained blue) and the endothelialized ends (white) showed that SMC proliferation reached a maximum at 48 hours in the media (46%) and at 96 hours in the intima (73%). Subsequently, the thymidine index declined to baseline (0.06%) by 4 weeks throughout the media and by 8 weeks in the intima covered by endothelium. SMC proliferation persisted at a high level (3.8%) at the surface of the intima lacking endothelium even at 12 weeks. Despite continued proliferation of luminal SMC, total arterial SMC number was the same at 2 and 12 weeks. These results support the concept that intimal SMC proliferation after arterial injury is an acute event related to the initial injury process. Persistent proliferation of luminal SMC does not result in an increase in intimal cell number.

Failure of long-term synthetic arterial bypass grafts has been attributed in part to anastomotic stenosis, but the pathologic basis for this has not been determined. Which cells participate in the formation of the stenosis and the relationship between normal healing and the pathologic development of anastomotic narrowing have not been delineated. In this study we have examined early wound healing in 4-mm polytetrafluorethylene arterial bypass grafts placed in baboons. In this primate model, endothelium and smooth muscle cells (SMCs) derived from the cut ends of adjacent artery form the new intima and migrate together along the luminal surface of the graft at approximately 0.2 mm/day. Both cell types proliferate in association with the growing edge. In addition, both endothelium and SMCs located discretely over anastomoses continue to proliferate despite complete endothelial coverage. Intimal cross-sectional area in this region is always greater than over adjacent graft. Fibroblasts are invariably found in graft matrix and adventitia and do not contribute to formation of intima. It is hypothesized that anastomotic narrowing might be due to chronic endothelial injury and turnover associated with continued SMC proliferation and intimal thickening.

Heparin inhibits intimal thickening after arterial injury. Whether this effect is due to inhibition of medial smooth muscle cell (SMC) migration, SMC proliferation in the intima, or synthesis and deposition of connective tissue has not been evident. In this study we have investigated these possibilities in a rat carotid balloon injury model. Heparin (0.3 mg/kg/hour) was administered intravenously by means of osmotic pumps to experimental animals, and controls received lactated Ringer's solution. Smooth muscle proliferation (thymidine index), intimal smooth muscle accumulation, and endothelial regeneration were measured at intervals between 0 and 28 days. Total smooth muscle growth as determined biochemically at 14 days was markedly inhibited by heparin if the pumps were placed 24 hours before or at the time of injury and less so if inserted 48 or 96 hours after injury. SMC thymidine indices were maximal in the media at 4 days and in the intima at 7 days for injured arteries of both heparin-treated and control rats; at each time point SMC proliferation and intimal thickening were less in heparin-treated rats. The volume of connective tissue in the intima was the same in both groups at 28 days. Medial SMC migration into the intima was diminished by heparin treatment, but endothelial regeneration was not affected. These results support the hypothesis that heparin is a specific inhibitor of SMC migration and proliferation and is most effective if started before SMC enter S-phase.

Endothelium was removed from the carotid artery of both rat and rabbit using a 2F balloon catheter. The regrowth of endothelium progressed from either end of the vessels but had stopped after 2 weeks in the rabbit and 6 weeks in the rat. Total outgrowth along the vessel was 3 mm in the rabbit and approximately 10 mm in the rat, and by 12 weeks a large zone of the vessel was still devoid of endothelium. Autoradiographic data showed that 12 weeks after the injury endothelial cell replication was reduced to background values. When these vessels were reinjured with a nylon catheter, endothelial cell replication was initiated and the wound rehealed, but these cells did not progress further into the denuded region. Morphologic examination of arteries 12 weeks after balloon denudation showed that endothelial cells, abutting the luminal smooth muscle cells, were larger and not aligned with flow. The luminal smooth muscle cells formed a loose junction with the leading edge of the endothelium and exhibited an extracellular material on their luminal aspect. These results demonstrate that endothelial regeneration will stop before cellular regrowth is complete and that cell senescence is not responsible. The presence of smooth muscle cells on the luminal surface appears to be related to this inhibition of growth.

The effect of aspirin, reserpine, and flurbiprofen on in vivo platelet function and intimal smooth muscle cell hyperplasia in rat carotid arteries subjected to endothelial injury was investigated and related to the effect of these drugs on in vitro platelet aggregation. Endothelial injury was achieved by infusing air briefly into a segment of right common carotid artery. Beginning before or after surgery, experimental animals were given sufficient drug to suppress platelet aggregation in vitro in response to collagen, adenosine diphosphate, or thrombin. The carotid arteries were fixed by perfusion at 5 and 14 days after injury and examined by light, scanning electron, and transmission electron microscopy for platelet activity and intimal smooth muscle cell proliferation in the denuded segment. Platelets in platelet-rich plasma from control animals aggregated in response to collagen, adenosine diphosphate, and thrombin; platelets from aspirin-, flurbiprofen- and reserpine-treated rats showed markedly diminished aggregation in response to collagen and normal or slightly diminished aggregation in response to ADP and thrombin. At 5 days, platelets from control animals formed a dense layer in the denuded segment: at 14 days, marked intimal thickening due to smooth muscle cell hyperplasia was observed. In experimental animals, the platelets were morphologically identical with controls and covered the denuded segment; serotonin granules were missing in platelets of reserpine-treated rats. Intimal thickening at 14 days was the same as controls. We conclude that in the rat no correlation may be made between the effect of aspirin, reserpine, and flurbiprofen on in vitro platelet aggregation and the effect of these drugs on the function of platelets on an arterial wall denuded of endothelium, as judged by morphology; furthermore, even when these drugs are used in sufficient dose to inhibit in vitro aggregation of platelets, myointimal thickening is not inhibited.