Myointimal Thickening Research Articles

Inhibition of myointimal proliferation of the rat carotid artery by the peptides, angiopeptin and BIM 23034

Proliferation of vascular smooth muscle is an early and major event in the formation of an atherosclerotic lesion. Here we report for the first time the inhibitory effects on myointimal proliferation of the rat carotid artery by a synthetic peptide, angiopeptin, and its closely related congener, BIM 23034. Proliferation was initiated in the carotid artery of anesthetized rats by air-drying of the endothelium. After 15 days the rats were killed and the carotid artery was pressure-fixed and subjected to morphologic analysis for evaluation of the degree of myointimal thickening. Five synthetic somatostatin-like peptides were tested by pretreating rats (20 and 50 μg/kg/rat s.c. daily) for 2 days prior to and for 5 days after the endothelial injury. Angiopeptin and the closely related octapeptide (BIM 23034) significantly inhibited myointimal thickening. Angiopeptin was also effective when the pretreatment period was reduced from 2 days to 30 min. The inhibitory effect of angiopeptin was further confirmed in an additional experiment involving [ 3H]thymidine incorporation. In this experiment angiopeptin (100 μg/kg/day s.c.) was also administered for 2 days prior to and five days following the endothelial injury and it significantly inhibited thymidine uptake. All the peptides tested inhibit the release of growth hormone. However, only angiopeptin and BIM 23034 inhibited myointimal proliferation. Thus the effect of angiopeptin and its congener is unlikely to be mediated through growth hormone. Since angiopeptin inhibits myointimal proliferation it may have clinical utility in preventing restenosis following angioplasty and coronary artery by-pass procedures.

Porcine coronary arteries with regenerated endothelium have a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin.

To test the ability of regenerated endothelium to evoke endothelium-dependent relaxations, male Yorkshire pigs underwent balloon endothelial denudation of the proximal left anterior descending coronary artery. Endothelium-dependent responses were examined in vitro, in rings of coronary segments taken from the denuded area or from the proximal left circumflex coronary artery. The experiments were performed 8 days or 4 weeks after the denudation. Endothelial regrowth was confirmed by histologic examination 8 days after the denudation and by demonstrating the presence of endothelium-dependent relaxations to bradykinin; at that time aggregating platelets evoked normal endothelium-dependent responses. However, 4 weeks after the denudation, the relaxations to aggregating platelets were markedly depressed although continuous endothelial lining was present, and the endothelium-dependent responses to bradykinin, adenosine diphosphate, the Ca2+-ionophore A23187, platelet activating factor, and thrombin were unaltered. Four weeks after denudation, endothelium-dependent relaxations to serotonin were depressed. Higher concentration of serotonin induced endothelium-dependent contractions in quiescent rings with regenerated endothelium, suggesting that regenerated endothelial cells may produce endothelium-derived constricting factor(s) and release less endothelium-derived relaxing factor(s) when exposed to the monoamine. The endothelium-dependent relaxation to serotonin was not reduced by the S2-serotonergic antagonist ketanserin but prevented by the combined S1- and S2-serotonergic blocker methiothepin. The platelet-induced relaxation was due to released serotonin and adenine nucleotides in control left circumflex coronary arteries, but in left anterior descending coronary artery with regenerated endothelium, it was due solely to the latter. The platelet-induced contractions were due to activation of receptors on the smooth muscle cells. Four weeks after denudation, regenerated endothelial cells were morphologically different from native cells; they were elongated and cuboidal, and the number of the cells had increased twofold. At this state, eccentric myointimal thickening was present in the previously denuded portion. These experiments indicate that the protective role of endothelial cells against the vasoconstriction induced by aggregating platelets is depressed in the chronic regenerated state. A lack of responsiveness to serotonin appears to be the cause for the endothelial dysfunction.

Significance of quiescent smooth muscle migration in the injured rat carotid artery.

Cellular accumulation in the intima of injured artery has generally been attributed to smooth muscle cell proliferation. Since smooth muscle cells in normal artery are found mainly in the media, migration of smooth muscle cells into the intima has been considered a necessary prerequisite for subsequent myointimal thickening. The nondividing medial cells would appear to have no role in the reparative process. We have investigated in the rat ballooned carotid the possibility that nondividing cells might also contribute to injury-induced intimal thickening. All proliferating smooth muscle cells were labeled by 3H-thymidine given by continuous intraperitoneal infusion. The amounts of 3H-thymidine used were not toxic and did not inhibit smooth muscle cell proliferation. Autoradiograms performed on histological cross-sections showed a progressive decrease in the fraction of unlabeled cells at 3, 7, and 14 days after carotid injury. However, the actual number of nondividing cells remained constant. The calculated growth fraction for the 14-day period was 40%. A substantial number of unlabeled cells was observed in the intima. These data have led us to conclude that only a small fraction of smooth muscle cells in an artery proliferate in response to the injury stimulus, and do so shortly after injury, or not at all. Furthermore, nondividing, as well as proliferating smooth muscle cells, can migrate and contribute, in a substantial way, to the increase in intimal smooth muscle cell number.

Endothelial denudation and myointimal thickening in the rat carotid artery induced by the passage of bubbles

A new technique for the selective removal of endothelium in the rat carotid artery has been developed, and subsequent events in the vascular wall have been examined. To achieve de-endothelialization, more than 2,000 bubbles of nitrogen in phosphate-buffered saline are passed through a temporarily isolated segment of rat carotid over a period of 3 min. Bubbles are generated by a simple apparatus, consisting of a pressurized tilting chamber and catheter. Endothelium is removed while subendothelial basement membrane and other subjacent structures remain intact. Platelets attach to the denuded surface within minutes after re-establishment of blood flow. Myointimal thickening is found at 5 weeks and 4 months after de-endothelialization. The method is quite reliable and will facilitate further studies of reactions to carotid endothelial injury in young adult rats.

An evidence for “response to injury” hypothesis

Role of platelet-derived growth factor (PDGF) in myointimal thickening described in “response to injury” hypothesis was investigated with artery of rats in culture and with air-injured artery of rats. PDGF promoted cell growth in ring preparation of carotid artery in culture denuded with citrate. It did not promote any cell growth in preparations without denudation. Trapidil, a PDGF antagonist, inhibited the cell growth promoted by PDGF in the denuded arterial ring. Systemic injection of PDGF was performed for 8 days to rats with thrombocytopenia induced by injections of anti-platelet serum. This treatment caused myointimal thickening of carotid artery 10 days after denudation by means of air injury. Trapidil at oral intake levels of 1, 3 and 30 mg/kg/day inhibited this change observed in denuded site of artery. Trapidil at oral intake of 6 mg/kg/day also inhibited myointimal thickening observed 15 days after denudation of carotid artery by air injury in normotensive and spontaneous hypertensive rats both with normal platelet counts. These results evidenced the role of PDGF in myointimal thickening described in “response to injury” hypothesis and clinical use of trapidil may be a new approach to the treatment of atherosclerosis.

Ultrastructural evidence for collagen degradation in the walls of varicose veins

In the myointimal thickening of human varicose veins morphologically distinct collagen fibrils within the cytoplasm of intimal smooth muscle cells (SMC) were observed, which could be interpreted as a possible image of collagen phagocytosis. Human varicose veins were obtained surgically from five patients and studied both histologically and electronmicroscopically. A myointimal thickening of a different degree was established in all cases examined. In three cases a large number of intimal SMC displayed intracellular membrane-bound collagen-like fibrils. The fibrils were single or packed together in small groups, showed the typical periodicity of collagen, and were surrounded by an ill-defined homogeneous “matrix” material. Within other SMC cytoplasmic vacuoles were found, including collagen fibrils in a more advanced stage of lysosomal decomposition. Characteristically the collagen-containing intimal SMC were localized predominantly in close proximity to newly formed capillaries of the thickened intima, where a relatively great quantity of bloodborne monocytes was also observed. It is suggested that vascular SMC are capable of phagocytosis and decomposition of collagen. Thus they may be part of the cellular basis for collagen breakdown in the remodeling of the blood vessel wall.

O-145 - Effect of trapidil on myointimal thickening in the rat carotid artery

O-145 - Effect of trapidil on myointimal thickening in the rat carotid artery

Suppression by heparin of injury-induced myointimal thickening

To ascertain the effect of heparin upon intimal smooth muscle cell proliferation after arterial endothelial injury, we subjected young male Sprague-Dawley rats to right carotid air injury and infused them with heparin intravenously for 5, 10, and 14 days after surgery. Control animals received lactated Ringer's solution. Marked inhibition of intimal smooth muscle cell proliferation was observed in the heparinized rats as compared to controls. This profound heparin effect may be due to the enhancement by heparin of antithrombin III inhibition of thrombin and suggests a hitherto unsuspected role of the clotting system in an injury-induced smooth muscle cell proliferation.

Dysbaric Osteonecrosis

Dysbaric osteonecrosis appears to be independent of decompression sickness. The 2 conditions, however, may share etiologic and pathogenetic factors. The incidence of osteonecrosis is influenced by the number of hyperbaric exposures, extent of pressure, decompression profile and possibly by the rate of compression and degree of obesity. Though etiology and pathogenesis are unclear, osteonecrosis is probably due to ischemia, with gas bubbles causing direct or indirect circulatory impairment. In vitro experiments, as well as human and animal studies, suggest multiple pathogenetic mechanisms: intraosseous vessel compression by extravascular bubbles; vessel obstruction by bubbles, fibrin thrombi, platelet aggregates, clumped erythrocytes or coalesced lipids; and narrowing of arterial lumina by bubble-induced myointimal thickening. Obstructing materials, whether autochthonous or embolic, may result from blood-bubble interface reactions. Rheologic changes and blood flow redistribution could play contributing roles. It seems likely that multiple pathogenetic factors act in concert or sequentially. Proposed nonischemic changes, such as hyperoxic injury gas-induced osmosis, or autoimmunity, lack sufficient supporting evidence. The peculiar vulnerability of bone may be related to gas supersaturation of the fatty marrow; sensitivity to extravascular gas pressure because of tissue rigidity; poor vascularization; and the presence of uranium 238 which promotes nucleation and subsequent gas bubble formation.

Suppression by heparin of smooth muscle cell proliferation in injured arteries.

INTIMAL smooth muscle cell (SMC) proliferation dominates the early phase of healing after arterial endothelial injury1–6; what growth factors are responsible for this mitotic activity of an otherwise quiescent cell is not known. SMC growth in vitro is enhanced by platelet factors, insulin and lipoproteins7–11 and, according to some reports, can be diminished in vivo with various antiplatelet agents. Harker et al.6 have reported decreased myointimal thickening in homocystinaemic baboons treated with dipyridamole, and Friedman et al.11 and Moore et al.12 have reported suppression of SMC proliferation in the injured arteries of rabbits injected with anti-platelet serum. Intimal thickening was not diminished, however, in the injured carotid arteries of rats given various anti-platelet drugs13. Because the clotting system is inextricably linked to arterial injury and because thrombin is used to generate the platelet growth factor9 and may itself be a mitogen14,15, we speculated that heparin might inhibit myointimal thickening. We report here the use of heparin to suppress intimal SMC hyperplasia in a rat model of arterial endothelial injury and suggest possible mechanisms for the heparin effect.

Repair in arterial tissue. Electron microscopy of Evans blue vital stained embolectomy catheter lesion of the rabbit thoracic aorta.

The rabbit thoracic aorta was studied by EM and vital staining with Evans blue at vaired points of time after a single lesion produced by an embolectomy catheter. EM of white areas: the surface cells of the myo-intimal thickening resembled endothelial cells with a discrete occurrence of microfilaments inside the plasma-membranes and rather differentiated flap-like junctions. EM of blue areas: the surface cells resembled modified smooth muscle cells with a heavily contrasted zone just inside the luminal plasma membrane containing closely packed microfilaments. The cell contacts either were missing, or they presented undifferentiated side-to-side contacts.