Michael L. Klyachkin

The integrity of experimental vein graft endothelium--implications on the etiology of early graft failure.

INTRODUCTION the vascular endothelium serves as a functional barrier between the circulating blood and the vessel wall. It is an essential element for the maintenance of vascular homeostasis and is implicated in the pathogenesis of vascular disease. Reversed vein bypass grafting is considered to be a devastating procedure for the endothelial cell layer of the graft during the first 7 days. At this time, smooth muscle cell proliferation, the forerunner of intimal hyperplasia, begins. Loss of endothelial cell integrity is cited as an important factor in this smooth muscle cell response. The integrity of the vein graft endothelial lining after grafting was examined in this study. METHODS reversed vein bypass grafting of the common carotid artery using external jugular vein was performed in 24 New Zealand white rabbits. All grafts were pressure fixed (80 mmHg) in situ, at 0 and 10 min, 6 h and 1, 3, 5, 7 and 14 days postoperatively. The endothelial cell layer was examined by light microscopy (LM), scanning (SEM) and transmission electron microscopy (TEM) and immunohistochemistry (Factor VIII) using standard histological procedures. RESULTS the endothelium was observed by SEM and confirmed by both LM, Factor VIII and TEM in all specimens. It covered almost the entire surface examined. At 0 and 10 min, endothelial cells were present and displayed minimal evidence of injury. At 6 h and 1 day, numerous red cells, polymorphonucleocytes (PMNs), platelets and fibrin were adherent to the luminal surface. Blood cells were also seen beneath the endothelium. At day 3, the adherent fibrin and cellular elements were reduced with most of the endothelial lining intact. Within 10 min, TEM demonstrated that these cells were stretched, very thin with few microvesicles and a blurred cytoplasm, which would indicate viability but a degree of cellular injury. By day 1, the endothelial cells were lifted from their underlying structures by subendothelial oedema and an infiltrate predominantly of PMNs. By day 5, the blood cells and fibrin which were adherent to the endothelium had been dispersed and the subendothelial infiltrate was to a large extent replaced by disintegrated PMNs. On days 7 and 14, a viable confluent endothelial cell layer was present and a degree of intimal hyperplasia was noted. The endothelial cells appeared to have enlarged nucleoli and cytoplasms filled with a considerable quantity of rough endoplasmic reticulum. CONCLUSION the endothelium of reversed vein grafts is preserved at the time of implantation and at all time intervals studied in this model. These findings do not support the assumption that endothelial denudation is a prerequisite for intimal hyperplasia. Endothelial cell dysfunction and morphological changes are maximal within the first 3 days after grafting but appear to recover by the 5th postoperative day. The gross preservation of the endothelial cell layer implies that therapeutic approaches, to mitigate endothelial cell injury and its consequences, should be focused on the preoperative period and the first 5 days following implantation.

Photodynamic therapy of vein grafts: Suppression of intimal hyperplasia of the vein graft but not the anastomosis

PURPOSE There is no clinically useful therapy for the suppression of vein bypass graft intimal hyperplasia (IH). Photodynamic therapy (PDT), a technique that uses light to activate otherwise biologically inert photosensitizers to produce cytotoxic effects, has been demonstrated to successfully inhibit experimental IH in balloon-injured arteries. The purpose of this study was to investigate the efficacy of PDT as a method to reduce vein graft IH. METHODS Reversed external jugular vein bypass grafts of the common carotid artery were performed in 28 male Sprague-Dawley rats. The animals received either chloroaluminum sulfonated phthalocyanine (2.5 mg/kg intravenously) 24 hours before the ex vivo irradiation of the vein grafts (VG) with 100 joule/cm2 at 675 nm (PDT VG) or saline solution as control (CON VG). Preharvest bromodeoxyuridine was administered to label proliferating cells. All vein grafts were perfusion fixed within 96 hours for a pilot study or at 2 and 4 weeks for the main study. Histology, immunohistochemistry, and morphometric analysis were performed. RESULTS There was no acute thrombus formation in the hypocellular PDT VG with occasional platelets but no leukocytes adherent to the luminal surface. Intimal areas of the PDT VG were 18% at 2 weeks and 53% at 4 weeks of the CON VGs (p < 0.05). Medial areas and percent of stenoses were also significantly less in PDT than in CON VG. However, intimal hyperplasia noted in the longitudinal sections within 2 mm of the anastomoses did not demonstrate a difference between PDT and CON VG. Intimal hyperplasia of both PDT and CON VG consisted of smooth muscle cells, verified by immunohistochemistry. Bromodeoxyuridine-labeled cells were more abundant in 2-week than in 4-week specimens, were found most frequently in the intimal areas of the CON VG body, and were equivalent in the anastomoses of PDT VG and CON VG. CONCLUSIONS These data suggest that PDT of vein grafts suppresses the development of IH in the body of the vein graft but does not affect IH adjacent to the anastomoses. The artery may be the source of proliferating smooth muscle cells that contribute to the anastomotic vein graft IH.

Endothelin and Vein Bypass Grafts in Experimental Atherosclerosis

Endothelin-1 (ET-1) is a potent vasoconstrictor whose serum concentration increases with the development of atherosclerosis. Coronary artery-vein bypass grafts are susceptible to vasospasm and to the development of accelerated atherosclerosis. Although ET-1 is thought to play a role in coronary vasospasm, the effect of ET-1 in atherosclerotic vein grafts is unknown. The responses of veins, arteries, and vein bypass grafts from normolipidemic and hyperlipidemic animals to ET-1 were therefore investigated. Vein bypass grafts were placed in the carotid position of 12 New Zealand White rabbits. Seven were fed a 1% cholesterol diet for 4 weeks before surgery and thereafter until harvest (hyperlipidemia), and five were fed a normal diet (normolipidemia). Vein grafts, contralateral common carotid arteries, and jugular veins were harvested 4 weeks after surgery. Whereas there were no histologic changes in veins or carotids, normolipidemic vein grafts developed intimal hyperplasia and hyperlipidemic vein grafts developed atherosclerosis. Isometric tension studies with ET-1 (10(-12) to 10(-6) M) showed that hyperlipidemia increased the maximal tension generated to ET-1 in the veins (660 +/- 80 to 1,110 +/- 140 mg, mean +/- SEM; p < 0.05), carotids (150 +/- 30 mg to 540 +/- 120 mg; p < 0.05), and vein grafts (180 +/- 20 to 450 +/- 60 mg; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of endothelium-derived relaxing factor-mediated relaxation and smooth muscle cell function in arterial and venous vein bypass grafts☆

The development of intimal hyperplasia in reversed vein grafts is associated with altered endothelial and vasomotor function. This study examines the effect of surgery on the morphology and vasomotor function of experimental arterial and venous vein bypass grafts. Twelve reversed vein grafts, 12 in situ vein grafts and 12 venovenous grafts were placed in 24 New Zealand White rabbits. All grafts remained patent and were harvested after 28 days. Isometric contraction to norepinephrine, histamine, bradykinin, serotonin and relaxation to acetylcholine and sodium nitroprusside following pre-contraction with prostaglandin F(2 alpha) were determined on the grafts and on contralateral jugular veins. Compared to the contralateral jugular veins, norepinephrine supersensitivity was induced in the reversed vein grafts, and venovenous vein grafts but not in the in situ vein grafts. Decrease in histamine sensitivity occurred in all grafted vessels. Bradykinin responses were significantly reduced in the in situ vein grafts and reversed vein grafts. Contractile responses to serotonin developed in the in situ vein grafts and reversed vein grafts only. Acetylcholine-induced endothelium-derived relaxing factor-mediated relaxation of the contralateral jugular veins was preserved in both venovenous grafts and in situ vein grafts but was lost in reversed vein grafts. All tissues relaxed to sodium nitroprusside in dose-dependent manner. The data suggest that norepinephrine supersensitivity in reversed vein grafts results from excision of the vessel. Attenuation of bradykinin responses and the enhanced contractile responses to serotonin appear predominantly to result from arterialization. Decreases in histamine sensitivity appear related both to excision and to arterialization. Neither the excision of the vein nor arterialization individually influences the alterations in endothelium-derived relaxing factor-mediated relaxation. However, a combination of excision and arterialization results in the altered endothelium-derived relaxing factor-mediated relaxation. This study suggests that the surgical preparation of the vein and the surgical procedure used have significantly different effects on endothelium-derived relaxing factor-mediated relaxation and smooth muscle contractility 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.

Prolonged Hypercholesterolemia Induces Reversible Alterations in Venous Vasomotor Function

Hypercholesterolemia is associated with altered arterial endothelial and smooth muscle cell function. This study examines the influence of hypercholesterolemia on external jugular venous endothelial and smooth muscle cell vasoreactivity. Eighteen New Zealand White rabbits received a 1% cholesterol diet: in nine animals, this diet was continued until harvest at 8 weeks (hypercholesterolemic group), but in the other nine animals, the diet was changed to standard rabbit chow after 4 weeks and continued for a further 4 weeks (cholesterol reduction group). The change in the diet resulted in a 70% decrease in serum cholesterol concentration. Eight animals received standard rabbit chow for 8 weeks. Hypercholesterolemia induced hypersensitivity and increased maximal contractions to norepinephrine and endothelin-1. In addition, the maximal response to bradykinin increased, and a contraction to serotonin was induced in the veins from the hypercholesterolemic animals. Cholesterol reduction induced bradykinin hypersensitivity but had no effect on endothelin-1 sensitivity. Norepinephrine hypersensitivity returned to normal and the serotonin response disappeared. A decrease in the maximal contractile responses to these agonists was also observed. Hypercholesterolemia interfered with dose-dependent, EDRF (endothelium derived relaxing factor)-mediated relaxation induced by acetylcholine but, following the reduction of serum cholesterol, normal acetylcholine-induced, endothelium-dependent relaxation returned. Non-endothelium-dependent relaxation to sodium nitroprusside of precontracted veins was unaffected by the presence of high cholesterol concentrations. There were no morphological changes apparent in the veins of either the hypercholesterolemic or the cholesterol reduction groups. In conclusion, this study suggests that hypercholesterolemia induces reversible functional abnormalities in venous tissue and this ability of the jugular veins to recover may be, in part, linked to the lack of morphological changes.

The morphology of venovenous bypass graft endothelium.

Venovenous bypass grafts are commonly used in the repair of vascular trauma to large- and small-caliber veins. This study examines the morphology of the venous wall in an experimental model of the venovenous bypass graft. The morphology of the venous endothelium from unmanipulated jugular veins and from jugular veins implanted as a venovenous bypass graft in the external jugular venous system for 10 min, 6 h, and 1, 3, 5, 7 and 28 days was examined. Veins and venovenous grafts were pressure fixed in situ at 80 mmHg and were examined by scanning and transmission electron microscopy. The endothelial cell lining remained confluent and intact over the 28-day period with evidence of endothelial cell contraction (spindle-shaped cells) for the first 72 h. Pinocytotic activity in endothelial cells and underlying smooth muscle cells was observed throughout the study, strongly indicating physiologically active cells. There was some accumulation of blood cells, predominantly polymorphonuclear leukocytes on the endothelial surface. Polymorphonuclear leukocytes were observed to infiltrate into the subendothelium through endothelial cell junctions within 6 h but by day 3, none was noted in the subendothelial space. There was no major disruption of the graft wall at any time point. By day 28, there was evidence of intimal thickening in the venovenous bypass grafts but no well-demarcated intimal hyperplasia. This study shows that there is no significant endothelial injury in the venovenous bypass grafts and that the endothelial cells remain physiologically active. Short-term failure of venovenous bypass grafts, therefore, appears not be due to significant endothelial cell damage in the graft.

Arterial geometric abnormalities produce two-dimensional compliance disturbances

PURPOSE This study describes the two-dimensional compliance changes that develop in low-grade hemodynamically insignificant stenoses. METHODS Twenty-four male Sprague-Dawley rats were used in the study. In 14 rats, balloon injury and endothelial denudation of the common carotid artery was followed by the application of a ligature, which produced a 20% narrowing of the outer diameter; five other rats were similarly injured without creating a stenosis, and five were stenosed without injury. The latter two groups served as control. Two-dimensional measures of arterial wall motions were obtained 30 minutes and 3 days after surgery by use of a video motion analyzer. Subsequently, circumferential (Cc) and longitudinal (Cl) compliance values were calculated and analyzed. RESULTS At 30 minutes, in the injured stenosed arteries, overall Cc was significantly greater than in the nonstenosed injured arteries at all measured points along the artery. Both stenosed injured and stenosed noninjured arteries also displayed increased Cc both proximal and distal to the stenoses compared with Cc values furthest from the stenosis. Cl values in the stenosed arteries, both injured and noninjured, became negative across the stenosis. All two-dimensional compliance changes occurred within the area of arterial narrowing and gradually resolved approaching the nonstenotic region of the artery. At systole, in the adjacent to the stenosis arterial segments, circumferential distension was accompanied by simultaneous longitudinal compression. Three days after surgery, although Cc increased proximal and distal to the stenosis and Cl decreased in the areas adjacent to the stenoses, no statistically significant difference was found. CONCLUSIONS Low-grade stenosis results in a longitudinal compression accompanied by a relatively increased circumferential distension of arterial segments adjacent to the stenotic region. This pattern is not initiated or influenced by the presence or absence of endothelial cells.