Mark G. Davies

Pathophysiology of Vein Graft Failure: A Review

Vein bypass grafting is an integral component of cardiovascular surgical practice for both arterial and venous diseases. However, many of these grafts will eventually fail due to either intrinsic or extrinsic causes. This review examines the current understanding and knowledge of venous histology, vein graft pathology and the associated endothelial and smooth muscle cell physiology and pharmacology. In addition, the status of research on the therapeutic control of vein graft intimal hyperplasia and accelerated atherosclerosis is assessed.

Experimental hypercholesterolemia in rabbits induces cavernosal atherosclerosis with endothelial and smooth muscle cell dysfunction

Hypercholesterolemia and other vascular risk factors for atherosclerosis are commonly associated with impotence. To characterize cavernosal smooth muscle reactivity in hypercholesterolemia, we performed isometric tension studies (with norepinephrine, acetylcholine, papaverine and electrical field stimulation) on isolated strips of corpus cavernosum from rabbits fed a 1% cholesterol diet. To assess the impact of cholesterol reduction, a group of rabbits was fed a cholesterol diet for 4 weeks and was then returned to a normal diet for 4 weeks before testing. Potential structure-function relationships were delineated by ultrastructural evaluation with transmission electron microscopy (TEM). All forms of cavernosal relaxation, including papaverine relaxation, were impaired with hypercholesterolemia, and norepinephrine contraction was augmented. In addition, ultrastructural evidence of an early atherosclerotic process in the cavernosal sinusoids was detected. Importantly, reduction of elevated serum cholesterol normalized cavernosal relaxation, including that of papaverine, and decreased the sensitivity to norepinephrine, thereby suggesting that cavernosal smooth muscle dysfunction in hypercholesterolemia is reversible.

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

INTRODUCTIONnthe 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.nnnMETHODSnreversed 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.nnnRESULTSnthe 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.nnnCONCLUSIONnthe 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.

Investigative Urology: Modulation of Vasoactive Intestinal Polypeptide (VIP)-Mediated Relaxation by Nitric Oxide and Prostanoids in the Rabbit Corpus Cavernosum

The polypeptide VIP has been proposed as an inhibitory nonadrenergic, noncholinergic (NANC) neurotransmitter involved in the relaxation of cavernosal smooth muscle during erection. However, the specific mechanism(s) by which VIP exerts its effect is unknown. To determine whether VIP is involved in NANC-mediated cavernosal relaxation, strips of corpus cavernosum from New Zealand White rabbits were hung in tissue baths; atropine and guanethidine were added to block cholinergic and adrenergic neurotransmission, respectively. The tissue was then submaximally precontracted with norepinephrine (NE) and relaxed by electrical field stimulation (EFS; 10 V square waves, 0.5 msec. duration, 10 second trains at 5, 15 and 40 Hz) before and after preincubation with the VIP antagonist, VIP 10-28. To evaluate the role of nitric oxide (NO) and prostaglandins on the relaxant effect of VIP in the corpus cavernosum, the strips were contracted with NE and subsequently relaxed with cumulative doses of VIP (10(-10) to 10(-6) M.). After VIP dose-response curves were obtained, the strips were preincubated with N omega-nitro-L-arginine (NOARG, an inhibitor of nitric oxide synthase, 10(-4) M.) and the VIP dose-response curve was repeated. Indomethacin (10(-5) M.) was added to one-half of the NOARG treated strips to inhibit prostaglandin synthesis. VIP 10-28 inhibited EFS-induced relaxation (p < 0.05) and produced dose-dependent relaxation, which was inhibited by NOARG (p < 0.05). In contrast, the VIP-induced relaxation was more pronounced in the presence of indomethacin (p < 0.05). Moreover, indomethacin substantially reversed the NOARG inhibition of VIP relaxation (p < 0.05). These results suggest that VIP is a mediator of NANC cavernosal relaxation and that NO synthesis is involved in VIP-induced NANC relaxation in the corpus cavernosum. In addition, the presence of vasoconstrictive prostanoids modulates this VIP-mediated NANC relaxation. Therefore, VIP appears to contribute to NANC neurally mediated cavernosal relaxation, and its mechanisms of relaxation are dependent on prostanoid and involve the generation of NO.

Alterations in wall tension and shear stress modulate tyrosine kinase signaling and wall remodeling in experimental vein grafts

PURPOSEnHemodynamic alterations have been implicated as major stimuli for the development of intimal hyperplasia in vein grafts that are implanted in the arterial circulation. Tyrosine kinase is known to mediate cell signaling. However, its role with in vivo mechanotransduction is not yet well defined. We used a novel bioprosthetic collagen tube to provide an external support to vein grafts and examined the subsequent changes in hemodynamics, tyrosine kinase signaling, wall remodeling, and vasomotor function.nnnMETHODSnCarotid interposition bypass grafting was performed with the reversed jugular vein in New Zealand white rabbits. In the experimental group (n = 15), after the completion of the proximal anastomosis, the vein was passed through a 4-mm collagen tube and the distal anastomosis was performed. The tube support was fashioned to completely cover the vein grafts. The control animals (n = 14) had no tube support. After surgery, the blood pressure and flow rate were measured and the wall tension and shear stress were calculated in the vein grafts on day 3 or day 28 (n = 5 per group). Tyrosine phosphorylation was assessed with the Western blot test in vein grafts at day 3 (n = 4 per group). The intimal and medial dimensions of the vein grafts were assessed with videomorphometry on day 28 (n = 5 per group). The cumulative dose response curves of the vein grafts to contractile and relaxant agonists were determined in isometric tension studies on day 28 (n = 5 per group).nnnRESULTSnThe use of tube support reduced wall tension 1.7-fold (P <.01) and increased shear stress 4.8-fold (P <.001) without altering the flow rate or blood pressure. The tyrosine kinase activity was reduced 15-fold (P <.001) in the tube-supported vein grafts. The intimal thickness was reduced by 45% in the tube-supported vein grafts as compared with the control grafts (46 +/- 2 mm vs 84 +/- 5 mm, respectively; P <.0001), and the media thickness was reduced by 20% (63 +/- 8 mm vs 79 +/- 4 mm, respectively; P <.05). Isometric tension studies showed preservation of contractile function and modulation of endothelial-dependent dysfunctional relaxation in tube-supported vein grafts.nnnCONCLUSIONnThese results show that reduced wall tension and increased shear stress with an external tube support can effectively modulate the signaling, functional, and hyperplastic responses in vein grafts. We conclude that this simple strategy deserves further study and clinical consideration.

G protein signaling and vein graft intimal hyperplasia: Reduction of intimal hyperplasia in vein grafts by a G(βγ) inhibitor suggests a major role of G protein signaling in lesion development

Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.

Modulation of Tissue Factor Protein Expression in Experimental Venous Bypass Grafts

Vein graft failure is a major limitation of coronary artery and peripheral vascular surgery. Tissue factor (TF), a transmembrane glycoprotein, generates thrombin by initiating the extrinsic coagulation cascade and plays a major role in the response to arterial injury. This study was designed to examine changes in TF protein expression in response to venous bypass grafting. New Zealand White rabbits underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. The contralateral control jugular veins (n = 6), early vein grafts (1 or 3 days after grafting, n = 18), and late vein grafts (14 or 28 days after grafting, n = 8) were examined by immunohistochemistry. The presence or absence of TF immunostaining in the intima was assessed in each vessel quadrant. In control veins, intimal TF staining was present in 5 of 24 vessel quadrants. In early vein grafts, TF staining was markedly increased in the intima (72 of 72 quadrants, P < .001 vs control veins), and TF immunostaining colocalized with CD18-positive leukocytes but not with endothelial cells, vascular smooth muscle cells, or RAM11-positive macrophages. In late vein grafts with intimal hyperplasia, TF expression was low or absent in the intima (6 of 32 quadrants, P < .001 vs early vein grafts; P = NS vs control veins), although medial smooth muscle cells expressed TF. Marked changes in TF expression occur in vein grafts. In early vein grafts, TF protein was greatly increased in the intima for at least 3 days and was associated with CD18-positive leukocytes. In late vein grafts with intimal hyperplasia, however, TF protein was not seen in the intima. These findings may have important implications for the development of therapeutic strategies to limit vein graft failure.

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

OBJECTIVESnThe 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.nnnMATERIALSnForty New Zealand white rabbits underwent jugular vein interposition grafting of the common carotid artery.nnnDESIGNnTen 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.nnnRESULTSnAll 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.nnnCONCLUSIONnThese 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.

G Protein Signaling and Vein Graft Intimal Hyperplasia: Reduction of Intimal Hyperplasia in Vein Grafts by a Gbg Inhibitor

Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.

Characterization and Function of Histamine Receptors in Corpus Cavernosum

Widespread clinical use of H2 antagonists for peptic ulcer disease has been associated with reports of impotence. Histamine is a vasoactive amine which is endogenously produced in many organs including the penis. To date, 3 histamine receptor subtypes (H1, H2 and H3) have been identified. However, the role and function of histamine in the corpus cavernosal physiology are poorly understood. This study evaluates the in vitro functional characteristics of the 3 histamine receptor subtypes in the isolated corpus cavernosal strips from New Zealand White rabbits. The isometric responses to histamine and specific histamine receptor subtype agonists were assessed, following cyclooxygenase (indomethacin, 10(-5) M.), adrenergic (guanethidine, 5 x 10(-6) M.) and cholinergic (atropine, 5 x 10(-6) M.) blockade, at resting tension and after submaximal precontraction with norepinephrine (NE, 2 x 10(-5) M.). Histamine (10(-8) M. to 10(-3) M.) produced concentration-dependent contraction from basal and precontracted states and did not relax precontracted tissue. The H1 agonist, 2-(2-thiazolyl)ethylamine (10(-8) M. to 10(-3) M.), produced a contractile response from both basal and precontracted states, while the corporal tissue did not respond to either dimaprit, an H2 agonist (10(-8) M. to 10(-5) M.) or R(-)-alpha-methyl-histamine, an H3 agonist (10(-8) M. to 10(-5) M.). The response to histamine was progressively attenuated by an H1 antagonist (mepyramine; 10(-8) M. to 10(-5) M.), while neither an H2 antagonist (cimetidine; 10(-4)M.) nor an H3 antagonist (thioperamide; 10(-4)M.) had any inhibitory effects. H1 antagonism enhanced relaxation induced by electrical field stimulation (neurally mediated). Such relaxation increased after preincubation with 10(-6) M. or greater of mepyramine (p < 0.05). This study suggests that the principal histamine receptor subtype that mediates smooth muscle cell contraction in the corpus cavernosum is the H1 subtype. Since histamine H1 receptor antagonism increased NANC neurally mediated corporal relaxation, it possesses potential as an intracavernosal pharmacotherapeutic agent for the treatment of erectile dysfunction. This study, therefore, strongly indicates that H2 receptor antagonists are unlikely to have direct effects on penile erection.