Arterial pO2 stimulates intimal hyperplasia and serum stimulates inward eutrophic remodeling in porcine saphenous veins cultured ex vivo

Ex vivo culture of arteries and veins is an established tool for investigating mechanically induced remodeling. Porcine saphenous veins (PSV) cultured ex vivo with a venous mechanical environment, serum-supplemented cell-culture medium and standard cell-culture conditions (5% CO₂ and 95% balance air ~140 mmHg pO₂) develop intimal hyperplasia (IH), increased cellular proliferation, decreased compliance and exhibit inward eutrophic remodeling thereby suggesting that nonmechanical factors stimulate some changes observed ex vivo. Herein we explore the contribution of exposure to greater than venous pO₂ and serum to these changes in cultured veins. Removing serum from culture medium did not inhibit development of IH, but did reduce cellular proliferation and inward eutrophic remodeling. In contrast, veins perfused using reduced pO₂ (75 mmHg) showed reduced IH. Among the statically cultured vessels, veins cultured at arterial pO₂ (95 mmHg) and above showed IH as well as increase in proliferation and vessel weight compared to fresh veins; veins cultured at venous pO₂ did not. Taken together, these data suggest that exposure of SV to arterial pO₂ stimulates IH and cellular proliferation independent of changes in the mechanical environment, which might provide insight into the etiology of IH in SV used as arterial grafts.     

Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-dependent pathway

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~ 3-fold increase in proliferation and ~ 3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O₂^??) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.     

Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide ⁴³gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment.     

Shear stress and pressure modulate saphenous vein remodeling ex vivo

Vein graft failure remains an important clinical challenge, but factors contributing to vein graft failure have not clearly been defined. We investigated the role of the mechanical environment in vein remodeling in an ex vivo perfusion system. Porcine saphenous veins were subjected to five different ex vivo hemodynamic environments, including one mimicking an arterial bypass graft, for one week in order to independently assess the effects of shear stress and pressure on vein remodeling. The extent of intimal hyperplasia decreased with culture under increasing shear stress, with veins cultured under the lowest levels of shear stress exhibiting the greatest ratio of intimal/medial area, 0.15+/-0.03, which was greater than that of fresh veins (0.06+/-0.01, p<0.05). All perfused veins displayed characteristics of both medial hypertrophy and eutrophic remodeling, with those veins cultured under elevated pressures showing greater increases in mass and area than those cultured under venous pressures. Medial area correlated with the average pressure under which veins were cultured (R2=0.95, p<0.001), with veins cultured under bypass graft conditions, which were exposed to the greatest pressure during the one week culture, exhibiting the largest medial area (1.69+/-0.15 mm2), which was significantly greater than that of fresh veins (1.08+/-0.05 mm2, p<0.05). However, pulsatility was not a necessary stimulus for medial growth, as increases in medial area were observed in culture conditions in which steady flow and pressure were present. Our results suggest that pressure and shear stress act independently to regulate vein remodeling, influencing changes in vessel size as well as the nature of the remodeling.     

The clopidogrel after surgery for coronary artery disease (CASCADE) randomized controlled trial: clopidogrel and aspirin versus aspirin alone after coronary bypass surgery [NCT00228423]

Background

Saphenous vein graft disease remains a major limitation of coronary artery bypass graft surgery. The process of saphenous vein intimal hyperplasia begins just days after surgical revascularization, setting the stage for graft atherosclerotic disease and its sequalae. Clopidogrel improves outcomes in patients with atherosclerotic disease, and is effective at reducing intimal hyperplasia in animal models of thrombosis. Therefore, the goal of this study will be to evaluate the efficacy of clopidogrel and aspirin therapy versus aspirin alone in the prevention of saphenous vein graft intimal hyperplasia following coronary artery bypass surgery.

Methods

Patients undergoing multi-vessel coronary artery bypass grafting and in whom at least two saphenous vein grafts will be used are eligible for the study. Patients will be randomized to receive daily clopidogrel 75 mg or placebo, in addition to daily aspirin 162 mg, for a one year duration starting on the day of surgery (as soon as postoperative bleeding has been excluded). At the end of one year, all patients will undergo coronary angiography and intravascular ultrasound assessment of one saphenous vein graft as selected by randomization. The trial will be powered to test the hypothesis that clopidogrel and aspirin will reduce vein graft intimal hyperplasia by 20% compared to aspirin alone at one year following bypass surgery.

Discussion

This trial is the first prospective human study that will address the question of whether clopidogrel therapy improves outcomes and reduces saphenous vein graft intimal hyperplasia following cardiac surgery. Should the combination of clopidogrel and aspirin reduce the process of vein graft intimal hyperplasia, the results of this study will help redefine modern antiplatelet management of coronary artery bypass patients.     

The effects of aging on the intimal region of the human saphenous vein: insights from multimodal microscopy and quantitative image analysis

We hypothesized that structural remodeling associated with advancing age occurs in human saphenous veins. To address this hypothesis, we have identified structural remodeling in human saphenous veins by applying histochemistry, fluorescence staining and quantitative image analysis to specifically assess intimal area, intimal cellularity and intimal collagen content and organization. Saphenous veins were collected from patients undergoing coronary artery bypass graft surgery. Area measurements and cellularity were quantified using the image analysis software Stereo Investigator, employing planimetry and counting frames, respectively. Collagen content and organization were quantified in MetaMorph image analysis software based on measurements of color (hue, saturation, and intensity) from polarized light images. Intimal area and cellularity showed no statistically significant increases with age; in contrast, total collagen content showed a significant decrease with advancing age. Furthermore, collagen fiber types also demonstrated a statistically significant alteration with age; increases in age resulted in decreases in larger collagen fibers. No significant changes in small collagen fibers were identified. These results raise the possibility that age-associated structural alterations in total collagen content, specifically collagen fiber size, could be a factor in the etiology of age-associated venous diseases.     

Arterial Shear Stress Reduces Eph-B4 Expression in Adult Human Veins

Vein graft adaptation to the arterial environment is characterized by loss of venous identity, with reduced Ephrin type-B receptor 4 (Eph-B4) expression but without increased Ephrin-B2 expression. We examined changes of vessel identity of human saphenous veins in a flow circuit in which shear stress could be precisely controlled. Medium circulated at arterial or venous magnitudes of laminar shear stress for 24 hours; histologic, protein, and RNA analyses of vein segments were performed. Vein endothelium remained viable and functional, with platelet endothelial cell adhesion molecule (PECAM)-expressing cells on the luminal surface. Venous Eph-B4 expression diminished (p = .002), Ephrin-B2 expression was not induced (p = .268), and expression of osteopontin (p = .002) was increased with exposure to arterial magnitudes of shear stress. Similar changes were not found in veins placed under venous flow or static conditions. These data show that human saphenous veins remain viable during ex vivo application of shear stress in a bioreactor, without loss of the venous endothelium. Arterial magnitudes of shear stress cause loss of venous identity without gain of arterial identity in human veins perfused ex vivo. Shear stress alone, without immunologic or hormonal influence, is capable of inducing changes in vessel identity and, specifically, loss of venous identity.     

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.     

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.     

Lyophilized veins as arterial interposition allografts

Venous allografts were evaluated in two models. Lyophilized allograft veins used as interposition grafts in the infrarenal aorta of the canine were studied and found to be patent at 1 year. Pathologic examination of the grafts revealed mild intimal hyperplasia and persistence of the basic structure of the lyophilized vessel. The ability of venous tissue to elicit an antibody response when transplanted into an allogeneic recipient was studied in the rat using the lymphocyte cytotoxicity assay. Fresh and Me₂SO-cryoprotected frozen veins produced circulating antibody when used as interposition grafts in the infrarenal aorta of the rat. Lyophilized and noncryoprotected frozen veins did not induce measurable antibody. Lyophilized allograft veins are a nonimmunogenic vascular graft material with acceptable long-term patency.     

Nanoparticle-Mediated Local Delivery of an Antisense TGF-β1 Construct Inhibits Intimal Hyperplasia in Autogenous Vein Grafts in Rats

Background

Intimal hyperplasia is one of the most important causes of vascular graft failure. Numerous studies have correlated transforming growth factor-β1 (TGF-β1) with extracellular matrix (ECM) deposition, a hallmark of intimal thickening.

Principal Findings

In the present study, we performed immunohistochemistry, RT-PCR, and Western blot to examine the dynamic expression of TGF-β1, TGF-β1 receptor type I (TGF-β RI), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) during intimal hyperplasia in grafted veins of a rat model generated by grafting a portion of the right internal jugular vein to the ipisiliary caroid artery. Additionally, we determined whether nanoparticle-mediated delivery of a TGF-β1 antisense-expressing construct prevented TGF-β1 expression and intimal hyperplasia in grafted veins. In grafted veins, the expression of TGF-β1 significantly increased on day 3 after transplantation, peaked on day 7, slightly decreased on day 14, and returned to baseline levels on day 28. The positive expression of TGF-β RI in grafted veins remarkably increased on day 7, peaked on day 14, and decreased thereafter. MMP-1 expression decreased significantly, while TIMP-1 expression increased, significantly on days 14 and 28. Nanoparticle-mediated delivery of a TGF-β1 antisense-expressing construct down-regulated TGF-β1 expression and inhibited intimal hyperplasia in grafted veins.

Conclusions

Our findings provide further evidence that TGF-β1 plays an integral role in the development of intimal hyperplasia after vascular injury. Nanoparticle-mediated delivery of a TGF-β1 antisense-expressing construct is a feasible strategy to target TGF-β1-induced intimal thickening.     

Heparin and dibutyryl cAMP modulate gene expression in stimulated human saphenous vein smooth muscle cells

Summary Increased expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A chain, and tissue plasminogen activator (tPA) by smooth muscle cells (SMC) has been postulated to mediate the progression of intimal hyperplasia. We tested whether heparin would suppress the expression of these genes in stimulated human saphenous vein SMC. Quiescent cultured human saphenous vein SMC were stimulated for 4 h with heat-inactivated fetal bovine serum (10% by vol) in the presence or absence of heparin (1 to 250μg/ml). Heparin (50μg/ml) attenuated the induction by serum of bFGF mRNA, tPA mRNA, and tPA secretion. Nonanticoagulant heparin also attenuated serum induction of bFGF and tPA mRNA levels. To further study the role of second messenger signaling, a more specific mode of SMC stimulation was used with thrombin (3 U/ml) in the presence or absence of dibutyryl cyclic AMP (Bu₂-cAMP; 0.5 mM). In contrast to heparin, which had no effect on PDGF expression, Bu₂-cAMP decreased the induction by thrombin of PDGF-A chain mRNA levels. In thrombin-stimulated SMC, Bu₂-cAMP significantly decreased secretion of PDGF-AA protein. Thrombin, however, caused an increase in bFGF mRNA levels which was potentiated by Bu₂-cAMP with associated potentiation by Bu₂-cAMP of intracellular bFGF protein levels. The induction of tPA mRNA and tPA secretion by thrombin was sharply blocked by Bu₂-cAMP. These results suggest that heparin reduces intimal hyperplasia at least partly via partial inhibition of SMC gene expression.     

Influence of variations in pericellular oxygen tension on individual cell growth,muscle-characteristic proteins,and lactate dehydrogenase isoenzyme pattern in cultures of beating rat heart cells

Summary Primary cell cultures from neonatal rat ventricles were continuously exposed for 7 days in a modified roller apparatus to defined pericellular oxygen tension varying from 0.6 to 600 mm Hg. 5-Fluorodeoxyuridine was added to the medium to prevent over-growth of muscle cells by nonmuscle cells. A pericellular pO₂ of 600 mm Hg was lethal. The range of about 15 to 150 mm Hg was favorable, as indicated by increases in total and muscle-characteristic proteins. Between the 2nd and 8th day of cultivation at a pO₂ of 38 mm Hg, myosin content per cell increased 3.2-fold and creatine kinase activity 2.5-fold. At 0.6 mm Hg, myosin content increased only 1.3-fold and there was no increase in creatine kinase activity. The rate of myosin synthesis was diminished at this low pO₂. ATP level and beating rate at 0.6 mm Hg did not differ from values at 38 mm Hg. The isoenzyme pattern of lactate dehydrogenase remained unchanged during cultivation at 38 mm Hg, whereas at 0.6 mm Hg it shifted towards an M-type pattern. These experiments suggest that neonatal rat heart cells maintained in vitro can adapt themselves to low oxygen tensions.     

Prostacycin production by arterialized autogenous venous grafts in dogs

Arteries are capable of producing significantly larger quantities of protacyclin than are veins. To test the hypothesis, whether prostacyclin production by the vessel wall is related to blood pressure and flow, we measured the amounts of PGI₂ released and synthesized by venous segments transplanted for 6 weeks into the arterial circulation. These results were compared with the production of prostacyclin by normal veins and arteries. In 20 dogs a segment of jugular vein was interposed into the carotid system; a sham dissection was done on the opposite side. “Arterialized” vein grafts showed prominent intima lined by endothelium, medial smooth muscle cell proliferation and fibrotic proliferation in adventitia. Spontaneous and arachidonic acid- stimulated prostacyclin production (measured by radioimmunoassay for 6-keto-PGF_1α) was not significantly different between arterialized venous autografts and jugular veins. Significantly larger amounts of prostacyclin were synthesized by the carotid artery. Thus, histologic changes and rheologic effects occurring in vein grafts transposed to the arterial site do not affect prostacyclin production.     

Effect of the thromboxane receptor antagonist EP 092 on endotoxin shock in the sheep

The thromboxane receptor antagonist EP 092 inhibits the acute pulmonary vascular response to endotoxin in the anaesthetized, closed-chest sheep. The increase in the TXB₂ level in arterial blood was not suppressed by EP 092. Intravenous infusion of the thromboxane mimetic 11,9-epoxymethano PGH₂, but not PGF_2α, raises pulmonary artery pressure and lowers arterial pO₂ similar to the endotoxin. Isolated strips of lobar pulmonary veins but not lobar arteries are contracted by low concentrations of 11,9-epoxymethano PGH₂ - the effects are potently inhibited by EP 092.     

Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia

Vascular endothelial growth factor (VEGF) promotes neovascularization, microvascular permeability, and endothelial proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of 121, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, vascular permeability, and integrity.     

Scanning electron microscopy of pulmonary vascular endothelium in rats with hypoxia-induced hypertension

Scanning electron microscopy was used to study the endothelial surface of the pulmonary trunk, artery, and vein in normobaric control rats as well as in rats exposed to hypobaric hypoxia for 7 and 21 days. The individual endothelial cells of the normobaric pulmonary trunk and hilar artery were flat and slightly elongated with elevated nuclear regions, and those of the intermediate-sized artery were more elongated and had more microvilli than the large arteries studied. Their endothelial cell boundaries were outlined by beaded cytoplasmic projections. The surfaces of the normobaric hilar and intermediate-sized veins were smooth and demonstrated numerous longitudinal streaks. These venous endothelial cells were elongated and their cell boundaries were outlined by low discontinuous marginal folds. Exposure to hypobaric hypoxia caused the following changes on the arterial surface: elevation of the endothelial cells; formation of microvilli-rich cell clusters; formation of hollow defects; and the attachment of leukocytes. Hypobaric hypoxia also caused the disappearance of the longitudinal streaks and the occurrence of microvilli-rich cells in the hilar veins. The endothelial surface modifications in the hypobaric rats could be related to thickening of the endothelium, intimal edema, increased intimal connective tissue, luminal invasion of leukocytes, and increased endothelial cell proliferation, known to occur in systemic arteries of hypertensive animals.     

Effect of oxygen on galactose oxidase synthesis and secretion inDactylium dendroides

Dactylium dendroides mycelia exposed to different oxygen tensions secreted galactose oxidase in proportion to the pO₂. The intracellular levels of galactose oxidase, catalase, and superoxide dismutase increased 10.4-, 2.3-, and 2.1-fold, respectively, when the oxygen tension was raised from zero to 100%. Oxygen consumption was enhanced by increased partial pressure of O₂ and was higher than CO₂ production above 40% O₂. The results suggest that galactose oxidase could participate in an oxygen protection mechanism and/or as a microbicidal agent inD. dendroides.     

Venous Distensibility in Patients with Varicose Veins

Forearm veins were studied to determine whether patients with primary varicosity of the saphenous veins had a generalized abnormality of the venous system. Distensibility of the superficial forearm and hand veins was measured in 25 patients with varicosity of the saphenous veins, and in 25 control subjects. Patients with saphenous varicosity had a significantly greater distensibility of the undistorted forearm veins than control subjects. Hysteresis of distensibility curves was more prnounced in patients with varicosity than in control subjects; mean hysteresis index was 0.65 ± 0.06 versus 0.28 ± 0.02 in controls. These investigations suggest that an increased distensibility of the venous system is the predisposing factor in the development of varicose veins.