Vein Graft Atherosclerosis Research Articles

Immunohistochemical Localization of Fibroblast Activation Protein in Coronary Arteries with Different Forms of Atherosclerosis

Background: Fibroblast activation protein (FAP) is a cell surface glycoprotein expressed by myofibroblasts in areas of active tissue remodeling. It plays a potentially important role in cardiac remodeling, atherosclerotic plaque formation, and plaque rupture. Given the distinct pathophysiology and morphology of different forms of atherosclerosis, we analyzed FAP expression in human coronary vessels with no coronary artery disease, atherosclerotic plaques at different levels of progression, and other distinct forms of coronary disease in post bypass vein grafting and cardiac allograft vasculopathy after a heart transplant. Methods: Immunohistochemical staining with monoclonal F19 mouse anti-human FAP antibody was performed to identify FAP in human atherosclerotic plaques, coronary bypass atherosclerosis, and post-transplant arteriosclerosis. The presence and distribution of FAP in different types and stages of human atherosclerosis were compared. Results: There was no FAP staining in patients with no significant coronary disease. All different types of human atherosclerotic lesioning lesions showed the presence of FAP expression, with different staining patterns in advanced atherosclerotic plaque, vein graft atherosclerosis lesions, and arteriosclerosis after a heart transplant. Conclusions: These data suggest that FAP may be a potential diagnostic marker and target for interventions, not only in coronary atherosclerotic plaque, but also in other forms of coronary disease, which have distinct pathophysiologies and currently limited treatment options.

Therapeutic inhibition of biomechanically-activated YAP-TAZ signaling prevents vein graft atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rembrandt Institute for Cardiovascular Sciences Background Venous bypass grafts may have limited patency due to excessive intimal hyperplasia and accelerated atherosclerosis. YAP-TAZ signaling regulates cellular responses to biomechanical cues, such as disturbed flow or vessel distention as seen in vein grafts. YAP activation leads to inflammation and angiogenesis, which both contribute to vein graft atherosclerosis. Inhibition of YAP-TAZ attenuates naive atherogenesis, but its potential to inhibit intraplaque angiogenesis and reduce vein graft atherosclerosis has not been explored. Methods Saphenous vein progenitor cells (SVPs) were subjected to mechanical strain in vitro to assess YAP activation. IHC to assess YAP-expression was performed on vein grafts from pigs as well as ex vivo cultured human saphenous veins. Furthermore, YAP-IHC was performed on murine vein grafts from hypercholesterolemic ApoE3*Leiden mice harvested at early, mid- and late-stage disease timepoints to assess optimal treatment window. In a separate experiment, mice (n=11-13/group) underwent bypass surgery and were treated with the FDA-approved YAP-TAZ inhibitor Verteporfin (50 mg/kg) or vehicle 3 times/week from day 10 until sacrifice (day 28). Ultrasound was used to longitudinally quantify vascular remodeling. Vein grafts were harvested and processed for morphometric and compositional analysis. Results Mechanical strain induced activation of YAP and its downstream targets (Ctgf, Cyr61) in SVPs, which was inhibited by Verteporfin (confirmed by WB, IP and qPCR). Moreover, YAP was abundantly expressed in pig vein graft lesions, whilst Verteporfin blocked YAP expression in ex vivo cultured human saphenous veins after exposure to disturbed flow. In addition, YAP was increasingly expressed during vein graft lesion development in mice, reaching its peak around day 14, which coincides with the initiation of intraplaque angiogenesis. Furthermore, YAP was predominantly expressed in luminal and adventitial endothelial cells. Analysis of ultrasound demonstrated that Verteporfin prevented vein graft thickening in mice over time. End-point histology revealed that the intima/media ratio was significantly decreased by Verteporfin treatment (37%, p=0.001). Intraplaque angiogenesis was also reduced (34%, p=0.002), whilst neovessel maturation was improved upon verteporfin treatment. The vessel wall ACTA2 content was reduced (42%, p=0.023), whilst collagen was not altered between both groups, indicating that Verteporfin diminished the fibroproliferative response without affecting vessel wall stability. Conclusion Verteporfin inhibited YAP-TAZ activation induced by mechanical strain in vitro (SVPs) as well as ex vivo (cultured human saphenous veins). In vivo, Verteporfin treatment resulted in a significant decrease of vein graft atherosclerosis and intraplaque angiogenesis. Therefore, therapeutic targeting of YAP-TAZ using Verteporfin might be a new candidate to improve vein graft patency.

Apolipoprotein A-I vascular gene therapy reduces vein-graft atherosclerosis

Coronary artery venous bypass grafts typically fail because of atherosclerosis driven by lipid and macrophage accumulation. Therapy for vein-graft atherosclerosis is limited to statin drugs, which are only modestly effective. We hypothesized that transduction of vein-graft endothelium of fat-fed rabbits with a helper-dependent adenovirus expressing apolipoprotein AI (HDAdApoAI) would reduce lipid and macrophage accumulation. Fat-fed rabbits received bilateral external jugular vein-to-carotid artery interposition grafts. Four weeks later, one graft per rabbit (n= 23 rabbits) was infused with HDAdApoAI and the contralateral graft with HDAdNull. Grafts were harvested 12weeks later. Paired analyses of grafts were performed, with vein graft cholesterol, intimal lipid, and macrophage content as the primary endpoints. HDAd genomes were detected in all grafts. APOAI mRNA was median 63-fold higher in HDAdApoAI grafts versus HDAdNull grafts (p<0.001). HDAdApoAI grafts had a mean 15% lower total cholesterol (by mass spectrometry; p= 0.003); mean 19% lower intimal lipid (by oil red O staining; p= 0.02); and mean 13% lower expression of the macrophage marker CD68 (by reverse transcriptase-mediated quantitative PCR; p= 0.008). Invivo transduction of vein-graft endothelium achieves persistent APOAI expression and reduces vein-graft cholesterol, intimal lipid, and CD68 expression. Vascular gene therapy with APOAI haspromise for preventing vein-graft failure caused by atherosclerosis.

Abstract 482: Early Reduction of Blood Flow in Rabbit Jugular Vein-Carotid Artery Bypass Grafts is not Caused by Anastomotic Stenosis

Background: Obstructed arteries are often treated by surgical implantation of venous bypass grafts. Vein grafts can fail for many reasons, including thrombosis, atherosclerosis, or anastomotic stenoses (narrowings where the grafts connect to the artery). In a previous study aimed at developing a hyperlipidemic rabbit model of vein-graft atherosclerosis for testing gene therapy, vein grafts used to bypass carotid arteries were widely patent. However, vein graft blood flow decreased by a mean of 5.6 mL/minute (~23%) between 8 and 16 wks after grafting, with flow at 16 wks reduced to &lt;60% of flow in an unoperated carotid artery. We hypothesized that decreased flow was caused by anastomotic stenoses, raising concern that this might lead to graft occlusion, precluding use of the model to test gene therapy for atherosclerosis. Methods: We designed an experiment to test whether anastomotic stenoses were present and—if so—whether the stenoses were the cause of decreased blood flow. In a new set of grafted veins, we harvested the anastomoses, embedded, sectioned and stained them, and measured percent stenosis using computer-assisted image analysis. Percent stenosis was calculated by dividing intimal area by the stenosis-free lumen area (calculated from the length of the IEL, assuming circular geometry in vivo). We measured stenoses in both anastomoses of 22 patent vein grafts, harvested from 14 hyperlipidemic rabbits (44 total anastomoses). Blood flow (mL/min) in each vein graft was measured with a flow meter at 4 wks after grafting and again at 16 wks (at harvest). Results: The mean blood flow at the 4 wks measurement was 51 mL/minute (range: 28 to 90) and 36 mL/minute (range: 21 to 53) at the harvest measurement. The calculated mean change in blood flow was –16 mL/minute (range: –55 to +14). The mean anastomotic stenosis was 39% (range: 2.3% to 89%). Using Spearman’s rank-order correlation test, we found that percentage stenosis did not correlate with the change in blood flow (r=0.059; P=0.8). Conclusions: Decreased blood flow in our vein graft model is likely not caused by anastomotic stenoses. Other explanations, such as decreased cardiac output or increased downstream vascular resistance (caused by cerebral atherosclerosis or microemboli) should be considered.

Influence of cholesterol level on long-term survival and cardiac events after surgical coronary revascularization

ObjectiveStatins have been shown to delay the inevitable progression of atherosclerosis in native coronaries and saphenous vein grafts, thereby reducing ischemic events after surgical coronary revascularization. However, there is significant controversy as to whether titrating statin therapy to concrete cholesterol targets is appropriate. MethodsA single-center retrospective analysis of 309 consecutive patients who underwent isolated coronary artery bypass graft in 2007 and 2008 was performed. Measurements of lipid profile subcomponents, namely total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides, in mmol/L, were obtained by retrospective review of electronic health records. The primary end point was cardiac death. The secondary end point was the composite of cardiac events, including cardiac death, nonfatal myocardial infarction, hospitalization for unstable angina, and target lesion revascularization. Database lock date was August 15, 2020. ResultsThe median follow-up duration was 12.5 years. Cardiac death occurred in 6.8% of the cohort. Cardiac events occurred in 21.7% of the cohort. New-onset myocardial infarction occurred in 8.7% (n = 27), of which 48.1% (n = 13) underwent repeat revascularization. A 2-level nested Cox proportional hazards regression model was constructed to determine whether cholesterol target attainment was independently associated with cardiac events. After risk adjustment, LDL-C, non–HDL-C, total cholesterol (TC), and TC/HDL-C ratio were independently associated with cardiac death. In receiver operating characteristics analyses, the optimal cut-off values for non–HDL-C, LDL-C, and TC/HDL-C ratio were 3.2 mmol/L, 2.3 mmol/L, and 3.5, respectively. ConclusionsExposure to elevated LDL-C and non–HDL-C cholesterol levels independently predicted long-term cardiac death after coronary artery bypass graft.

Abstract 11919: Igg1 Phosphorylcholine Diminishes Murine Atherosclerosis and Ameliorates Plaque Stability via Reduced Intraplaque Angiogenesis and Hemorrhage

Introduction: Phosphorylcholine (PC), one of the main oxLDL epitopes, is an important pro-inflammatory damage associated molecular pattern. Epidemiologic data show that natural anti-PC IgM protect against cardiovascular disease. Within atherosclerotic lesions, inflammation and angiogenesis are linked via VEGFA secreting CD163 + macrophages. PC antibodies are recognized for their anti-inflammatory properties, the effect of PC antibodies on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH) is yet unknown. Hypothesis: Newly developed IgG1 PC antibody (PCmAb) inhibits lesion development via IPA and IPH reduction in murine vein graft atherosclerosis. Methods: Hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly ip injections of (5mg/kg) PCmAb (n=11) or vehicle (n=12) until sacrifice at day 28. Vein graft morphometry and lesion composition including IPA and IPH were evaluated via immunohistochemistry. In vitro PCmAb effects were investigated in Hemoglobin (Hb):Haptoglobin(Hp)-cultured THP-1 macrophages and HUVECs. Results: PCmAb significantly decreased vein graft media area (13%), intima lesion (25%), and increased lumen area with 53% compared to vehicle. PCmAb improved lesion stability by increasing collagen content (18%) and decreased macrophages presence (31%). VCAM-1 and ICAM-1 expression in the vessel wall were also reduced (29%, 36%). PCmAb resulted in 23% decreased CD163 + macrophages content in vein grafts whereas CD163 expression was reduced in Hb:Hp stimulated macrophages. PCmAb significantly reduced neovessel density (34%) and significant reduced EC proliferation and migration with and without oxLDL stimulation. Moreover, PCmAb enhanced maturation of intraplaque angiogenic vessels by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62%reduction of IPH. Conclusions: PCmAb effectively inhibits murine atherosclerotic lesion formation. PCmAb reduces IPA and IPH by decreased neovessel density and macrophages influx via reduced expression of VCAM-1 and ICAM-1, and increased neovessel maturation in vein graft atherosclerosis. PCmAb holds promise as a new therapeutic approach for plaque stability.

IgG1 phosphorylcholine ameliorates plaque stability via reduced intraplaque angiogenesis and intraplaque haemorrhage in a murine atherosclerosis model

Abstract Background Phosphorylcholine, (PC) the polar headgroup of the dominating membrane phospholipid phosphatidylcholine, is one of the main oxLDL epitopes and an important pro-inflammatory damage associated molecular pattern. Experimental and epidemiologic data show that natural anti-PC IgM protect against cardiovascular disease. Within atherosclerotic lesions, inflammatory and angiogenesis processes are interdependent and contribute to plaque destabilization. Atherosclerotic lesion resident CD163+ macrophages promote leukocyte infiltration but also induce angiogenesis and vessel permeability by secreting VEGFA. PC antibodies are recognized for their anti-inflammatory properties. However, the effect of PC antibodies on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH), the main entrance route for inflammatory cells in advanced lesions, is unknown. Purpose To investigate the therapeutic effect of a new IgG1 PC antibody (PCmAB) on lesion development, IPA and IPH in murine vein graft atherosclerosis. Methods All animal experiments were performed in compliance with Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery. Mice received weekly ip injections of (5mg/kg) PCmAb (n=11) or vehicle (n=12) until sacrifice at day 28. Immunohistochemistry was used to evaluate vein graft morphometry and lesion composition including IPA and IPH. PCmAB isolated effects on pro-angiogenic and pro-inflammatory behaviour was investigated in vitro in HUVECs and Hemoglobin (Hb):Haptoglobin (Hp)-cultured THP-1 macrophages. Results PCmAB treatment decreased vein graft media area (13%) and intima lesion (25%), but more importantly increased lumen area with 53% when compared to vehicle treatment. PCmAb improved lesion stability by increasing collagen content (18%) and by decreasing macrophages presence (31%). VCAM-1 and ICAM-1 expression in the vessel wall were also reduced (resp.29% and 36%) by PCmAb. PCmAb improved IPA by a significant reduction in neovessel density of 34%. This was supported in vitro by significant reduced EC proliferation and migration upon PCmAB with and without oxLDL stimulation. Moreover, PCmAb enhanced maturation of intraplaque angiogenic vessels by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a reduction of IPH of 62% in the PCmAB group. PCmAb resulted in decreased macrophages CD163+ content in vein grafts by 23% whereas CD163 expression was reduced by PCmAb in Hb:Hp stimulated macrophages. Conclusion PCmAB is an effective inhibitor of atherosclerotic lesion formation in ApoE3*Leiden mice. PCmAb reduces IPA and IPH by decreased neovessel density and (CD163+) macrophages influx via reduced expression of VCAM-1 and ICAM-1, and increased neovessel maturation in vein graft atherosclerosis. PCmAB holds a promise as a new therapeutic approach for plaque stability. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Leiden University Medical Center

Inhibition of BMP9 and BMP10 signalling by the ALK1-Fc Ligand Trap enhances systemic inflammation and increases vein graft atherosclerosis

Abstract Background Vein graft patency rapidly declines within 5 years due to extensive intimal hyperplasia (IH) and instable atherosclerosis. The transforming growth factor-β (TGF-β) superfamily is involved in vein graft failure by regulating cell activation and inflammation. Activin receptor–like kinase-1 (ALK1), a TGFβ superfamily type I receptor, induces signalling through the Smad1/5/8 pathway upon binding of BMP9/10. ALK1-Fc, a soluble chimeric protein, consisting of both the Fc-part of an antibody and the extracellular domain of ALK1, this functions as a ligand trap for BMP9/10 thereby inhibiting downstream signalling. Purpose To identify whether BMP signalling blockade enhances inflammatory responses and IH formation resulting in plaque destabilization. Methods All animal experiments were performed in compliance with Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Hypercholesterolemic male ApoE3*Leiden mice underwent an interposition of a donor caval vein in the carotid artery and were treated twice weekly with ip injections of 10 mg/kg control-Fc y or ALK1-Fc. Non-invasive ultrasound imaging was performed weekly until the day of sacrifice (d28). Flow cytometry was performed on blood on d7 and d28. Vein grafts were processed for histological analysis. Results The long-axis lumen diameter (relative to d7) in the control group decreased significantly at d21 with 5% and 11% at d28 compared to an increase of 5% (d21) and 2% (d28) in the ALK1-Fc group. D28 short-axis analyses demonstrated a significant difference in lumen area between a decrease of 8% (control-Fc) and a 30% increase (Alk1-Fc). Moreover, d28 short-axis analyses showed a significant difference in wall area between an 8% decrease in the controls and a 27% increase in the Alk1-Fc group. Histological assessment confirmed that ALK1-Fc resulted in increased outward remodelling and a 45% significant increase in lesion size. Lesion composition analyses showed a significant reduction in relative collagen, a trend in relative smooth muscle cell content reduction, and more destabilizing plaque dissections, characterized by blood filled gaps in the IH from the lumen towards the adventitia. At 7 and 28 days after surgery Ly6C monocytes in the blood were significantly increased in the ALK1-Fc group compared to control-FC. In the control-FC group the Ly6Chigh monocytes were significantly reduced at t28 in comparison to t7 whereas the Ly6Clow monocytes were significantly increased. In the ALK1-Fc treated mice, both Ly6C subpopulations stayed high at both time points. ALk1-Fc treatment thus results in a sustained pro-inflammatory response. Conclusions Ultrasound and histological assessment demonstrated an increase in outward remodelling, increased IH, a sustained inflammatory response and plaque destabilization upon ligand trapping of the ALK1-Smad1/5 signalling pathway. This identifies BMP9/10 signalling as an attractive target to treat vein graft failure. Funding Acknowledgement Type of funding source: Public hospital(s). Main funding source(s): Leiden University Medical Center

The impact of advances in percutaneous catheter interventions on redo cardiac surgery.

Toward the end of the twentieth century, redo cardiac surgery accounted for approximately 15-20% of total cardiac surgical volume. Major risk factors for redo cardiac surgery include young age at time of the first operation, progression of native coronary artery disease (CAD), vein graft atherosclerosis, bioprosthetic valve failure and endocarditis, and transplantation for end stage heart failure. Historically, redo coronary artery bypass grafting (CABG) alone carried a mortality risk of around 4%. Factors such as older age, female sex, comorbidities, combined procedures, hemodynamic instability, and emergency procedures contributed to even higher mortality and morbidity. These poor outcomes made it necessary to look for less invasive alternate methods of treatment. Advances in catheter-based interventions have made a major impact on redo cardiac surgeries, making it no longer the first option in a majority of cases. Percutaneous interventions for recurrence following CABG, transcutaneous aortic valve replacement (TAVR) for calcific aortic stenosis, valve in valve (VIV) implantations, device closure of paravalvular leaks (PVL), and thoracic endovascular aortic repair (TEVAR) for residual and recurrent aneurysms and mitral clip to correct mitral regurgitation (MR) in heart failure are rapidly developing or developed, obviating the need for redo cardiac surgery. Our intent is to review these advances and their impact on redo cardiac surgery.

A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis

Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases.

Pathophysiology of native coronary, vein graft, and in-stent atherosclerosis.

Plaque rupture, usually of a precursor lesion known as a 'vulnerable plaque' or 'thin-cap fibroatheroma', is the leading cause of thrombosis. Less-frequent aetiologies of coronary thrombosis are erosion, observed with greatest incidence in women aged <50 years, and eruptive calcified nodules, which are occasionally identified in older individuals. Various treatments for patients with coronary artery disease, such as CABG surgery and interventional therapies, have led to accelerated atherosclerosis. These processes occur within months to years, compared with the decades that it generally takes for native disease to develop. Morphological identifiers of accelerated atherosclerosis include macrophage-derived foam cells, intraplaque haemorrhage, and thin fibrous cap. Foam-cell infiltration can be observed within 1 year of a saphenous vein graft implantation, with subsequent necrotic core formation and rupture ensuing after 7 years in over one-third of patients. Neoatherosclerosis occurs early and with greater prevalence in drug-eluting stents than in bare-metal stents and, although rare, complications of late stent thrombosis from rupture are associated with high mortality. Comparison of lesion progression in native atherosclerotic disease, atherosclerosis in saphenous vein grafts, and in-stent neoatherosclerosis provides insight into the pathogenesis of atheroma formation in natural and iatrogenic settings.

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis.

AimsThis study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms.MethodsVein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression.ResultsSignificantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis.ConclusionsIncreased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.

Total Arterial Revascularization: Achievable and Prognostically Effective—A Multicenter Analysis

Total arterial revascularization (TAR) is adopted to overcome late vein graft atherosclerosis, and occlusion. Uptake of TAR remains low despite reports suggesting superior survival. Previous studies primarily involved single sites and short-term follow-up. We report the influence of TAR on long-term survival in a large multicenter patient cohort. We reviewed 63,592 cases from an audited collaborative multicenter database. Of those, 34,181 consecutive patients undergoing first-time isolated coronary artery bypass (CABG) from 2001 to 2012 were studied. The data were linked to the National Death Index. We compared outcomes in patients who underwent TAR (n = 12,271) with outcomes in those who did not (n = 21,910). The influence of TAR on 10-year all-cause late mortality was assessed by propensity score analyses in 6,232 matched pairs. The 30-day mortality was 0.8% (96/12,271) for TAR patients and 1.8% (398/21,910) for non-TAR patients (p < 0.001). Late mortality was 7.5% (918/12,271) for TAR patients and 8.9% (1,952/21,910) for non-TAR patients (p < 0.001). The mean follow-up time was 4.9 years. In the propensity-matched cohort, the perioperative mortality was 0.9% (53/6,232) for TAR patients versus 1.2% (76/6,232) for non-TAR patients (p < 0.001). Kaplan-Meier survival in the matched cohort at 1, 5, and 10 years was 97.2%, 91.3%, and 85.4% for TAR patients and 96.5%, 90.1%, and 81.2% for non-TAR patients (p < 0.001). Late mortality was 8.0% (n = 500) for TAR patients and 10.0% (n = 622) for non-TAR patients (p < 0.001). Stratified Cox proportional hazards models showed lower risk for all-cause late mortality in the TAR group (TAR:HR 0.80, 95% confidence interval 0.71 to 0.90, p < 0.001). TAR is associated with low perioperative mortality and, importantly, improved long-term survival and could be used more liberally.

Platelet adhesion on endothelium early after vein grafting mediates leukocyte recruitment and intimal hyperplasia in a murine model.

Intimal hyperplasia (IH) is the substrate for accelerated atherosclerosis and limited patency of vein grafts. However, there is still no specific treatment targeting IH following graft surgery. In this study, we used a mouse model of vein grafting to investigate the potential for early intervention with platelet function for later development of graft IH. We transferred the inferior vena cava (IVC) from donor C57BL/6 mice to the carotid artery in recipients using a cuff technique. We found extensive endothelial injury and platelet adhesion one hour following grafting. Adhesion of leukocytes was distinct in areas of platelet adhesion. Platelet and leukocyte adhesion was strongly reduced in mice receiving a function-blocking antibody against the integrin αIIbβ3. This was followed by a reduction of IH one month following grafting. Depletion of platelets using antiserum also reduced IH at later time points. These findings indicate platelets as pivotal to leukocyte recruitment to the wall of vein grafts. In conclusion, the data also highlight early intervention of platelets and inflammation as potential treatment for later formation of IH and accelerated atherosclerosis following bypass surgery.

Association of Angiotensin-Converting Enzyme Genotype, Insertion/Deletion Polymorphism and Saphenous Vein Graft Atherosclerosis in Iranian Patients.

OBJECTIVE The aim of this study was to evaluate possible interactions among Angiotensin-I converting enzyme genotype, insertion/deletion polymorphism and atherosclerosis of vein grafts in Iranian patients, and characterize their clinical and demographic profile.METHODS In this cross-sectional study, patients who underwent coronary artery bypass graft surgery more than five years ago, were included for angiographic analysis. Atherosclerosis was determined by quantitative angiography and adjusted Gensini score. The gene angiotensin converting enzyme I/D polymorphism was detected by polymerase chain reaction.RESULTS A total of 102 patients participated in this study. Eighty-four patients were male. The frequency distribution of DD, ID and II polymorphism were 23.6%, 62.7% and 13.7% respectively. There were no differences among genotypic groups in age, sex, number of risk factors, number of vein grafts and months since bypass surgery. According to adjusted Gensini score [0.18±0.12 (II) vs. 0.11±0.09 (ID) and 0.1±0.09 (DD) P=0.021] the II genotype was associated with severity of vein graft atherosclerosis.CONCLUSION Although there are conflicting results about gene angiotensin converting enzyme I/D polymorphism and the degree of venous bypass graft degeneration, this study suggests an association between ACE genotype II and atherosclerosis of saphenous vein grafts, however, large samples considering clinical, demographic and ethnic profile are necessary to confirm these results.

Efficient gene transfer and durable transgene expression in grafted rabbit veins.

Venous bypass grafts are useful treatments for obstructive coronary artery disease. However, their usefulness is limited by accelerated atherosclerosis. Genetic engineering of venous bypass grafts that prevented atherosclerosis could improve long-term graft patency and clinical outcomes. We used a rabbit model of jugular vein-to-carotid interposition grafting to develop gene therapy for vein-graft atherosclerosis. Rabbit veins were easily transduced in situ with a first-generation adenoviral vector; however, most transgene expression (∼80%) was lost within 3 days after arterial grafting. This rapid loss of transgene expression was not prevented by transducing veins after grafting or by prolonged ex vivo transduction. However, delaying vein-graft transduction for 28 days (after the vein had adapted to the arterial circulation) prevented this early loss of transgene expression. We used the delayed transduction approach to test the durability of expression of a therapeutic transgene (apolipoprotein A-I) expressed from a helper-dependent adenoviral (HDAd) vector. HDAd DNA and apolipoprotein A-I mRNA were easily detectable in transduced vein grafts. Vector DNA and mRNA declined by 4 weeks, and then persisted stably for at least 6 months. Delaying transduction for 28 days after grafting permitted initiation of vein-graft neointimal growth and medial thickening before gene transfer. However, vein-graft lumen diameter was not compromised, because of gradual outward remodeling of grafted veins. Our data highlight the promise of HDAd-mediated gene therapy, delivered to arterialized vein grafts, for preventing vein-graft atherosclerosis.

Abstract 19106: Diabetes Induced Actin S-glutathiolation Increases Vein Graft Accelerated Atherosclerosis Through Delayed Endothelial Regeneration

Endothelial cell (EC) loss and regeneration are important aspects of the response to vascular injury leading to atherosclerosis in vein grafts (VG). EC regeneration is known to be impaired in diabetes mellitus (DM) leading to an accelerated atherosclerosis, however the mechanism remains unclear. Given the increased oxidative stress in DM, we hypothesized that protein s-glutathiolation, the reversible covalent addition of glutathione to cysteine on target proteins was a candidate mechanism. Overall s-glutathiolation of proteins measured by immunoblot was significantly increased in human DM atherosclerotic plaque compared to non-DM (4-fold, P&lt;0.01; n =10) with the increase in protein intensity at ≈ 40kDa. Mass spectrometry peptide fingerprint screening identified actin (42Kda) and the actin regulatory protein annexin A2 (39Kda) as the s-glutathiolated proteins. In cultured ECs in which s-glutathiolation was induced, proliferation (22%, P&lt;0.05) and motility (37%, P&lt;0.05) were significantly decreased compared to control. Assessment of actin conformational state identified a significant decrease in the ratio of filamentous to globular actin (P&lt;0.05) supporting our findings of decreased motility. As an in vivo model of acute vascular injury we constructed VGs in apolipoprotein-E knockout (ApoE-KO) mice with expression of an EC reporter gene (LacZ) in which DM was induced by streptoztocin (n=6-10/group). In VGs harvested 28 days post-operatively, DM increased VG total vessel wall area by 51% (P=0.001) and reduced endothelialization by 44% (P=0.001). S-glutathiolation levels were increased 10-fold in primary cultured ECs from these mice. Although there was no differences in baseline EC content or number of isolated cells, following 3 days of culture, total EC number, colony size and colony number from ApoE-KO/LacZ DM mice were decreased (4-fold P=0.05; 3-fold P=0.02; 3-fold P=0.04 respectively, n=6) as were putative EC progenitor levels from bone-marrow derived mononuclear cells by culture (20%,P=0.03) and FACS (22%, P=0.02). These observations indicate an important role for actin s-glutathiolation in EC regeneration following vascular injury and identify a mechanism by which accelerated atherosclerosis develops in patients with DM.

Pleiotropic Effects of HDL: Towards New Therapeutic Areas for HDL-Targeted Interventions

Plasma levels of high density lipoprotein (HDL) cholesterol levels and of apolipoprotein A-I are inversely correlated with the incidence of coronary heart disease. According to the HDL hypothesis, raising HDL cholesterol is expected to lead to a decrease of coronary heart disease risk. The stringent requirement for proving or refuting this hypothesis is that the causal pathway between the therapeutic intervention and a hard clinical end-point obligatory passes through HDL. The lack of positive clinical results in several recent HDL trials should be interpreted in light of the poor HDL specificity of the drugs that were investigated in these trials. Nevertheless, the results of Mendelian randomization studies further raise the possibility that the epidemiological relationship between HDL cholesterol and coronary artery disease might reflect residual confounding. HDL are circulating multimolecular platforms that exert divergent functions: reverse cholesterol transport, antiinflammatory effects, anti-oxidative effects, immunomodulatory effects, improved endothelial function, increased endothelial progenitor cell number and function, antithrombotic effects, and potentiation of insulin secretion and improvement of insulin sensitivity. Pleiotropic effects of HDL might be translated in clinically significant effects in strategically selected therapeutic areas that are not directly related to native coronary artery disease. In this review, four new therapeutic areas for HDL-targeted diseases are presented: critical illness, allograft vasculopathy and vein graft atherosclerosis, type 2 diabetes mellitus, and heart failure. The strategic selection of these therapeutic areas is not only based on specific functional properties of HDL but also on significant pre-clinical and clinical data that support this choice.

Adventitial stem cells in vein grafts display multilineage potential that contributes to neointimal formation.

This study was designed to carry out the characterization of stem cells within the adventitia and to elucidate their functional role in the pathogenesis of vein graft atherosclerosis. A mouse vein graft model was used to investigate the functional role of adventitial stem/progenitor cells on atherosclerosis. The adventitia of vein grafts underwent significant remodeling during early stages of vessel grafting and displayed markedly heterogeneous cell compositions. Immunofluorescence staining indicated a significant number of stem cell antigen-1-positive cells that were closely located to vasa vasorum. In vitro clonogenic assays demonstrated 1% to 11% of growing rates from adventitial cell cultures, most of which could be differentiated into smooth muscle cells (SMCs). These stem cell antigen-1-positive cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro. In light of the proatherogenic roles of SMCs in atherosclerosis, we focused on the functional roles of progenitor-SMC differentiation, in which we subsequently demonstrated that it was driven by direct interaction of the integrin/collagen IV axis. The ex vivo bioreactor system revealed the migratory capacity of stem cell antigen-1-positive progenitor cells into the vessel wall in response to stromal cell-derived factor-1. Stem cell antigen-1-positive cells that were applied to the outer layer of vein grafts showed enhanced atherosclerosis in apolipoprotein E-deficient mice, which contributed to ≈ 30% of neointimal SMCs. We demonstrate that during pathological conditions in vein grafting, the adventitia harbors stem/progenitor cells that can actively participate in the pathogenesis of vascular disease via differentiation into SMCs.