Osteopontin Activation and Microcalcification in Venous Grafts Can Be Modulated by Dexamethasone.

BACKGROUND: Osteopontin has been implicated in vascular calcification formation and vein graft intimal hyperplasia, and its expression can be triggered by pro-inflammatory activation of cells. The role of osteopontin and the temporal formation of microcalcification in vein grafts is poorly understood with a lack of understanding of the interaction between haemodynamic changes and the activation of osteopontin. METHODS: We used a porcine model of vein interposition grafts, and human long saphenous veins exposed to ex vivo perfusion, to study the activation of osteopontin using polymerase chain reaction, immunostaining, and 18F-sodium fluoride autoradiography. RESULTS: The porcine model showed that osteopontin is active in grafts within 1 week following surgery and demonstrated the presence of microcalcification. A brief pretreatment of long saphenous veins with dexamethasone can suppress osteopontin activation. Prolonged culture of veins after exposure to acute arterial haemodynamics resulted in the formation of microcalcification but this was suppressed by pretreatment with dexamethasone. 18F-sodium fluoride uptake was significantly increased as early as 1 week in both models, and the pretreatment of long saphenous veins with dexamethasone was able to abolish its uptake. CONCLUSIONS: Osteopontin is activated in vein grafts and is associated with microcalcification formation. A brief pretreatment of veins ex vivo with dexamethasone can suppress its activation and associated microcalcification.

The Role of Preservation Solutions upon Saphenous Vein Endothelial Integrity and Function: Systematic Review and UK Practice Survey.

The long saphenous vein is the most used conduit in cardiac surgery, but its long-term patency is limited by vein graft disease (VGD). Endothelial dysfunction is a key driver of VGD; its aetiology is multi-factorial. However emerging evidence identifies vein conduit harvest technique and preservation fluids as causal in their onset and propagation. This study aims to comprehensively review published data on the relationship between preservation solutions, endothelial cell integrity and function, and VGD in human saphenous veins harvested for CABG. The review was registered with PROSPERO (CRD42022358828). Electronic searches of Cochrane Central Register of Controlled Trials, MEDLINE, and EMBASE databases were undertaken from inception until August 2022. Papers were evaluated in line with registered inclusion and exclusion criteria. Searches identified 13 prospective, controlled studies for inclusion in the analysis. All studies used saline as a control solution. Intervention solutions included heparinised whole blood and saline, DuraGraft, TiProtec, EuroCollins, University of Wisconsin (UoW), buffered, cardioplegic and Pyruvate solutions. Most studies demonstrated that normal saline appears to have negative effects on venous endothelium and the most effective preservation solutions identified in this review were TiProtec and DuraGraft. The most used preservation solutions in the UK are heparinised saline or autologous whole blood. There is substantial heterogeneity both in practice and reporting of trials evaluating vein graft preservation solutions, and the quality of existing evidence is low. There is an unmet need for high quality trials evaluating the potential for these interventions to improve long-term patency in venous bypass grafts.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

BACKGROUND: Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. METHODS: PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. RESULTS: PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. CONCLUSIONS: We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Large animal model of vein grafts intimal hyperplasia: A systematic review.

Coronary artery bypass grafting remains the treatment of choice for a large cohort of patients with significant coronary disease. Despite the increased use of arterial grafts, the long saphenous vein remains the most commonly used conduit. Long-term graft patency continues to be the Achilles heel of saphenous vein grafts. This is due to the development of intimal hyperplasia, a chronic inflammatory disease that results in the narrowing and occlusion of a significant number of vein grafts. Research models for intimal hyperplasia are essential for a better understanding of pathophysiological processes of this condition. Large animal models resemble human anatomical structures and have been used as a surrogate to study disease development and prevention over the years. In this paper, we systematically review all published studies that utilized large animal models of vein graft disease with a focus on the type of model and any therapeutic intervention, specifically the use of external stents/mesh.

Hybrid coronary revascularization versus coronary artery bypass grafting for multivessel coronary artery disease: A systematic review and meta-analysis.

BACKGROUND: Hybrid coronary revascularization (HCR) combines the benefits of a left internal mammary artery to left anterior descending artery anastomosis, via a mini thoracotomy, with percutaneous coronary intervention (PCI) for other diseased coronaries. AIMS: The aim of this meta-analysis is to compare the short- and long-term outcomes of HCR with those of coronary artery bypass grafting (CABG) for multi-vessel coronary artery disease (MCAD). METHODS: We performed a meta-analysis with a primary outcome of short-term mortality and secondary outcomes of mid-term survival, length of hospital stay, stroke, renal failure and mid-term MACE rate. RESULTS: 3399 patients (HCR = 1164, CABG = 2235) were included, with no significant difference in short-term mortality between groups (OR = 1.50, 95% CI = [0.90,2.49], p = 0.11), although a higher mortality rate was seen in the HCR group (0.73% vs 0.64%). The average length of stay in intensive care unit was significantly shorter following HCR than CABG (mean difference = -15.52 h, CI = [-22.47,-8.59], p˂0.001) and overall hospital stay was also shorter in this group, although not statistically significant (mean difference = -3.15 days, 95% CI = [-6.55, 0.25], p = 0.07). HCR was associated with a reduced odds of blood transfusion (OR = 0.34, 95% CI = [0.22,0.54], p < 0.001). There was not a significant difference in mid-term survival (OR = 0.86, 95% CI = [0.62,1.21], p = 0.39) or MACE rate (OR = 0.82, 95% CI = [0.55,1.23], p = 0.34). No differences were found between HCR and CABG for post-operative stroke (OR = 1.36, 95% CI = [0.87, 2.13], p = 0.16) or renal failure (OR = 0.71, 95% CI = [0.43,1.16], p = 0.14). CONCLUSIONS: HCR has a higher incidence of short-term mortality compared to CABG in patients with MCAD, although this difference is not statistically significant. Similar rates of mid-term survival and other short term post-operative complications were found between the two groups. HCR has a shorter ICU stays and reduced requirement for blood transfusion.

Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro.

Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.

Investigation of Atherosclerotic Plaque Vulnerability.

Histochemical and immunohistochemical approaches permit the detection and evaluation of proteins and cell types within murine brachiocephalic artery atherosclerotic plaques, that can be subsequently analyzed to provide inferences on atherosclerotic plaque vulnerability. Here we describe the specific histochemical techniques deployed to examine the expression of elastin, fibrillar collagens, and neutral lipids, alongside immunohistochemistry protocols for the identification of macrophages (CD68) and vascular smooth muscle cells (α-smooth muscle actin). We will also describe how analyses derived from these methods can be combined to determine evidence of previous plaque rupture and susceptibility to rupture.

Aneurysm severity is suppressed by deletion of CCN4.

Patients with abdominal aortic aneurysms are frequently treated with high-risk surgery. A pharmaceutical treatment to reverse aneurysm progression could prevent the need for surgery and save both lives and healthcare resources. Since CCN4 regulates cell migration, proliferation and apoptosis, processes involved in aneurysm progression, it is a potential regulator of aneurysm progression. We investigated the role of CCN4 in a mouse aneurysm model, using apolipoprotein-E knockout (ApoE-/-) mice fed high fat diet and infused with Angiotensin II (AngII). Blood pressure was similarly elevated in CCN4-/-ApoE-/- mice and CCN4+/+ApoE-/- mice (controls) in response to AngII infusion. Deletion of CCN4 significantly reduced the number of ruptured aortae, both thoracic and abdominal aortic area, and aneurysm grade score, compared to controls. Additionally, the frequency of vessel wall remodelling and the number of elastic lamina breaks was significantly suppressed in CCN4-/-ApoE-/- mice compared to controls. Immunohistochemistry revealed a significantly lower proportion of macrophages, while the proportion of smooth muscle cells was not affected by the deletion of CCN4. There was also a reduction in both proliferation and apoptosis in CCN4-/-ApoE-/- mice compared to controls. In vitro studies showed that CCN4 significantly increased monocyte adhesion beyond that seen with TNFα and stimulated macrophage migration by more than threefold. In summary, absence of CCN4 reduced aneurysm severity and improved aortic integrity, which may be the result of reduced macrophage infiltration and cell apoptosis. Inhibition of CCN4 could offer a potential therapeutic approach for the treatment of aneurysms.

Nonautologous Grafts in Coronary Artery Bypass Surgery: A Systematic Review.

BACKGROUND: Suitable autologous conduits may be lacking when performing coronary artery bypass grafting. The aim of this review is to determine the status of nonautologous grafts in coronary artery bypass grafting. METHODS: We conducted a literature search on MEDLINE All, Embase Classic, and Embase through Ovid from 1960 to April 2020. RESULTS: Of the 1579 records identified, 21 studies were included in the review. The following grafts were assessed for patency: 109 homologous saphenous veins (patency rates ranged from 66.7% at a median follow-up of 8.5 months to 0% at 6-12 months and 7-18 months, respectively), 29 expanded polytetrafluoroethylene grafts (from 80% at a median follow-up of 5 months to 14.3% at 45 months), 12 human umbilical veins (50% at a median follow-up of 6 months), 50 Bioflow bovine internal mammary arteries (from 15.8% to 0% at a mean follow-up of 9.5 months and 19 months, respectively), 39 Perma-Flow grafts (80% and 76.9% at 1-3 months and 12 months, respectively), 20 No-React bovine internal mammary arteries (57.1% at a median follow-up of 28 months and 23.1% at a mean follow-up of 7 months), 40 autologous venous endothelial cell-seeded expanded polytetrafluoroethylene grafts (94.7% and 81% at a mean follow-up of 27 months and 60 months, respectively), and 12 autologous venous endothelial cell-seeded cryopreserved homologous veins (83.3% at a mean follow-up of 8.5 months). CONCLUSIONS: The goal of an alternative conduit with patency and attributes that match those of autografts remains elusive. Autologous endothelial cell-seeded synthetic grafts have demonstrated promising results but require further investigation.

NF-κB inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium.

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.

Saphenous vein graft disease, pathophysiology, prevention, and treatment. A review of the literature.

BACKGROUND: The saphenous vein remains the most frequently used conduit for coronary artery bypass grafting, despite reported unsatisfactory long-term patency rates. Understanding the pathophysiology of vein graft failure and attempting to improve its longevity has been a significant area of research for more than three decades. This article aims to review the current understanding of the pathophysiology and potential new intervention strategies. METHODS: A search of three databases: MEDLINE, Web of Science, and Cochrane Library, was undertaken for the terms "pathophysiology," "prevention," and "treatment" plus the term "vein graft failure." RESULTS: Saphenous graft failure is commonly the consequence of four different pathophysiological mechanisms, early acute thrombosis, vascular inflammation, intimal hyperplasia, and late accelerated atherosclerosis. Different methods have been proposed to inhibit or attenuate these pathological processes including modified surgical technique, topical pretreatment, external graft support, and postoperative pharmacological interventions. Once graft failure occurs, the available treatments are either surgical reintervention, angioplasty, or conservative medical management reserved for patients not eligible for either procedure. CONCLUSION: Despite the extensive amount of research performed, the pathophysiology of saphenous vein graft is still not completely understood. Surgical and pharmacological interventions have improved early patency and different strategies for prevention seem to offer some hope in improving long-term patency.

Galectin-3 Identifies a Subset of Macrophages With a Potential Beneficial Role in Atherosclerosis.

OBJECTIVE: Galectin-3 (formerly known as Mac-2), encoded by the LGALS3 gene, is proposed to regulate macrophage adhesion, chemotaxis, and apoptosis. We investigated the role of galectin-3 in determining the inflammatory profile of macrophages and composition of atherosclerotic plaques. Approach and Results: We observed increased accumulation of galectin-3-negative macrophages within advanced human, rabbit, and mouse plaques compared with early lesions. Interestingly, statin treatment reduced galectin-3-negative macrophage accrual in advanced plaques within hypercholesterolemic (apolipoprotein E deficient) Apoe-/- mice. Accordingly, compared with Lgals3+/+:Apoe-/- mice, Lgals3-/-:Apoe-/- mice displayed altered plaque composition through increased macrophage:smooth muscle cell ratio, reduced collagen content, and increased necrotic core area, characteristics of advanced plaques in humans. Additionally, macrophages from Lgals3-/- mice exhibited increased invasive capacity in vitro and in vivo. Furthermore, loss of galectin-3 in vitro and in vivo was associated with increased expression of proinflammatory genes including MMP (matrix metalloproteinase)-12, CCL2 (chemokine [C-C motif] ligand 2), PTGS2 (prostaglandin-endoperoxide synthase 2), and IL (interleukin)-6, alongside reduced TGF (transforming growth factor)-ß1 expression and consequent SMAD signaling. Moreover, we found that MMP12 cleaves macrophage cell-surface galectin-3 resulting in the appearance of a 22-kDa fragment, whereas plasma levels of galectin-3 were reduced in Mmp12-/-:Apoe-/- mice, highlighting a novel mechanism where MMP12-dependent cleavage of galectin-3 promotes proinflammatory macrophage polarization. Moreover, galectin-3-positive macrophages were more abundant within plaques of Mmp12-/-:Apoe-/- mice compared with Mmp12+/+:Apoe-/- animals. CONCLUSIONS: This study reveals a prominent protective role for galectin-3 in regulating macrophage polarization and invasive capacity and, therefore, delaying plaque progression.

Changes in contractile protein expression are linked to ventricular stiffness in infants with pulmonary hypertension or right ventricular hypertrophy due to congenital heart disease.

Background: The right ventricle (RV) is not designed to sustain high pressure leading to failure. There are no current medications to help RV contraction, so further information is required on adaption of the RV to such hypertension. Methods: The Right Ventricle in Children (RVENCH) study assessed infants with congenital heart disease undergoing cardiac surgery with hypertensive RV. Clinical and echocardiographic data were recorded, and samples of RV were taken from matched infants, analysed for proteomics and compared between pathologies and with clinical and echocardiographic outcome data. Results: Those with tetralogy of Fallot (TOF) were significantly more cyanosed than those with ventricular septal defect (median oxygen saturation 83% vs 98%, P=0.0038), had significantly stiffer RV (tricuspid E wave/A wave ratio 1.95 vs 0.84, P=0.009) and had most had restrictive physiology. Gene ontology in TOF, with enrichment analysis, demonstrated significant increase in proteins of contractile mechanisms and those of calmodulin, actin binding and others associated with contractility than inventricular septal defect. Structural proteins were also found to be higher in association with sarcomeric function: Z-disc, M-Band and thin-filament proteins. Remaining proteins associated with actin binding, calcium signalling and myocyte cytoskeletal development. Phosphopeptide enrichment led to higher levels of calcium signalling proteins in TOF. Conclusion: This is the first demonstration that those with an RV, which is stiff and hypertensive in TOF, have a range of altered proteins, often in calcium signalling pathways. Information about these alterations might guide treatment options both in terms of individualised therapy or inotropic support for the Right ventricle when hypertensive due to pulmoanry hypertension or congenital heart disease.

Activation and inflammation of the venous endothelium in vein graft disease.

The long saphenous vein is the most commonly used conduit in coronary artery bypass graft (CABG) surgery when bypassing multiple diseased arteries; however, its use is complicated by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis leading to compromised graft efficacy. Despite refinement of surgical techniques to improve graft patency, late vein graft failure remains a significant problem. Moreover, there is a lack of pharmacological interventions proven to be effective in the treatment of late vein graft failure. A greater understanding of the molecular nature of the disease and the interactions between endothelial and smooth muscle cells as a result of alterations in local haemodynamics may assist with designing future beneficial pharmacological interventions. Venous endothelial cells (ECs) are physiologically adapted to chronic low shear stress; however, once the graft is implanted into the arterial circulation, they become suddenly exposed to acute high levels of shear stress. A small number of in vitro and ex vivo studies have demonstrated that acute high shear stress is associated with the activation of a pro-inflammatory profile in saphenous vein ECs, which may be mediated by mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signalling pathways. The impact of acute changes in shear stress on venous ECs and the role of ECs in the development of intimal hyperplasia remains incomplete and is the subject of this review.

Targeting Wnt/ß-Catenin Activated Cells with Dominant-Negative N-cadherin to Reduce Neointima Formation.

Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/ß-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/ß-catenin/TCF pathway. Infection with Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis (3 ± 0.2% versus 9 ± 0.7%; p < 0.05, n = 6) and significantly inhibited VSMC migration by 83 ± 15% (p < 0.05, n = 6), but did not affect VSMC proliferation (p > 0.05, n = 5). In an ex vivo human saphenous vein organ culture model, luminal delivery of Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis after 7 days of culture (4 ± 1.4% versus 9 ± 1.6%; p < 0.01, n = 6) and suppressed intimal thickening by 75 ± 7% (p < 0.05, n = 5), without a detrimental effect on endothelial cell coverage. In vivo, Ad-TOP-dn-N-cadherin significantly reduced intimal thickening at day 21 (n = 10) in comparison to the Ad-ß-galactosidase (Ad-ß-gal) control virus (n = 12, p < 0.05) in the mouse carotid artery ligation model. In summary, we have developed a novel approach to selectively reduce intimal thickening, which may be beneficial in reducing late vein graft failure.

Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration.

OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.

Differential effects of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 on atherosclerosis and monocyte/macrophage invasion.

AIMS: MMPs contribute to atherosclerotic plaque progression and instability, but the relative potency of their endogenous tissue inhibitors of metalloproteinases (TIMPs) as protective factors has not been defined. We therefore investigated the impact of TIMP-1 and TIMP-2 knockout on atherosclerotic plaque burden and composition in apolipoprotein E-knockout (Apoe(-/-)) mice and studied the underlying effects on monocyte/macrophage behaviour. METHODS AND RESULTS: Analysis of brachiocephalic artery plaques revealed comparable atherosclerotic lesion areas between TIMP-1(-/-) Apoe(-/-) or TIMP-2(-/-) Apoe(-/-) double deficient mice and relevant age-matched, strain-matched Apoe(-/-) controls after 8 weeks of high-fat feeding. However, lesions from TIMP-2(-/-) Apoe(-/-) mice had higher levels of markers associated with plaque vulnerability, including increased macrophage: vascular smooth muscle cell ratios, larger necrotic core areas, reduced collagen contents, increased macrophage proliferation, and apoptosis frequencies, compared with TIMP-1(-/-)Apoe(-/-) and controls. In contrast, TIMP-1(-/-) Apoe(-/-) animals only had a significant reduction in vascular smooth muscle cell content compared with Apoe(-/-) controls. In vitro and in vivo findings implicated heightened monocyte/macrophage invasion in the detrimental effects observed on atherosclerotic plaque composition in TIMP-2(-/-) Apoe(-/-) mice. Moreover, TIMP-2 specifically decreased MMP-14-dependent monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions. CONCLUSION: Our data demonstrate that TIMP-2 plays a greater protective role than TIMP-1 during the pathogenesis of atherosclerosis, in part by suppressing MMP-14-dependent monocyte/macrophage accumulation into plaques.

Sustained reduction of vein graft neointima formation by ex vivo TIMP-3 gene therapy.

BACKGROUND: Coronary artery vein graft failure, resulting from thrombosis, intimal thickening, and atherosclerosis, is a significant clinical problem, with approximately 50% of vein grafts failing within 10 years. Intimal thickening is caused by migration of vascular smooth muscle cells from the media to the intima, where they proliferate. Interventions using gene transfer to inhibit vascular smooth muscle cells proliferation and migration are attractive because ex vivo access to the graft is possible. The involvement of matrix-degrading metalloproteinases in intimal thickening is well established, and we previously showed that adenoviral-delivered overexpression of an endogenous inhibitor, the tissue inhibitor of metalloproteinases-3 (TIMP-3), significantly retarded intimal thickening in short-term autologous porcine arteriovenous interposition grafts (28 days). However, it is essential to determine whether this approach will provide longer-term benefits. METHODS AND RESULTS: We assessed whether a recombinant adenovirus that overexpresses TIMP-3 (RAdTIMP-3) affects vein graft intimal thickening in the longer term (at 3 months). Porcine saphenous veins were subjected to luminal infection with 2.5×10(10) pfu/mL RAdTIMP-3 or RAd60 (control virus) or vehicle control, for 30 minutes before implantation into the carotid artery. Analysis of grafts harvested 3 months after delivery revealed that RAdTIMP-3-infected grafts had significantly reduced intimal areas compared with both controls (3.2 ± 0.4 mm(2) versus 5.6 ± 0.7 mm(2) and 5.9 ± 0.5 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). Medial areas were also significantly decreased by TIMP-3 (3.8 ± 0.3 mm(2) versus 6.7 ± 1.0 mm(2) and 5.2 ± 0.4 mm(2), RAdTIMP-3, RAd60, and vehicle, respectively). CONCLUSIONS: Overexpression of TIMP-3 provides a sustained retardation of vein graft intimal thickening and highlights the translational potential for ex vivo TIMP-3 gene therapy.

Prevention of post-cardiopulmonary bypass acute kidney injury by endothelin A receptor blockade.

OBJECTIVE: The aim of this study was to determine whether administration of a specific endothelin A receptor antagonist, sitaxsentan sodium, would prevent the development of post-cardiopulmonary bypass acute kidney injury in swine. DESIGN: Experimental study. SETTING: Cardiovascular Research Institute. INTERVENTIONS: Adult pigs (n = 8 per group) were randomized to undergo a sham procedure, cardiopulmonary bypass, or cardiopulmonary bypass plus administration of endothelin A receptor antagonist (RA), with recovery and reassessment at 24 hrs. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary bypass resulted in a significant reduction in creatinine clearance relative to sham pigs (mean difference for cardiopulmonary bypass vs. sham, -50.3 mL/min [95% confidence interval -89.2 to -11.4 mL/min], p = .008). This was reversed by the administration of endothelin A RA during cardiopulmonary bypass (mean difference for cardiopulmonary bypass + endothelin A RA vs. cardiopulmonary bypass, +43.3 mL/min [95% confidence interval +3.3 to +83.4 mL/min], p = .030). Cardiopulmonary bypass also resulted in a significant rise in the specific urinary biomarker of acute kidney injury interleukin-18 compared to sham procedures (mean difference +209 pg/mL [95% confidence interval +119 to +299 pg/mL], p < .001) that was reversed by endothelin A receptor antagonist administration. Post-cardiopulmonary bypass kidney injury was associated with vascular endothelial injury and dysfunction, reduced nitric oxide bioavailability, inflammation, and a significant increase in the expression of the paracrine vasoconstrictors adenosine and endothelin-1. In post-cardiopulmonary bypass kidneys at 24 hrs there was persistent hypoxia at the level of the outer medulla, cortical adenosine triphosphate depletion, and evidence of proximal tubule epithelial cell stress manifest as phenotypic change. There was no evidence of acute tubular necrosis. Administration of endothelin A RA to cardiopulmonary bypass pigs reversed endothelial dysfunction, regional hypoxia, inflammation, and tubular changes. CONCLUSION: In this model, post-cardiopulmonary bypass acute kidney injury is associated with endothelial dysfunction, regional tissue hypoxia, and proximal tubular epithelial cell stress but not acute tubular necrosis. Antagonism of the endothelin-1 A receptor reversed these changes and may represent a therapeutic target for the prevention of post-cardiac surgery acute kidney injury.