Murine Vein Graft Research Articles

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.

Exploring PD-1+ and tissue resident memory T cells in atherosclerotic diseases

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Health Holland Visual Sonics Background Checkpoint inhibitor therapy (ICI), a potent oncologic treatment, has been associated with an enhanced risk for atherosclerotic cardiovascular events and increased plaque progression. Among the key players affected by ICI are tissue resident memory T cells (Trm), a subset residing in tissues and characterized by CD44, CD69, PD-1, and CD103. Although Trms have been associated with chronic inflammatory conditions, their role in atherosclerosis and accelerated atherosclerosis, as observed in postinterventional diseases such as vein graft disease, remains elusive Here, we present an investigation into the role of PD-1 positive cells and Trms in accelerated atherosclerosis as found in vein graft disease. Methods Human carotid atherosclerotic plaques were stained with a MOVAT stain and scored to determine plaque stability. 25 Stable and 25 unstable plaques were stained for CD3+ T cells and PD-1. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery and were sacrificed at t7, t14, t28 (n=6 per time point). The vein grafts were processed for flow cytometry to quantify the Trms population, using markers including CD4+, CD8+, CD44+, CD25-, CD69+, CD103+ and PD-1+. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery. Biweekly, mice received ICI (anti-PD-L1) or control antibody (n=7). After 28 days vein graft morphology was assed using MOVAT staining and vein graft inflammation by staining MAC3 and CXCL10. Results Within human atherosclerotic plaques, PD-1-expressing T cells were predominantly located in the shoulder regions of the necrotic core. Unstable lesions exhibited significantly higher levels of PD-1 expressing CD3+ T cells compared to stable lesions (p=0.001). Furthermore, the percentage of PD-1 expression positively correlated with immune cell infiltration scores (p=0.019). In murine vein grafts, Trms were identified through flow cytometry. A trend was observed indicating an increase in the number of CD4 and CD8 Trms at t14 compared to t7 Trms (CD8+, CD44+, CD69+/CD103+). At t28, the number of CD4 and CD8 Trms decreased compared to t14, with PD1+ expressing Trms showing a similar trend, peaking at t14. Vein grafts in mice receiving anti-PD-L1 mAb showed no morphological differences compared to the control group. However, a significant difference in inflammation was noted in the anti-PD-L1 treated group, characterized by a higher number of macrophages (p=0.02) and increased expression of CXCL10 (p=0.008). Conclusion Here we demonstrate the association of PD-1+ T cells with unstable lesions and reveals that Trms increase during the inflammatory phase and decrease during the stabilizing phase of accelerated atherosclerosis. Trms emerge as potential detrimental targets of ICI therapy, which is characterized by increased inflammation.

Phosphorylcholine Monoclonal Antibody Therapy Decreases Intraplaque Angiogenesis and Intraplaque Hemorrhage in Murine Vein Grafts.

Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease.

Visualization of Murine Vascular Remodeling and Blood Flow Dynamics by Ultra-High-Frequency Ultrasound Imaging.

Vein grafts (VGs) are used to bypass atherosclerotic obstructions and arteriovenous fistulas (AVFs) as vascular access for hemodialysis. Vascular remodeling governs post-interventional arterialization, but may also induce VG and AVF failure. Although the endpoint characteristics of vascular remodeling are known, the in vivo process and the role of blood flow dynamics has not been fully studied. Therefore, here we non-invasively quantify vascular remodeling and blood flow alterations over time in murine VG and AVF models. C57BL/6J (n = 7, chow diet) and atherosclerosis-prone ApoE3*Leiden (n = 7) mice underwent VG surgery. Ultrasound imaging was performed at 3, 7, 14, 21, and 28 days post-surgery. C57BL/6J mice (n = 8) received AVF surgery. Ultrasound imaging was performed at 7 and 14 days post-surgery. The luminal volume increased by 42% in the VGs of C57BL/6J and 38% in the VGs of ApoE3*Leiden mice at 28 days relative to 3 days post-surgery. Longitudinally, an 82% increase in wall volume and 76% increase in outward remodeling was found in the ApoE3*Leiden mice, with a constant wall size in C57BL/6J mice. Proximally, the pulsatility index, resistive index, and peak systolic velocity decreased longitudinally in both groups. Distally, the maximum acceleration increased with 56% in C57BL/6J VGs. Among the AVFs, 50% showed maturation after 7 days, based on a novel flow-criterium of 23 mL/min. Distinct flow patterns were observed at the anastomotic site and inflow artery of the AVFs relative to the control carotid arteries. Vascular remodeling can be quantified by ultra-high-frequency ultrasound imaging over time in complex animal models, via three-dimensional structural parameters and site-specific hemodynamic indices.

Systemic Delivery of Clopidogrel Inhibits Neointimal Formation in a Mouse Vein Graft Model.

Clopidogrel inhibits platelet aggregation and has beneficial effects on patients undergoing coronary artery bypass grafting surgery, but it is unknown whether clopidogrel inhibits the neointima formation of grafted veins. In this study, we used a murine vein graft model to study the effect of clopidogrel on intima hyperplasia of the vein graft. Vein grafting was performed among C57BL/6J mice, immediately after surgery; 1 mg/kg clopidogrel and vehicle control were used to inject mice peritoneally daily for 2 weeks. As compared with the vehicle, clopidogrel significantly inhibited the neointima formation of vein grafts at 4 weeks after surgeries. The immunohistochemistry study showed that as compared with the vehicle, clopidogrel significantly decreased the rate of proliferating cell nuclear antigen-positive cells in the wall of vein grafts and significantly increased the expression of vascular smooth muscle cell (VSMC) contractile protein markers (α-smooth muscle actin, calponin, and SM22) within the neointima area of vein grafts. Clopidogrel significantly decreased the plasma interleukin 6 (IL-6) level at 1 week after surgery as compared with the vehicle. We isolated VSMCs from mouse aortic arteries. As compared with the vehicle, clopidogrel significantly inhibited thrombin-induced VSMC proliferation and migration, significantly decreased IL-6 mRNA expression and protein secretion, and increased intracellular cyclic adenosine monophosphate generation in a dose-dependent manner. In conclusion, systemic delivery of clopidogrel inhibits neointima formation of the mouse vein graft, the mechanisms of which are associated with its inhibitory effects on VSMC proliferation, migration, and the tendency to synthetic phenotype after vein graft surgery, reducing the expression of IL-6 and increasing the intracellular cyclic adenosine monophosphate level.

Co-stimulatory treatment enhances the protective role of CD8 and CD4 t cells in murine vein grafts

Background and Aims : Background Vein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency that rapidly declines due to extensive intimal hyperplasia (IH). T-cells demonstrate both pro-and anti-atherogenic effects. Therefore, we investigated T-cell involvement in a mouse-model for VGD using pharmacological targeting of the co-stimulatory factor CD137.

Interfering in the ALK1 Pathway Results in Macrophage-Driven Outward Remodeling of Murine Vein Grafts.

AimsVein grafts are frequently used to bypass coronary artery occlusions. Unfortunately, vein graft disease (VGD) causes impaired patency rates. ALK1 mediates signaling by TGF-β via TGFβR2 or BMP9/10 via BMPR2, which is an important pathway in fibrotic, inflammatory, and angiogenic processes in vascular diseases. The role of the TGF-β pathway in VGD is previously reported, however, the contribution of ALK1 signaling is not known. Therefore, we investigated ALK1 signaling in VGD in a mouse model for vein graft disease using either genetic or pharmacological inhibition of the Alk1 signaling.Methods and ResultsMale ALK1 heterozygous (ALK1+/−), control C57BL/6, as well as hypercholesterolemic ApoE3*Leiden mice, underwent vein graft surgery. Histologic analyses of ALK1+/− vein grafts demonstrated increased outward remodeling and macrophage accumulation after 28 days. In hypercholesterolemic ApoE3*Leiden mice receiving weekly ALK1-Fc injections, ultrasound imaging showed 3-fold increased outward remodeling compared to controls treated with control-Fc, which was confirmed histologically. Moreover, ALK1-Fc treatment reduced collagen and smooth muscle cell accumulation, increased macrophages by 1.5-fold, and resulted in more plaque dissections. No difference was observed in intraplaque neovessel density. Flow cytometric analysis showed increased systemic levels of Ly6CHigh monocytes in ALK1-Fc treated mice, supported by in vitro increased MCP-1 and IL-6 production of LPS-stimulated and ALK1-Fc-treated murine monocytes and macrophages.ConclusionReduced ALK1 signaling in VGD promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype.Translational PerspectiveVein graft disease (VGD) severely hampers patency rates of vein grafts, necessitating research of key disease-driving pathways like TGF-β. The three-dimensional nature of VGD together with the multitude of disease driving factors ask for a comprehensive approach. Here, we combined in vivo ultrasound imaging, histological analyses, and conventional in vitro analyses, identifying the ambiguous role of reduced ALK1 signaling in vein graft disease. Reduced ALK1 signaling promotes outward remodeling, increases macrophage influx, and promotes an unstable plaque phenotype in murine vein grafts. Characterization of in vivo vascular remodeling over time is imperative to monitor VGD development and identify new therapies.

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

Early Inhibition of P-Selectin/P-Selectin Glycoprotein Ligand-1 Reduces Intimal Hyperplasia in Murine Vein Grafts through Platelet Adhesion.

Inflammatory processes contribute to intimal hyperplasia (IH) and long-term failure of vein grafts used in bypass surgery. Leukocyte recruitment on endothelial cells of vessels during inflammation is regulated by P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), which also mediates the interaction between platelets and endothelial cells in vein grafts transferred to arteries. However, how this pathway causes IH in vein grafts is unclear. In this study, we used a murine model of vein grafting to investigate P-selectin-mediated platelet adhesion, followed by IH. On the luminal surface of the vein graft, leukocyte recruitment occurred mainly in areas with adhered platelets rather than on endothelial cells without adherent platelets 1 hour after vein grafting. Blockage of either P-selectin or PSGL-1 reduced platelet adhesion and leukocyte recruitment on the luminal surface of vein grafts. Inhibition of the P-selectin pathway in vein grafts significantly reduced platelet-mediated leukocyte recruitment and IH of vein grafts 28 days after surgery. The study demonstrates that functional blockage of the P-selectin/PSGL-1 pathway in the early inflammatory phase after vein grafting reduced leukocyte invasion in the vein graft wall and later IH development. The findings imply an attractive early time window for prevention of vein graft failure by manipulating platelet adhesion.

PC236. Cav-1 Regulates EphB4-Mediated Arteriovenous Fistula Maturation

Arteriovenous fistula (AVF) continues to be the most common access created for hemodialysis. However, many AVFs fail to mature, suggesting a need to improve AVF maturation. Caveolin1 (Cav-1) is the major scaffolding protein of caveolae, a distinct microdomain that serves as a flow-activated mechanosensor at the membrane of endothelial cells; Eph-B4 is the embryonic venous determinant. We have previously shown that Cav-1 is a mechanism of Eph-B4-mediated vessel remodeling in the murine vein graft model. We have also shown that Eph-B4 expression increases during AVF maturation and Eph-B4 activity inhibits venous remodeling in the murine AVF model. Then we hypothesized that Cav-1 is a critical regulator of Eph-B4-mediated AVF maturation through inhibition of eNOS activity. We used a mouse aortocaval fistula model. The venous AVF limb and control (sham) inferior vena cava (IVC) of wild-type C57BL/6 (WT) were compared for Cav-1 expression. AVFs of WT mice, Cav-1 knockout (KO) mice, Cav-1 endothelial reconstituted (RC) mice, and Eph-B4 heterozygous (Eph-B4 het) mice were analyzed. A Cav-1 scaffolding domain peptide (cavtratin) was administrated to stimulate Cav-1 signaling; ephrinB2/Fc was used to stimulate Eph-B4 signaling. Vessel remodeling was assessed postoperatively by serial ultrasound measurements of the IVC/AVF diameter. AVFs were harvested at day 21 and examined with histology; IVC wall thickness was measured by computerized morphometry. Both Cav-1 mRNA and protein were increased in the fistula veins compared with control veins. Cav-1 KO mice showed enhanced venous remodeling with increased eNOS activity compared with WT mice. Inhibition of remodeling by ephrin-B2/Fc in WT mice was abolished in Cav-1 KO mice, but maintained in Cav-1 RC mice (Fig A). Administration of cavtratin decreased the fistula wall thickness in WT mice as well as in Eph-B4 het mice (Fig B). Endothelial Cav-1 is a critical regulator of Eph-B4-mediated AVF maturation. Manipulation of Cav-1 function may improve arteriovenous maturation within the fistula environment.

Abstract 303: Increased Endogenous Hydrogen Sulfide Protects Against Vein Graft Disease

Objective: Vein graft failure secondary to intimal hyperplasia remains a challenge. Hydrogen sulfide (H 2 S) is produced endogenously by cystathionine γ-lyase (CGL) in endothelial cells, and as a gasotransmitter holds numerous beneficial vascular effects. We thus hypothesized that increased endogenous H 2 S would attenuate the vascular response to injury. Here, we leveraged a CGL transgenic overexpressing mouse model to test the potential of increased endogenous H 2 S to attenuate the vascular response to injury in a vein graft model. Approach and Results: A murine carotid-interposition cuff technique vein graft model was employed, including an artificial bacterial chromosome-based CGL transgenic (Tg) strain with an extra copy of the CGL gene locus randomly inserted in the genome. CGL Tg mice were fed a high-fat/high-cholesterol diet, implanted with a vein graft from CGL Tg donors (n=8), and compared to wild-type (WT) controls (n=7; WT/ WT conduits); all on C57BL/6 background. Grafts were imaged in vivo with ultrasound biomicropscopy and harvested after 28 days. CGL Tg mice demonstrated an approximate two-fold increase in serum H 2 S production capacity (lead acetate assay) compared to controls. The CGL Tg mice exhibited a significant decrease in their intimal thickness (p=<0.05, Fig.A) and intimal/medial+adventia ratios (p=<0.05, Fig.B) 28 days after implantation. In vivo biomicroscopy was supportive: CGL tg mice had a larger mean luminal diameter relative to WT controls (p=<0.05, Fig.C). Conclusion: Elevated endogenous H 2 S production reduces the fibroproliferative response to vein graft arterialization. Manipulation of this gasotransmitter’s biology stands as a novel approach to impact the durability of vascular reconstructions.

Abstract 256: Differential Biomechanical Regulation of Vein Graft Endothelial CD39 by Pulsatile Radial Forces

Saphenous veins are frequently used bypass grafts in coronary & peripheral artery bypass surgery, but are more prone to failure than arterial grafts due to adverse remodeling. The endothelium senses radial and linear biomechanical forces exerted by blood flow. Thin-walled venous blood flow in situ is non-pulsatile until transposed as an arterial graft. The impact of cyclic stretch from pulsatile distention on the vein wall is not well elucidated. CD39 is a potent ecto-enzyme at the blood:vessel interface regulating vascular thrombo-inflammation by dissipating extracellular ATP & ADP. We recently identified that vascular CD39 expression can be induced by laminar shear stress. However, the response of CD39 to cyclic stretch during vein graft arterialization remains unknown. We hypothesized that the vasculo-protective enzyme CD39 is induced by the venous endothelium in response with pulsatile stretch. Methods: Primary human saphenous vein endothelial cells (HSVEC) were exposed to cyclic stretch in vitro , mimicking arterial & vein graft patterns. CD39 transcript & protein expression were quantified. Ecto-ATPase and -ADPase activity of CD39 on HSVEC were quantified with a malachite green assay. CD39 haploinsufficient ( Cd39 +/- ) & WT mice underwent carotid bypass surgery with a vein graft, & subsequent neointimal hyperplasia was compared. Results: High (16%, 1 Hz) and low (5%, 1 Hz) levels of cyclic stretch of HSVEC elicited distinct patterns of CD39 expression. Cd39 mRNA remained unchanged after 48h of low or high stretch (n=6,each). Low-level cyclic stretch did not alter CD39 protein expression (n=6,each). In stark contrast, 16% cyclic stretch induced increases in CD39 protein expression after 24h (60%) and 48h (130%) compared with static controls (n=6 each, P <0.05). CD39-dependent ATP & ADP hydrolysis increased by 60% & 100% in HSVEC following high-level cyclic stretch (n=4, p<0.005). Preliminary murine vein graft model studies suggest that CD39-deficiency exacerbates vein graft neointimal hyperplasia compared to WT controls. Conclusions: We have identified CD39 as a novel, stretch-responsive endothelial ecto-enzyme in human saphenous veins, which may represent a protective response to venous distention from arterial stretch patterns.

Recombinant soluble apyrase APT102 inhibits thrombosis and intimal hyperplasia in vein grafts without adversely affecting hemostasis or re‐endothelialization

Background Occlusion of vein grafts (VGs) after bypass surgery, owing to thrombosis and intimal hyperplasia (IH), is a major clinical problem. Apyrases are enzymes that scavenge extracellular ATP and ADP, and promote adenosine formation at sites of vascular injury, and hence have the potential to inhibit VG pathology. Objectives To examine the effects of recombinant soluble human apyrase, APT102, on platelets, smooth muscle cells (SMCs) and endothelial cells (ECs) in vitro, and on thrombosis and IH in murine VGs. Methods SMC and EC proliferation and migration were studied in vitro. Inferior vena cava segments from donor mice were grafted into carotid arteries of recipient mice. Results APT102 potently inhibited ADP-induced platelet aggregation and VG thrombosis, but it did not impair surgical hemostasis. APT102 did not directly inhibit SMC or EC proliferation, but significantly attenuated the effects of ATP on SMC and EC proliferation. APT102 significantly inhibited SMC migration, but did not inhibit EC migration, which may be mediated, at least in part, by inhibition of SMC, but not EC, migration by adenosine. At 4 weeks after surgery, there was significantly less IH in VGs of APT102-treated mice than in control VGs. APT102 significantly inhibited cell proliferation in VGs, but did not inhibit re-endothelialization. Conclusions Systemic administration of a recombinant human apyrase inhibits thrombosis and IH in VGs without increasing bleeding or compromising re-endothelialization. These results suggest that APT102 has the potential to become a novel, single-drug treatment strategy to prevent multiple pathologic processes that drive early adverse remodeling and occlusion of VGs.

Abstract 172: Arteriovenous Fistula Adaptation Requires Caveolin1 Signaling

Background: Arteriovenous fistula (AVF) is the preferred access for hemodialysis. However, many AVFs fail to mature and require intervention, showing a need to improve AVF maturation. Caveolin1 (Cav1) is the major scaffolding protein of caveolae, a distinct microdomain that serves as a flow activated mechanosensor at the membrane of endothelial cells. We have previously shown that stimulation of embryonic venous determinant EphB4 associates with Cav1, mediating vessel remodeling in the murine vein graft model. Since Cav1 is a mechanism of EphB4 mediated vein graft adaptation, we examined the role of Cav1 in mediating AVF maturation, a different and distinct hemodynamic and molecular conditions from vein graft. We hypothesized that Cav1 function is critical for AVF maturation. Methods: We used a mouse aortocaval fistula model; the venous AVF limb and control (sham) inferior vena cava of wild type (WT) C57BL/6 mice were analyzed and compared for Cav1 expression. Vessel remodeling was assessed in WT and Cav1 knockout (KO) mice by serial ultrasound measurements of the IVC/AVF diameter. AVF were harvested at day 21 and examined with histology; IVC wall thickness was measured using computerized morphometry. Results: Both Cav1 mRNA and protein were increased in the fistula veins compared to control veins (Figure A; mRNA: *p=0.019, ANOVA, n=8; protein: *p=0.005, t-test, n=5). Cav1 KO mice had increased fistula vein diameter compared with WT mice (Figure B; *p<0.0001, ANOVA, n=7-10). The fistula vein wall thickness was increased in Cav1 KO mice as compared with WT mice (Figure B; 15.6 vs 30.9μm, *p=0.0005, t-test, n=7, bar 50μm). Conclusions: Cav1 expression is increased during AVF maturation, and loss of Cav1 leads to increased fistula vein diameter and wall thickness, similar to the function of Cav1 during vein graft adaptation. These results suggest that Cav1 is a regulator of AVF maturation and manipulation of Cav1 function may enhance venous adaptation to the arterial environment.

Abstract 15140: VEGFR2 Blockade in Murine Vein Grafts Reduces Intraplaque Hemorrhage due to Enhanced Plaque Neovessel Maturation

Introduction: Vein graft patency rates are poor due to the development of extensive neointimal thickening, atherosclerosis and lesion instability. We have previously shown that murine vein graft lesions exhibit leaky neovessels and that intraplaque hemorrhage induces lesion growth. Immature plaque neovessels contribute to atherosclerotic plaque instability by leakage of erythrocytes and leukocytes (intraplaque haemorrhage) in the plaque. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), together with the angiopoietin (Ang)-Tie2 system, regulates the maturation of neovessels. Hypothesis: VEGFR2 blockade induces plaque neovessel maturation resulting in reduced intraplaque hemorrhage and plaque instability. Methods: Donor caval veins were engrafted in carotid arteries of recipient hypercholesterolemic ApoE3*Leiden mice (n=14/group). Mice were treated at day 14, 17, 21 and 25 with VEGFR2 blocking antibodies (DC101) or control IgG antibodies. At day 28 vein grafts were harvested for histological and RNA analysis. Results: Morphometric analysis revealed a striking 50% decrease in vein graft segments that expressed intraplaque hemorrhage in the DC101 treated group. This was accompanied by a significant 25-fold decrease in extravasated erythrocytes. DC101 treatment resulted in a decrease in VEGF expression whereas VEGFR1 and VEGFR2 expression was comparable between the two groups. Connexin 37 and 43 expression was also comparable between the groups, indicating that gap junctions were not affected upon DC101 treatment. However, lesions that exhibit intraplaque hemorrhage showed a strong increase in destabilizing Ang-2. The vein graft lesion area was significantly reduced in the DC101 group, which was accompanied by an increased plaque stability phenotype as a result of a significant reduction in macrophage content and increases in smooth muscle cells and collagen content. Conclusions: Blockade of VEGFR2 leads to reduced intraplaque hemorrhage, decreased vein graft lesion area and increased plaque stability as a result of increased neovessel maturation. This identifies plaque neovascularization as an attractive target for the treatment of unstable atherosclerotic diseases.

CARD9 mediates necrotic smooth muscle cell-induced inflammation in macrophages contributing to neointima formation of vein grafts.

Inflammation plays an important role in the neointima formation of grafted veins. However, the initiation of inflammation in grafted veins is still unclear. Here, we investigated the role and underlying mechanism of an innate immunity signalling protein, caspase-associated recruitment domain 9 (CARD9) in vein grafts in mice. In early murine vein grafts, we observed robust death of smooth muscle cells (SMCs), which was accompanied by infiltration of macrophages and expression of pro-inflammatory cytokines. Meanwhile, SMC necrosis was associated with the expression of pro-inflammatory cytokines in macrophages in vitro. To explore the mediators of necrotic SMC-induced inflammation in grafted veins from mice, we examined the expression of CARD family proteins and found CARD9 highly expressed in infiltrated macrophages of grafted veins. CARD9-knockout (KO) inhibited necrotic SMC-induced pro-inflammatory cytokine expression and NF-κB activation. Furthermore, CARD9-KO suppressed necrotic SMC-induced expression of VEGF in macrophages. Finally, CARD9-KO decreased neointima formation of grafted veins in mice. The innate immune protein CARD9 in macrophages may mediate necrotic SMC-induced inflammation by activating NF-κB and contributed to neointima formation in the vein grafts.

Abstract 188: Selective Inhibition of Vascular Smooth Muscle Cell Migration by Targeting Plasminogen Activator Inhibitor-1

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions. Methods: We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs. Conclusions: Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.

Ephrin type-B receptor 4 activation reduces neointimal hyperplasia in human saphenous vein in vitro

Vein bypass is an essential therapy for patients with advanced peripheral and coronary artery disease despite development of neointimal hyperplasia. We have shown that stimulation of the receptor tyrosine kinase ephrin type-B receptor 4 (Eph-B4) with its ligand ephrin-B2 prevents neointimal hyperplasia in murine vein grafts. This study determines whether Eph-B4 in adult human veins is capable of phosphorylation and activation of downstream signaling pathways, as well as functional to release nitric oxide (NO) and prevent neointimal hyperplasia in vitro. Discarded human saphenous veins were taken from the operating room and placed in organ culture without or with ephrin-B2/Fc (2 μg/mL) for 14 days, and the neointima/media ratio was measured in matched veins. Primary human umbilical vein endothelial cells were treated with ephrin-B2/Fc (2 μg/mL) and examined with quantitative polymerase chain reaction, Western blot, immunoassays, and for release of NO. Ephrin-B2/Fc (2 μg/mL) was placed on the adventitia of saphenous veins treated with arterial shear stress for 24 hours in a bioreactor and activated Eph-B4 examined with immunofluorescence. The baseline intima/media ratio in saphenous vein rings was 0.456 ± 0.097, which increased to 0.726 ± 0.142 in untreated veins after 14 days in organ culture but only to 0.630 ± 0.132 in veins treated with ephrin-B2/Fc (n = 19, P = .017). Ephrin-B2/Fc stimulated Akt, endothelial NO synthase and caveolin-1 phosphorylation, and NO release (P = .007) from human umbilical vein endothelial cells (n = 6). Ephrin-B2/Fc delivered to the adventitia stimulated endothelial Eph-B4 phosphorylation after 24 hours of arterial stress in a bioreactor (n = 3). Eph-B4 is present and functional in adult human saphenous veins, with intact downstream signaling pathways capable of NO release and prevention of neointimal hyperplasia in vitro. Adventitial delivery of ephrin-B2/Fc activates endothelial Eph-B4 in saphenous veins treated with arterial shear stress in vitro. These results suggest that stimulation of Eph-B4 function may be a candidate strategy for translation to human clinical trials designed to inhibit venous neointimal hyperplasia.

Vegfr2 blockade in murine vein graft results in reduced intraplaque hemorrhage and stable atherosclerotic lesions

Introduction: A third of patients with critical limb ischaemia (CLI) eventually require amputation. In spite of clinically successful revascularisation patients rarely return to their pre-morbid status, and often report no improvement in their functional outcomes, which may be due to an underlying musculopathy. Non-haematopoietic EPO-derivatives have been designed to retain only tissue-protective functions of EPO. We hypothesised that ARA-290 (EPO-derivative) may have tissue-protective potential that would represent a novel therapeutic adjunct in patients with CLI. Methods: The effect of EPO and ARA-290 in mediating cytoprotection was assessed firstly in vitro using skeletal myoblasts isolated from CLI and control donors, and a model of simulated ischaemia. Characterisation of CLI myoblasts was also performed, to assess their contractile, migratory and proliferative ability. Subsequently, an in vivo murine model of hindlimb ischaemia, which recapitulates the muscular pathology observed in CLI patients, was used to assess the potential of ARA-290 to improve functional, histological and perfusion outcomes. Results: Skeletal myoblasts were successfully isolated from CLI patients for the first time. CLI myoblasts and myotubes exhibited increased proliferative capacity but reduced migratory and contractile function and importantly a reduced susceptibility to a second ischaemic-insult compared with control myoblasts and myotubes. EPO and ARA-290 treatment led to significant improvements in myoblasts and myotube function and survival via the JAK2/ STAT3, PI3k/Akt and NFkB signalling pathways. In vivo, animals treated with EPO and ARA-290 demonstrated improved functional, histological and perfusion outcomes compared to vehicle-control treated animals. Conclusion: These studies demonstrate the potential of EPO and its derivatives to protect tissues and cells from ischaemic-injury and encourages the development of novel pharmacological therapies for use in patients with “no option” CLI or severe functional deficit.