Myeloid PHD2 Conditional Knockout Improves Intraplaque Angiogenesis and Vascular Remodeling in a Murine Model of Venous Bypass Grafting.

BACKGROUND: Intraplaque angiogenesis occurs in response to atherosclerotic plaque hypoxia, which is driven mainly by highly metabolically active macrophages. Improving plaque oxygenation by increasing macrophage hypoxic signaling, thus stimulating intraplaque angiogenesis, could restore cellular function and neovessel maturation, and decrease plaque formation. Prolyl hydroxylases (PHDs) regulate cellular responses to hypoxia. We therefore aimed to elucidate the role of myeloid PHD2, the dominant PHD isoform, on intraplaque angiogenesis in a murine model for venous bypass grafting. METHODS AND RESULTS: Myeloid PHD2 conditional knockout (PHD2cko) and PHD2 wild type mice on an Ldlr-/- background underwent vein graft surgery (n=11-15/group) by interpositioning donor caval veins into the carotid artery of genotype-matched mice. At postoperative day 28, vein grafts were harvested for morphometric and compositional analysis, and blood was collected for flow cytometry. Myeloid PHD2cko induced and improved intraplaque angiogenesis by improving neovessel maturation, which reduced intraplaque hemorrhage. Intima/media ratio was decreased in myeloid PHD2cko vein grafts. In addition, PHD2 deficiency prevented dissection of vein grafts and resulted in an increase in vessel wall collagen content. Moreover, the macrophage proinflammatory phenotype in the vein graft wall was attenuated in myeloid PHD2cko mice. In vitro cultured PHD2cko bone marrow-derived macrophages exhibited an increased proangiogenic phenotype compared with control. CONCLUSIONS: Myeloid PHD2cko reduces vein graft disease and ameliorates vein graft lesion stability by improving intraplaque angiogenesis.

Short-term Pre-operative Methionine Restriction Induces Browning of Perivascular Adipose Tissue and Improves Vein Graft Remodeling in Mice.

Short-term preoperative methionine restriction (MetR) shows promise as a translatable strategy to modulate the body's response to surgical injury. Its application, however, to improve post-interventional vascular remodeling remains underexplored. Here, we find that MetR protects from arterial intimal hyperplasia in a focal stenosis model and adverse vascular remodeling after vein graft surgery. RNA sequencing reveals that MetR enhances the brown adipose tissue phenotype in arterial perivascular adipose tissue (PVAT) and induces it in venous PVAT. Specifically, PPAR-α was highly upregulated in PVAT-adipocytes. Furthermore, MetR dampens the post-operative pro-inflammatory response to surgery in PVAT-macrophages in vivo and in vitro . This study shows for the first time that the detrimental effects of dysfunctional PVAT on vascular remodeling can be reversed by MetR, and identifies pathways involved in browning of PVAT. Furthermore, we demonstrate the potential of short-term pre-operative MetR as a simple intervention to ameliorate vascular remodeling after vascular surgery.

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.

Short-Term Pre-Operative Protein Caloric Restriction in Elective Vascular Surgery Patients: A Randomized Clinical Trial.

(1) Background: Vascular surgery operations are hampered by high failure rates and frequent occurrence of peri-operative cardiovascular complications. In pre-clinical studies, pre-operative restriction of proteins and/or calories (PCR) has been shown to limit ischemia-reperfusion damage, slow intimal hyperplasia, and improve metabolic fitness. However, whether these dietary regimens are feasible and safe in the vascular surgery patient population remains unknown. (2) Methods: We performed a randomized controlled trial in patients scheduled for any elective open vascular procedure. Participants were randomized in a 3:2 ratio to either four days of outpatient pre-operative PCR (30% calorie, 70% protein restriction) or their regular ad-libitum diet. Blood was drawn at baseline, pre-operative, and post-operative day 1 timepoints. A leukocyte subset flow cytometry panel was performed at these timepoints. Subcutaneous/perivascular adipose tissue was sampled and analyzed. Follow-up was one year post-op. (3) Results: 19 patients were enrolled, of whom 11 completed the study. No diet-related reasons for non-completion were reported, and there was no intervention group crossover. The PCR diet induced weight loss and BMI decrease without malnutrition. Insulin sensitivity was improved after four days of PCR (p = 0.05). Between diet groups, there were similar rates of re-intervention, wound infection, and cardiovascular complications. Leukocyte populations were maintained after four days of PCR. (4) Conclusions: Pre-operative PCR is safe and feasible in elective vascular surgery patients.

Endothelial Barrier Function and Leukocyte Transmigration in Atherosclerosis.

The vascular endothelium is a highly specialized barrier that controls passage of fluids and migration of cells from the lumen into the vessel wall. Endothelial cells assist leukocytes to extravasate and despite the variety in the specific mechanisms utilized by different leukocytes to cross different vascular beds, there is a general principle of capture, rolling, slow rolling, arrest, crawling, and ultimately diapedesis via a paracellular or transcellular route. In atherosclerosis, the barrier function of the endothelium is impaired leading to uncontrolled leukocyte extravasation and vascular leakage. This is also observed in the neovessels that grow into the atherosclerotic plaque leading to intraplaque hemorrhage and plaque destabilization. This review focuses on the vascular endothelial barrier function and the interaction between endothelial cells and leukocytes during transmigration. We will discuss the role of endothelial dysfunction, transendothelial migration of leukocytes and plaque angiogenesis in atherosclerosis.

Periprocedural Hydrogen Sulfide Therapy Improves Vascular Remodeling and Attenuates Vein Graft Disease.

Background Failure rates after revascularization surgery remain high, both in vein grafts (VG) and arterial interventions. One promising approach to improve outcomes is endogenous upregulation of the gaseous transmitter-molecule hydrogen sulfide, via short-term dietary restriction. However, strict patient compliance stands as a potential translational barrier in the vascular surgery patient population. Here we present a new therapeutic approach, via a locally applicable gel containing the hydrogen sulfide releasing prodrug (GYY), to both mitigate graft failure and improve arterial remodeling. Methods and Results All experiments were performed on C57BL/6 (male, 12 weeks old) mice. VG surgery was performed by grafting a donor-mouse cava vein into the right common carotid artery of a recipient via an end-to-end anastomosis. In separate experiments arterial intimal hyperplasia was assayed via a right common carotid artery focal stenosis model. All mice were harvested at postoperative day 28 and artery/graft was processed for histology. Efficacy of hydrogen sulfide was first tested via GYY supplementation of drinking water either 1 week before VG surgery (pre-GYY) or starting immediately postoperatively (post-GYY). Pre-GYY mice had a 36.5% decrease in intimal/media+adventitia area ratio compared with controls. GYY in a 40% Pluronic gel (or vehicle) locally applied to the graft/artery had decreased intimal/media area ratios (right common carotid artery) and improved vessel diameters. GYY-geltreated VG had larger diameters at both postoperative days 14 and 28, and a 56.7% reduction in intimal/media+adventitia area ratios. Intimal vascular smooth muscle cell migration was decreased 30.6% after GYY gel treatment, which was reproduced in vitro. Conclusions Local gel-based treatment with the hydrogen sulfide-donor GYY stands as a translatable therapy to improve VG durability and arterial remodeling after injury.

bFGF blockade reduces intraplaque angiogenesis and macrophage infiltration in atherosclerotic vein graft lesions in ApoE3*Leiden mice.

Intraplaque angiogenesis increases the chance of unstable atherosclerotic plaque rupture and thrombus formation leading to myocardial infarction. Basic Fibroblast Growth Factor (bFGF) plays a key role in angiogenesis and inflammation and is involved in the pathogenesis of atherosclerosis. Therefore, we aim to test K5, a small molecule bFGF-inhibitor, on remodelling of accelerated atherosclerotic vein grafts lesions in ApoE3*Leiden mice. K5-mediated bFGF-signalling blockade strongly decreased intraplaque angiogenesis and intraplaque hemorrhage. Moreover, it reduced macrophage infiltration in the lesions by modulating CCL2 and VCAM1 expression. Therefore, K5 increases plaque stability. To study the isolated effect of K5 on angiogenesis and SMCs-mediated intimal hyperplasia formation, we used an in vivo Matrigel-plug mouse model that reveals the effects on in vivo angiogenesis and femoral artery cuff model to exclusively looks at SMCs. K5 drastically reduced in vivo angiogenesis in the matrigel plug model while no effect on SMCs migration nor proliferation could be seen in the femoral artery cuff model. Moreover, in vitro K5 impaired endothelial cells functions, decreasing migration, proliferation and tube formation. Our data show that K5-mediated bFGF signalling blockade in hypercholesterolemic ApoE3*Leiden mice reduces intraplaque angiogenesis, haemorrhage and inflammation. Therefore, K5 is a promising candidate to stabilize advanced atherosclerotic plaques.