Ultrastructural Study of Platelet Behavior and Interrelationship in Sprouting and Intussusceptive Angiogenesis during Arterial Intimal Thickening Formation

Platelets in atherosclerosis, bypass stenosis, and restenosis have been extensively assessed. However, a sequential ultrastructural study of platelets in angiogenesis during the early phases of these lesions has received less attention. Our objective was the study of platelets in angiogenesis and vessel regression during intimal thickening (IT) formation, a precursor process of these occlusive vascular diseases. For this purpose, we used an experimental model of rat occluded arteries and procedures for ultrastructural observation. The results show (a) the absence of platelet adhesion in the de-endothelialized occluded arterial segment isolated from the circulation, (b) that intraarterial myriad platelets contributed from neovessels originated by sprouting angiogenesis from the periarterial microvasculature, (c) the association of platelets with blood components (fibrin, neutrophils, macrophages, and eosinophils) and non-polarized endothelial cells (ECs) forming aggregates (spheroids) in the arterial lumen, (d) the establishment of peg-and-socket junctions between platelets and polarized Ecs during intussusceptive angiogenesis originated from the EC aggregates, with the initial formation of IT, and (e) the aggregation of platelets in regressing neovessels (‘transitory paracrine organoid’) and IT increases. In conclusion, in sprouting and intussusceptive angiogenesis and vessel regression during IT formation, we contribute sequential ultrastructural findings on platelet behavior and relationships, which can be the basis for further studies using other procedures.

Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodeling

Aims Intimal hyperplasia is a common feature of vascular remodeling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo. Methods and results RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodeling of cultured human saphenous vein segments. Conclusions Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signaling, and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease. Translational perspective Coronary artery disease is currently the leading cause of death worldwide. SMCs are a major contributor to angioplasty restenosis, graft stenosis, and accelerated atherosclerosis. Current therapeutic approaches including drug-eluting stents targeting cell growth still have limitations. By combining studies on cultured SMCs in vitro, animal surgical models in vivo, and a human organ culture model ex vivo, we revealed an important role of PDE10A in modulating SMC proliferation and injury-induced intimal thickening. Given that PDE10A has been proven to be a safe drug target, its inhibition may represent a novel therapeutic strategy for vascular diseases associated with intimal hyperplasia.

Accumulation of Plasma-Derived Lipids in the Lipid Core and Necrotic Core of Human Atheroma: Imaging Mass Spectrometry and Histopathological Analyses

Objective: To clarify the pathogenesis of human atheroma, the origin of deposited lipids, the developmental mechanism of liponecrotic tissue, and the significance of the oxidation of phospholipids were investigated using mass spectrometry-aided imaging and immunohistochemistry. Approach and Results: Atherosclerotic lesions in human coronary arteries were divided into 3 groups: pathological intimal thickening with lipid pool, atheroma with lipid core, and atheroma with necrotic core. The lipid pool and lipid core were characterized by the deposition of extracellular lipids. The necrotic core comprised extracellular lipids and liponecrotic tissue. The proportion of cholesteryl linoleate in cholesteryl linoleate+cholesteryl oleate fraction in the extracellular lipid and liponecrotic regions differed significantly from that of the macrophage foam cell–dominant region, and the plasma-derived components (apoB and fibrinogen) were localized in the regions. The liponecrotic region was devoid of elastic and collagen fibers and accompanied by macrophage infiltration in the surrounding tissue. Non–oxidized phospholipid (Non-OxPL), OxPL, and Mox macrophages were detected in the three lesions. In the atheroma with lipid core and atheroma with necrotic core, non-OxPL tended to localize in the superficial layer, whereas OxPL was distributed evenly. Mox macrophages were colocalized with OxPL epitopes. Conclusions: In human atherosclerosis, plasma-derived lipids accumulate to form the lipid pool of pathological intimal thickening, lipid core of atheroma with lipid core, and necrotic core of atheroma with necrotic core. The liponecrotic tissue in the necrotic core appears to be developed by the loss of elastic and collagen fibers. Non-OxPL in the accumulated lipids is oxidized to form OxPL, which may contribute to the lesion development through Mox macrophages.

Recent insights into atherosclerotic plaque cell autophagy

Cardiovascular and cerebrovascular diseases, such as coronary heart disease and stroke, caused by atherosclerosis have become the “number one killer”, seriously endangering human health in developing and developed countries. Atherosclerosis mainly occurs in large and medium-sized arteries and involves intimal thickening, accumulation of foam cells, and formation of atheromatous plaques. Autophagy is a cellular catabolic process that has evolved to defend cells from the turnover of intracellular molecules. Autophagy is thought to play an important role in the development of plaques. This review focuses on studies on autophagy in cells involved in the formation of atherosclerotic plaques, such as monocytes, macrophages, endothelial cells, dendritic cells, and vascular smooth muscle cells, indicating that autophagy plays an important role in plaque development. We mainly discuss the roles of autophagy in these cells in maintaining the stability of atherosclerotic plaques, providing a reference for the next steps to unravel the mechanisms of atherogenesis.

TET2 Protects Against VSMC Apoptosis and Intimal Thickening in Transplant Vasculopathy

Background: Coronary allograft vasculopathy (CAV) is a devastating sequelae of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET methylcytosine dioxygenase 2 (TET2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. Methods: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also employed the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild type and inducible smooth muscle-specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNу, a cytokine elaborated by T cells that drives CAV progression. Results: In the present study, we found that TET2 expression and activity is negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNу was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro . TET2 depletion mimicked the effects of IFNу, and TET2 overexpression rescued IFNу-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro , TET2 deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Notably, enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. Conclusions: TET2 is repressed in CAV and GA, likely mediated by IFNу. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNу stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

Intimal thickening and disruption of the media occur in the arterial walls of coronary arteries not associated with coronary arterial aneurysms in patients with Kawasaki disease

Background Coronary artery aneurysm (CAA) is an important complication of Kawasaki disease (KD) that is associated with arterial structure damage. However, few studies have examined structural changes in coronary arteries that are not associated with CAA. Methods We examined coronary arteries in KD patients with CAAs who underwent follow-up coronary angiography (CAG) and optical coherence tomography (OCT). Coronary arterial branches with no abnormal findings during the most recent CAG were classified into two groups. Arteries with an acute-phase CAA that later regressed were classified as group R; arteries with no abnormal findings on either acute or convalescent phase CAG were classified as group N. Coronary arterial wall structural changes were compared between groups using OCT. Results Fifty-seven coronary arterial branches in 23 patients were evaluated by OCT. Thirty-six branches showed no abnormality during the most recent CAG. Both groups R and N comprised 18 branches. Maximum intimal thicknesses in groups R and N were 475 and 355 µm, respectively (p = 0.007). The incidences of media disruption were 100% and 67%, respectively (p = 0.02). Calcification, macrophage accumulation, and thrombus were not found in either group. Conclusions Intimal thickening and disruption of the media occur in coronary arteries with acute phase CAAs that later regress in the convalescent phase, as well as in arteries with normal CAG findings in the acute and convalescent phases.

Intimal Thickening and Disruption of The Media Occur in The Arterial Walls of Coronary Arteries Not Associated With Coronary Arterial Aneurysms in Patients With Kawasaki Disease

Background Coronary artery aneurysm (CAA) is an important complication of Kawasaki disease (KD) that is associated with arterial structure damage. However, few studies have examined structural changes in coronary arteries not associated with CAA.Methods We examined coronary arteries in KD patients with CAAs who underwent follow-up coronary angiography (CAG) and optical coherence tomography (OCT). Coronary arterial branches with no abnormal findings on most recent CAG were classified into two groups. Arteries with an acute-phase CAA that later regressed were classified as group R and arteries with no abnormal findings on either acute or convalescent phase CAG were classified as group N. Coronary arterial wall structural changes were compared between groups using OCT.ResultsFifty-seven coronary arterial branches in 23 patients were evaluated by OCT. Thirty-six branches showed no abnormality on most recent CAG. Both groups R and N comprised 18 branches. Maximum intimal thickness in groups R and N was 475 and 355 µm, respectively (p = 0.007). The incidence of disruption of the media was 100% and 67%, respectively (p = 0.02). Calcification, macrophage accumulation, and thrombus were not found in either group.ConclusionsIntimal thickening and disruption of the media occur not only in coronary arteries with acute phase CAAs that later regress in the convalescent phase, but also in arteries with normal CAG findings in the acute and convalescent phases.

Infection of expanded polytetrafluoroethylene and Dacron-coated stents with Staphylococcus epidermidis: an experimental study in pigs

Background Diagnosis of the etiologic agent of endoprosthesis infections is essential to enable treatment, since these infections constitute important complications of endovascular procedures. Sonication of explanted tissue and materials is a technique that can be used to facilitate detection of biofilm-producing bacteria. Objectives To evaluate infection of pigs' aortas after implantation of nitinol stents coated with polytetrafluoroethylene (ePTFE) or Dacron, previously infected with biofilm-producing Staphylococcus epidermidis. Intimal thickening and the inflammatory response in the aortic wall were also evaluated. Methods 11 ePTFE-coated nitinol stents and 10 Dacron stents infected with S. epidermidis strains were implanted in the infrarenal aorta of 21 8-week-old pigs. After 2 weeks, the aorta containing the stents was removed. A vortex mixer and ultrasound were used to homogenize the samples and remove the biofilm. Subsequently, the number of colony-forming units was counted. Results There were no significant differences between the two groups in terms of the number of colony-forming units or of inflammation in the arterial wall. With the exception of one specimen from the Dacron group, all aortic stent cultures were positive for S. epidermidis. Conclusions There were no significant differences in the inflammatory response or infection rate between ePTFE and Dacron-coated stents actively infected with biofilm-producing S. epidermidis. Intimal thickening and the inflammatory response to infection of endoprostheses were similar. These results suggest that the two most widely used stent lining materials have a similar infection rate.

Abstract 14108: Does Primary Graft Dysfunction Increase First Year Intimal Thickness After Heart Transplantation?

Introduction: Primary graft dysfunction (PGD) has been reported in between 7-30% of heart transplant recipients with severe PGD known to have poor outcome. Due to graft dysfunction and potential for endothelial cell injury, it is not known whether all grades of PGD can increase first year intimal thickening, which may be a harbinger for clinical cardiac allograft vasculopathy (CAV). Methods: Between 2010 and 2017, we assessed 769 heart transplant patients and divided them into those patients with mild PGD (n=12), moderate PGD (n=35), severe PGD (n=36) and no PGD (n=686). Endpoints included 3-year survival and freedom from angiographic CAV (stenosis ≥30%) and non-fatal major adverse cardiac events (NF-MACE: myocardial infarction, percutaneous coronary intervention, new congestive heart failure, pacemaker/implantable cardioverter-defibrillator placement, stroke); and 1-year freedom from any treated rejection (ATR), acute cellular rejection (ACR), antibody mediated rejection (AMR). First year intravascular ultrasound (IVUS) was performed at baseline 4-8 weeks and at 1 year after transplant. IVUS parameters included first-year average change in MIT and change in MIT ≥0.5mm. Results: First year change in average MIT and MIT ≥0.5mm and 3-year freedom from CAV were not significantly different in all groups. The severe PGD group compared to all groups had significantly lower 3-year survival and freedom from NF-MACE. There was lower freedom from various types of rejection in the PGD groups compared to the no PGD group. Specifically, 1-year freedom from ACR was significantly lower in the mild PGD group compared to the moderate PGD group and the no PGD group (see table). Conclusions: PGD grades do not appear to lead to increased first year intimal thickening or angiographic CAV at 3 years. Lower freedom from ACR in the mild PGD group and ATR in the severe PGD group will need further study. Larger numbers of patients are needed to confirm these findings.