Thomas S. Monahan

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

MARCKS silencing differentially affects human vascular smooth muscle and endothelial cell phenotypes to inhibit neointimal hyperplasia in saphenous vein

Intimal hyperplasia (IH) limits the patency of all cardiovascular vein bypass grafts. We previously found the myristoylated alanine‐rich C kinase substrate (MARCKS), a key protein kinase C (PKC) substrate, to be up‐regulated in canine models of IH. Here, we further characterize the role of MARCKS in IH and examine the phenotypic consequences of MARCKS silencing by small interfering RNA (siRNA) transfection in human vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) in vitro and use a rapid 10‐min nonviral siRNA transfection technique to determine the effects of MARCKS silencing in human saphenous vein cultured ex vivo. We demonstrate MARCKS silencing attenuates VSMC migration and arrests VSMC proliferation in part through the up‐regulation of the cyclin‐dependent kinase inhibitor p27kip1. Conversely, MARCKS silencing had little or no effect on EC migration or proliferation. These phenotypic changes culminated in reduced neointimal formation in cultured human saphenous vein. These data identify MARCKS as a pathogenic contributor to IH and indicate therapeutic MARCKS silencing could selectively suppress the “atherogenic,” proliferative phenotype of VSMCs without collateral harm to the endothelium. This approach could be readily translated to the clinic to silence MARCKS in vein bypass grafts prior to implantation.— Monahan, T. S., Andersen, N. D., Martin, M. C, Malek, J. Y., Shrikhande, G. V., Pradhan, L., Ferran, C, LoGerfo, F. W. MARCKS silencing differentially affects human vascular smooth muscle and endothelial cell phenotypes to inhibit neointimal hyperplasia in saphenous vein. FASEB J. 23, 557–564 (2009)

Endothelial Cells Are Susceptible to Rapid siRNA Transfection and Gene Silencing Ex Vivo

BACKGROUND Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room. METHODS Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mm Hg hyperbaric pressure, or (iii) 120 mm Hg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry. RESULTS Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared with hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods. CONCLUSION Endothelial gene silencing is possible within the time frame and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease.