The myristoylated alanine-rich C kinase substrate differentially regulates kinase interacting with stathmin in vascular smooth muscle and endothelial cells and potentiates intimal hyperplasia formation.

OBJECTIVE: Restenosis limits the durability of all cardiovascular reconstructions. Vascular smooth muscle cell (VSMC) proliferation drives this process, but an intact, functional endothelium is necessary for vessel patency. Current strategies to prevent restenosis employ antiproliferative agents that affect both VSMCs and endothelial cells (ECs). Knockdown of the myristoylated alanine-rich C kinase substrate (MARCKS) arrests VSMC proliferation and paradoxically potentiates EC proliferation. MARCKS knockdown decreases expression of the kinase interacting with stathmin (KIS), increasing p27kip1 expression, arresting VSMC proliferation. Here, we seek to determine how MARCKS influences KIS protein expression in these two cell types. METHODS: Primary human coronary artery VSMCs and ECs were used for in vitro experiments. MARCKS was depleted by transfection with small interfering RNA. Messenger RNA was quantitated with the real-time reverse transcription polymerase chain reaction. Protein expression was determined by Western blot analysis. Ubiquitination was determined with immunoprecipitation. MARCKS and KIS binding was assessed with co-immunoprecipitation. Intimal hyperplasia was induced in CL57/B6 mice with a femoral artery wire injury. MARCKS was knocked down in vivo by application of 10 µM of small interfering RNA targeting MARCKS suspended in 30% Pluronic F-127 gel. Intimal hyperplasia formation was assessed by measurement of the intimal thickness on cross sections of the injured artery. Re-endothelialization was determined by quantitating the binding of Evans blue dye to the injured artery. RESULTS: MARCKS knockdown did not affect KIS messenger RNA expression in either cell type. In the presence of cycloheximide, MARCKS knockdown in VSMCs decreased KIS protein stability but had no effect in ECs. The effect of MARCKS knockdown on KIS stability was abrogated by the 26s proteasome inhibitor MG-132. MARCKS binds to KIS in VSMCs but not in ECs. MARCKS knockdown significantly increased the level of ubiquitinated KIS in VSMCs but not in ECs. MARCKS knockdown in vivo resulted in decreased KIS expression. Furthermore, MARCKS knockdown in vivo resulted in decreased 5-ethynyl-2'-deoxyuridine integration and significantly reduced intimal thickening. MARCKS knockdown enhanced endothelial barrier function recovery 4 days after injury. CONCLUSIONS: MARCKS differentially regulates the KIS protein stability in VSMCs and ECs. The difference in stability is due to differential ubiquitination of KIS in these two cell types. The differential interaction of MARCKS and KIS provides a possible explanation for the observed difference in ubiquitination. The effect of MARCKS knockdown on KIS expression persists in vivo, potentiates recovery of the endothelium, and abrogates intimal hyperplasia formation.

Considerations beyond Stenosis for Carotid Endarterectomy in Treating Free-Floating Thrombus of the Carotid Artery.

BACKGROUND: Free-floating thrombus (FFT) of the carotid artery is an uncommon condition that can present with neurologic symptoms, often in the setting of ischemic stroke. The literature pertaining to the incidence and optimal treatment of this condition is limited. Herein, we report our contemporary experience with FFT across a range of degrees of carotid stenosis. METHODS: Medical records and imaging studies from a single academic medical center from January 2016 to July 2018 were retrospectively reviewed. Patient demographics, presentation, treatment, and follow-up were abstracted. RESULTS: Six cases of FFT of the carotid artery with and without hemodynamically significant atherosclerotic disease were identified. All cases presented with ischemic stroke; one case had a hemorrhagic conversion. In each case, the FFT was visualized by at least one imaging modality including computed tomography angiography, magnetic resonance angiography, and duplex ultrasound. Three patients had >50% carotid stenosis and three had <50%. All cases were treated with endarterectomy. Four of the six patients received preoperative anticoagulation. There were no postoperative complications. Median follow-up was 252 days, with one case lost to follow-up. Four of the six patients have been without restenosis, recurrence of the thrombus, nor worsening or recurrent stroke on follow-up. The fifth patient developed restenosis but remained clinically stable. CONCLUSIONS: Although current society guidelines do not recommend carotid endarterectomy as first-line treatment for symptomatic patients with <50% stenosis, it may be indicated in the context of FFT.

A Unique Bailout Method for the Repair of Abdominal Aortic Aneurism with a Narrow Iliac Bifurcation.

BACKGROUND: Endovascular aneurysm repair (EVAR) became the procedure of first choice for the repair of the abdominal aortic aneurysms (AAAs) in the last decades. However, narrow distal aorta remains to be the main limiting factor for the use of EVAR. A limited number of bail-out procedures have been described in the literature to overcome this problem. METHODS: A 69-year-old male was transferred to our institution for the repair of a ruptured AAA. His initial presentation mimicked an acute coronary syndrome, provoking a cardiac catheterization that documented a ruptured AAA. RESULTS: The patient was brought to the operating room for EVAR, but his distal aorta was severely narrowed, preventing the use of a bifurcated graft. We had to convert the bifurcated graft to a unigraft and place two additional grafts extending into the iliac arteries to fix the type I endoleak that we encountered at the distal end of the unigraft. He recovered well postoperatively, and his repair was found to be stable at 6-month follow-up. CONCLUSION: The surgical technique that we are presenting here is a unique bail-out procedure that can be used as an alternative solution to the narrow distal aortas.

Small GTPases and Their Role in Vascular Disease.

Over eighty million people in the United States have cardiovascular disease that can affect the heart causing myocardial infarction; the carotid arteries causing stroke; and the lower extremities leading to amputation. The treatment for end-stage cardiovascular disease is surgical-either endovascular therapy with balloons and stents-or open reconstruction to reestablish blood flow. All interventions damage or destroy the protective inner lining of the blood vessel-the endothelium. An intact endothelium is essential to provide a protective; antithrombotic lining of a blood vessel. Currently; there are no agents used in the clinical setting that promote reendothelialization. This process requires migration of endothelial cells to the denuded vessel; proliferation of endothelial cells on the denuded vessel surface; and the reconstitution of the tight adherence junctions responsible for the formation of an impermeable surface. These processes are all regulated in part and are dependent on small GTPases. As important as the small GTPases are for reendothelialization, dysregulation of these molecules can result in various vascular pathologies including aneurysm formation, atherosclerosis, diabetes, angiogenesis, and hypertension. A better understanding of the role of small GTPases in endothelial cell migration is essential to the development for novel agents to treat vascular disease.

Surgical Scarring after Arterial Bypass, an Etiology of Venous Hypertension.

Venous ulcers can be a chronic debilitating condition with a high rate of recurrence. Herein, we describe a case of a patient who successfully underwent an arterial bypass for rest pain but returned with lower extremity swelling and venous ulcers. Venography demonstrated a focal common femoral vein stenosis due to scarring from the surgical exposure. This was treated with endovenous stenting and resulted in resolution of the swelling and ulceration.

A Defined Protocol to Resolving Cannulation Failure during Endovenous Ablation Procedures.

During endovenous ablation for the treatment of insufficient veins, failure to cannulate the entirety of the refluxing vein with the treatment catheter prevents technically successful ablation. In this technique report, we describe a defined protocol to overcome cannulation failure of axial veins for endovenous ablation. This protocol utilizes commonly available adjunctive techniques including ultrasound-guided digital compression, the use of a guidewire, the use of a guide catheter, and placement of a second puncture site in a step-wise fashion to overcome varying degrees of tortuosity or obstruction. The sequential application of these techniques as described in this report allows endovenous ablation to be applied to patients with challenging venous anatomy.

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.

Long-term follow-up of neck expansion after endovascular aortic aneurysm repair.

OBJECTIVE: This study determined the rate, extent, and clinical significance of neck dilatation after endovascular aneurysm repair (EVAR). METHODS: The study included 46 patients who underwent elective EVAR using bifurcated Zenith stent grafts (Cook, Bloomington, Ind) and had at least 48 months of clinical and radiographic follow-up. Computed tomography images were analyzed on a 3-dimensional workstation (TeraRecon, San Mateo, Calif). Neck diameter was measured 10 mm below the most inferior renal artery in planes orthogonal to the aorta. Nominal stent graft diameter was obtained from implantation records. RESULTS: Median follow-up was 59 months (range, 48-120 months). Neck dilation occurred in all 46 patients. The rate of neck dilation was greatest at early follow-up intervals. At 48 months, median neck dilation was 5.3 mm (range, 2.3-9.8 mm). The extent of neck dilation at 48 months correlated with percentage of stent graft oversizing (Spearman rho = 0.61, P < .001). No type I endoleak or migration >5 mm occurred. CONCLUSIONS: After EVAR with the Zenith stent graft, the neck dilates until its diameter approximates the diameter of the stent graft. Neck dilation was not associated with type I endoleak or migration of the stent graft.

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 p27(kip1). 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.

Prospective study of cryopreserved placental tissue wound matrix in the management of chronic venous leg ulcers.

OBJECTIVE: Chronic venous leg ulcers (VLUs) affect up to 2% of the general population, resulting in a significant socioeconomic burden. Placental tissue that contains mesenchymal stem cells and active growth factors has been shown to be beneficial in healing of chronic wounds. We compared the efficacy of a human viable wound matrix (hVWM) of cryopreserved placental tissue for the treatment of refractory VLUs with standard therapy. METHODS: This prospective single-center open-label single-arm study enrolled patients with Clinical, Etiology, Anatomy, and Pathophysiology clinical class C6 VLUs. The ulcers of all enrolled patients had failed to heal after a trial of standard therapy of at least 12 weeks, which included weekly multilayer compression therapy along with local wound care. The same patients subsequently received application of hVWM (Grafix; Osiris Therapeutics, Columbia, Md) every 1 to 2 weeks in addition to standard therapy. Healing with hVWM therapy was then compared with standard therapy, with each patient serving as his own control. RESULTS: There were 30 VLUs in 21 consecutive eligible patients who were enrolled in the study. All patients were men with an average age of 67 years (standard deviation [SD], ±10.8 years), and the average area of venous ulcers before hVWM initiation was 12.2 cm2 (SD, ±14.6 cm2; range, 3.3-12.3 cm2). Duplex ultrasound confirmed superficial or deep system venous reflux in all patients. Complete ulcer healing was achieved in 53% (16/30) of VLUs refractory to standard therapy after application of hVWM. There was a mean reduction in wound surface area by 79% (SD, ±27.3%; P < .001 compared with standard therapy) after a mean treatment time of 10.9 weeks. Eighty percent of VLUs were reduced in size by half compared with 25% with standard therapy (P < .001). The mean rate of reduction in ulcer area after hVWM applications was 1.69% per day vs 0.73% per day with standard therapy (P = .01). CONCLUSIONS: Cryopreserved placental tissue (hVWM) improves healing processes to achieve complete wound closure in a significant proportion of chronic VLUs refractory to standard therapy. Adjunctive therapy with hVWM provides superior healing rates in refractory VLUs.

Teleconferencing surgery enhances effective communication and enriches medical education.

Medical student surgical training has traditionally occurred in the operating room (OR). Present technology allows real time communication between the OR and a remote classroom. We seek to evaluate the value of the teleconferencing (TC) environment compared with the traditional OR environment. Students enrolled in the third-year core clerkship in surgery participated in both TC and OR teaching sessions. A total of 23 sessions were conducted and observed (TC=8; OR=15). In TC sessions, students asked over 4 times as many questions (17.4+/-8.5 vs. 3.4+/-3.0; P<0.01) and faculty asked 4 times as many questions of the students (13.1+/-6.8 vs. 3.1+/-2.7; P<0.01). In TC sessions, students felt more able to ask questions (P<0.01), left with fewer unanswered questions (P<0.01), and more felt it was a good use of their time (P=0.04). These findings demonstrate that TC is a valuable modality for medical student education.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

BACKGROUND: Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. RESULTS: Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. CONCLUSIONS: Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

Comparison of gene silencing in human vascular cells using small interfering RNAs.

BACKGROUND: Gene silencing achieved through small interfering RNA (siRNA) transfection represents a promising approach to vascular gene therapy. Here we characterize the behavior of RNA interference (RNAi) in vascular biology by comparing the RNAi response to single- and multigene siRNA transfections in vitro in human vascular cells. STUDY DESIGN: The strength and specificity of multigene silencing in cultured human coronary artery smooth muscle and human coronary artery endothelial cells (HCASMC/HCAEC) were assessed by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and Western blot after transfection singly or simultaneously with siRNAs targeting glyceraldehyde-3-phosphate dehydrogenase, the myristoylated alanine-rich C kinase substrate, and cadherin 11. RNAi response to low-dose (0.25 to 10 nM) siRNA transfection was characterized between the two cell types by QRT-PCR and fluorescence-activated cell sorter analysis. RESULTS: Powerful and specific silencing of all targets was observed in both cell types after multigene siRNA transfections, but with a reduction in effect compared with single-gene siRNA transfections. Multigene messenger RNA (mRNA) reductions in HCAECs exceeded those achieved in HCASMCs, and superior mRNA silencing and siRNA delivery were observed in HCAECs after low-dose siRNA transfections. CONCLUSIONS: Multigene silencing by siRNA stands as a promising nonviral approach for manipulating gene expression in human vascular cells. Under our in vitro conditions, endothelial cells were more susceptible to siRNA transfection and gene silencing than vascular smooth muscle cells. RNAi technology could potentially find use in the development of siRNA cocktails for application to vein bypass grafts or for modulating endothelial cell function in other forms of vascular disease.

Double prepuncture as a valuable adjunctive technique for complex endovenous ablation.

OBJECTIVE: The objective of this study was to characterize the technique and to report the results of double prepuncture used during complex radiofrequency ablation (RFA) in cases of treating multiple incompetent veins or encountering focal obstruction to catheter advancement. METHODS: A double prepuncture technique was applied in patients requiring endovascular ablation of multiple veins and patients with great saphenous vein cannulation failure. We treated 13 limbs in 12 patients during a 24-month period with RFA in which the double prepuncture technique was used. Clinical history, operative reports, outcomes, and follow-up were reviewed. RESULTS: RFA was performed with the double puncture technique on, collectively, 10 great saphenous veins, 5 small saphenous veins, and 5 anterior accessory saphenous veins. Mean preoperative Clinical, Etiology, Anatomy, and Pathophysiology score was 4.38 ± 1.6. Three limbs required prepuncture because of difficulty in advancing the catheter cephalad through tortuosity and focal obstruction after failure with techniques such as a guidewire, a guide catheter, and manual compression with ultrasound guidance. Ten limbs received planned double prepuncture for multiple adjacent incompetent veins, for which venipuncture and cannulation of the second target vein would be difficult after tumescent application to the first vein. Postoperative ultrasound demonstrated successful closure of all target veins in which the double prepuncture technique was used. One patient had a deep venous thrombosis (7.7%) that resolved without complications. CONCLUSIONS: Double prepuncture is a useful technical adjunct both for simultaneous endovenous ablation of multiple adjacent incompetent veins and when catheter passage is impeded. This technique aids in efficient and successful application of endovenous ablation to complex venous anatomy.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Development of an infection-resistant, bioactive wound dressing surface.

Trauma, whether caused by an accident or in an intentional manner, results in significant morbidity and mortality. The goal of this study was to develop a novel biomaterial surface in vitro and ex vivo that provides both localized infection resistance nd hemostatic properties. Our hypothesis is that a combination of specific surface characteristics can be successfully incorporated into a single biomaterial. Functional groups were created with woven Dacron (Cntrl) material via exposure to ethylenediamine (C-EDA). The antibiotic ciprofloxacin (Cipro) was then applied to the C-EDA material using pad/autoclave technique (C-EDA-AB) followed by surface immobilization of the coagulation cascade enzyme thrombin (C-EDA-AB-Thrombin). Antimicrobial activity by the C-EDA-AB surface persisted for 5 days compared with Cntrl and dipped controls, which lasted <1 h. C-EDA-AB-Thrombin surfaces had 2.6- and 105-fold greater surface thrombin activity compared with nonspecifically bound thrombin and Cipro-dyed surfaces, respectively. Surface thrombus formation ex vivo was evident after 1 min of exposure, with thrombus organization evident by 2.5 min. In contrast, C-EDA-AB and Cntrl segments showed only blood protein adsorption on the fibers. Thus, this study demonstrated that Cipro and thrombin can be simultaneously incorporated onto a biomaterial surface while maintaining their respective biological activities.

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

BACKGROUND: Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo. METHODS AND RESULTS: siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization. CONCLUSIONS: MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

Myristoylated Alanine-Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation.

BACKGROUND: Transcription of the myristoylated alanine-rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS-mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo. METHODS AND RESULTS: MARCKS knockdown blocked platelet-derived growth factor (PDGF)-induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP2, restored membrane-associated PIP2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation. CONCLUSIONS: MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP2 at the plasma membrane. This study delineates a novel mechanism for MARCKS-mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.

Results of radiofrequency ablation of the small saphenous vein in the supine position.

OBJECTIVES: To report the results of a novel approach using supine positioning for radiofrequency ablation (RFA) of the small saphenous vein (SSV) with combined ablation of the great saphenous vein (GSV). METHODS: Over a 24-month period, we identified patients with symptomatic SSV incompetence. Access to the SSV was accomplished by ultrasound-guided venipuncture with the patient in the supine position. RESULTS: Small saphenous vein ablation was performed on 27 limbs in 26 patients. Median follow-up was 94 days (interquartile range [IQR] 26, 171). Mean clinical-etiologic-anatomic-pathophysiologic (CEAP) score was 3.5 ± 1.3. Small saphenous vein ablation was performed in conjunction with GSV ablation in 17 patients and with phlebectomy in 14 patients. Postoperative ultrasound was performed after 26 of 27 procedures. The SSV was sealed in all 26 cases. Two patients (8%) had a deep venous thrombosis (DVT). CONCLUSIONS: The SSV can be effectively sealed by RFA from the supine position and combined SSV/GSV ablation can be carried out in a single setting.

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 PKCßII, 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-PKCßII, 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.