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.

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.

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.

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.

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.

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.

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.

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.

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.

Risk factors for lower-extremity vein graft failure.

Lower-extremity vein graft failure causes significant morbidity, increases health care costs, and negatively impacts patient quality of life. Identification of risk factors is essential for patient selection, risk factor modification, and identifying individuals who would benefit from more stringent surveillance protocols. Risk factors can be considered as either patient-related or technical. Here we discuss the patient-related risk factors for vein graft failure. Nontechnical factors related to the indication for operation include operation after a previously failed graft, or redo bypass, critical limb ischemia, and infection. Risk factors for vein graft failure are distinct from the risk factors for cardiovascular events. Young age and African American and Hispanic race are risk factors for lower-extremity vein graft failure. Hypercoaguable and inflammatory states also increase risk for vein graft failure. Therapy with statins is indicated in patients with peripheral atherosclerosis and may have beneficial effects on vein graft function, although further studies are needed in this area.

Fenestrated and branched stent grafts for repair of complex aortic aneurysms.

Open surgical repair of complex aortic aneurysms, such as juxtarenal or thoracoabdominal aortic aneurysms, is a highly demanding procedure. They frequently require major surgical exposure through both the thoracic and the abdominal cavities, suprarenal or supraceliac aortic cross-clamping, and exposure of the visceral and renal arteries. Endovascular aortic repair and thoracic endovascular aortic repair have become the mainstay of treatment for infrarenal abdominal aortic aneurysms and descending thoracic aneurysms. However, the obvious need to maintain perfusion of the visceral and renal arteries has limited application of endovascular techniques to treatment of more complex aneurysms. Fenestrated and branched stent grafts are being developed to address this need and enable repair of complex aneurysms involving branch vessels exclusively using minimally invasive techniques. Although these devices remain investigational in the United States, they have recently become commercially available in other countries and play an increasing role in the management of complex aortic aneurysms.

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.

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.

Low ejection fraction predicts shortened survival in patients undergoing infrainguinal arterial reconstruction.

BACKGROUND: With the advent of endovascular therapy for lower extremity ischemia it is important to better determine what factors may affect the outcome. The goal of the present study was to evaluate whether ejection fraction (EF) is predictive of outcome in infrainguinal arterial reconstruction. METHODS: We retrospectively reviewed 736 patients undergoing 897 infrainguinal arterial reconstructions from July 1999 to February 2002. Patients were divided into two groups: group I contained 54 patients with an EF<35% and group II had 216 patients with an EF > or =35%. The outcome evaluated was major adverse clinical events (MACEs), defined as postoperative myocardial infarction (MI), arrhythmia, congestive heart failure (CHF), and perioperative mortality. RESULTS: Major adverse clinical events occurred in 20.3% of patients (11/54) in group I and 10.6% patients (23/216) in group II (p = 0.068). Group I had a trend toward a greater incidence of MACEs compared to group II. Two-year survival for group I was 61.7%, whereas survival for group II was 78.4% (p = 0.0085). CONCLUSIONS: Low EF predicts a significantly shortened 2-year survival after infrainguinal arterial reconstruction and a trend toward increased perioperative complications. This is another factor to be considered in choosing open versus endovascular options.

A novel function for cadherin 11/osteoblast-cadherin in vascular smooth muscle cells: modulation of cell migration and proliferation.

OBJECTIVE: Intimal hyperplasia is a common cause of vein graft failure in cardiovascular surgery. The molecular basis for intimal hyperplasia remains poorly defined. We have previously identified, by gene chip analysis of vein grafts, increased messenger (mRNA) for the adhesion molecule cadherin 11/osteoblast-cadherin (CDH11). The function of CDH11 in vascular cells is unknown. The aim of the present study is to confirm CDH11 expression in vein grafts and characterize its role in vascular remodeling. METHODS: Cephalic vein interposition grafts were implanted in a canine model and harvested at predetermined time points. CDH11 protein expression was determined by immunohistochemistry. Early passage human coronary artery smooth muscle cells (SMCs) were used for in vitro studies. Real-time polymerase chain reaction was used to assess cellular CDH11 mRNA levels. CDH11 signaling was inhibited by either transfection with silencing RNA targeting CDH11 or with a blocking antibody to CDH11. Cellular migration was evaluated and cellular proliferation was assessed. RESULTS: Expression of CDH11 was increased in medial SMCs of vein grafts recovered at 7, 14, and 30 days after surgery compared with control veins from the same animals. In vitro CDH11 mRNA was up-regulated 1.8 +/- 0.2-fold (P = .003) in SMCs after treatment with tumor necrosis factor-alpha. Cellular migration was attenuated by inhibition of CDH11 both with a blocking antibody (0.67 +/- 0.09; P = .063) and gene knockdown mediated by small interfering RNA (0.67 +/- 0.14; P = .036). SMC proliferation decreased by 3.1-fold (P = .006) in the presence of CDH11-blocking antibody. Knockdown of CDH11 mediated by small interfering RNA resulted in a 1.3-fold (P = .018) decrease in proliferation. CONCLUSIONS: CDH11 is up-regulated in SMC in vivo and in vitro as part of the response to injury. Inhibition of CDH11 decreases SMC migration and proliferation, two pathogenic effectors of intimal hyperplasia.

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.

In vivo testing of an infection-resistant annuloplasty ring.

BACKGROUND: An infection-resistant surface incorporated into a prosthetic cardiac valve has great potential clinical applications. STUDY DESIGN: A sewing ring construct was created using ciprofloxacin-treated polyester. Then ciprofloxacin-treated and untreated constructs were implanted subcutaneously on the dorsum of rats and inoculated with Staphylococcus aureus. At 7, 14, and 30 days animals were sacrificed and the implants were retrieved. Each implant was assessed for frank purulence and gross tissue incorporation by a blinded observer. The implants were processed for conventional histology and examined by a blinded Pathologist. Ciprofloxacin-treated rings were also implanted in the absence of a bacterial challenge. At explantation, a maximal zone of inhibition, if present, was measured. Finally, ciprofloxacin was eluted with methanol from the explanted segments and the concentration of ciprofloxacin eluted was determined. RESULTS: Ciprofloxacin-treated sewing rings had greater gross tissue incorporation than untreated rings in the presence of a bacterial challenge (P=0.005). Ciprofloxacin-treated rings also had a lower incidence of frank purulence, but this did not reach statistical significance. After 14 days of implantation, ciprofloxacin treated rings had fewer neutrophils (P=0.018) and greater histological tissue incorporation (P=0.017) than untreated rings. The explanted ciprofloxacin-treated rings maintained a zone of inhibition of 3.0+/-1.0 mm after 1 day of implantation and 1.3+/-0.6 mm after 2 days. Ciprofloxacin could be eluted in significant quantities from the explanted rings after 7 days of implantation. CONCLUSIONS: Ciprofloxacin treated polyester can be incorporated into an annuloplasty ring construct that demonstrates excellent tissue incorporation and infection resistance. This study supports the use of this construct in the mitral position in a large animal model.