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Small-diameter vascular grafts perform poorly as arterial bypasses. We developed a cell-free, resorbable graft intended to remodel in situ into a living vessel. The graft consisted of a soft electrospun poly(glycerol sebacate) (PGS) core, a PGS prepolymer (pPGS) coating, and a reinforcing electrospun poly(e-caprolactone) (PCL) sheath. The core contained 4.37 ± 1.95 μm fibers and had a porosity of 66.4 ± 3.2%, giving it large pores to encourage cellular infiltration and pro-healing macrophages. The sheath contained 6.63 ± 0.89 μm fibers and had a porosity of 80.5 ± 2.1%. in vitro testing suggested that the stress achieved at arterial pressure would be 13-fold lower than the yield stress of the graft. Grafts were implanted as 7 cm carotid interpositions in two sheep. Sheep were maintained on dual antiplatelet therapy and followed with duplex ultrasound. One graft ruptured at 13 days. The second animal was euthanized with a dilated graft at 15 days. Histology showed near-total degradation of the core and a robust inflammatory response within the sheath. Little neotissue had formed within the graft wall or lumen, but the graft had become surrounded by fibroblast-rich and vascularized connective tissue. Because PCL is commonly used in resorbable grafts, this mechanical destabilization was unexpected. We speculate that the inflammatory response instigated by the rapidly degrading PGS intensified degradation of the PCL and that the large pores enabled a prolonged acute host-graft reaction which attacked the entire bulk of the material, speeding weakening. Future work will focus on how to moderate inflammation and improve remodeling of grafts in large animals.

Importance Smoking is a key modifiable risk factor in the development and progression of peripheral artery disease, which often manifests as intermittent claudication (IC). Smoking cessation is a first-line therapy for IC, yet a minority of patients quit smoking prior to elective revascularization. Objective To assess if preprocedural smoking is associated with an increased risk of early postprocedural complications following elective open and endovascular revascularization. Design, Setting, and Participants This retrospective cohort study used nearest-neighbor (1:1) propensity score matching of 2011 to 2019 data from the Veterans Affairs Surgical Quality Improvement Program, including all cases with a primary diagnosis of IC and excluding emergent cases, primary procedures that were not lower extremity revascularization, and patients with chronic limb-threatening ischemia within 30 days of the intervention. All data were abstracted June 18, 2020, and analyzed from July 26, 2020, to June 30, 2021. Exposures Preprocedural cigarette smoking. Main Outcomes and Measures Any and organ system-specific (ie, wound, respiratory, thrombosis, kidney, cardiac, sepsis, and neurological) 30-day complications and mortality, overall and in prespecified subgroups. Results Of 14 350 included cases of revascularization, 14 090 patients (98.2%) were male, and the mean (SD) age was 65.7 (7.0) years. A total of 7820 patients (54.5%) were smoking within the preprocedural year. There were a total of 4417 endovascular revascularizations (30.8%), 4319 hybrid revascularizations (30.1%), and 5614 open revascularizations (39.1%). A total of 1594 patients (11.1%) had complications, and 57 (0.4%) died. Among 7710 propensity score-matched cases (including 3855 smokers and 3855 nonsmokers), 484 smokers (12.6%) and 34 nonsmokers (8.9%) experienced complications, an absolute risk difference (ARD) of 3.68% (95% CI, 2.31-5.06; P

Objective Assess the relationships between case total work relative value units (wRVU), patient frailty, and the physiologic stress of surgical interventions. Summary of background data Surgeon reimbursement is frequently apportioned by wRVU. These subjective, procedure-specific valuations generated by physician survey estimate the intensity and time for typical patient care services. We hypothesized wRVU would not adequately account for patient-specific factors, such as frailty, that modify the required physician work, regardless of procedural complexity. Methods Using National and Veterans Affairs Surgical Quality Improvement Programs (2015-2018), we evaluated the correlation between case total wRVU, patient frailty (risk analysis index) and physiologic surgical stress (operative stress score). Results Of 4,111,371 (86%) cases, the correlation between total wRVU and operative stress was moderate [ρs = 0.587 (95% confidence interval, 0.586-0.587)], but negligible with frailty ρ = 0.177 (95% confidence interval, 0.176-0.178)]. Very high operative stress procedures [n = 34,047 (1%)] generated a mean total wRVU of 55.1 (standard deviation, 12.9), comprising 7%, 2%, and 1% of thoracic, vascular, and general surgical cases, respectively. Very frail patients [n = 152,535 (4%)] accounted for 9% of thoracic, 9% of vascular, 4% of general, 5% of urologic, and 4% of neurologic surgical cases, generating 21.0 (standard deviation, 12.4) mean total wRVU. Some nonfrail patients undergoing low operative stress procedures [n = 60,128 (2%)] nonetheless generated the highest quintile wRVU; these comprised >15% of plastic, gynecologic, and urologic surgical cases. Conclusions Surgeon reimbursement correlates with operative stress but not patient frailty. The total wRVU does not adequately reflect patient-specific factors that increase the physician workload required to render optimal care to complex patients.

Background While the perioperative stroke rate after carotid endarterectomy (CEA) is low, “silent” microinfarctions identified by magnetic resonance imaging (MRI) are common and have been correlated with postoperative neurocognitive decline. Our study will investigate the role of remote ischemic preconditioning (RIPC) as a potential neuroprotective mechanism. RIPC is a well-tolerated stimulus that, through neuronal and humoral pathways, generates a systemic environment of greater resistance to subsequent ischemic insults. We hypothesized that patients undergoing RIPC before CEA will have improved postoperative neurocognitive scores compared with those of patients undergoing standard care. Methods Patients undergoing CEA will be randomized 1:1 to RIPC or standard clinical care. Those randomized to RIPC will undergo a standard protocol of 4 cycles of RIPC. Each RIPC cycle will involve 5 min of forearm ischemia with 5 min of reperfusion. Forearm ischemia will be induced by a blood pressure cuff inflated to 200 mm Hg or at least 15 mm Hg higher than the systolic pressure if it is >185 mm Hg. This will occur after anesthesia induction and during incision/dissection but before manipulation or clamping of the carotid; thus, patients will be blinded to their assignment. Before carotid endarterectomy, all patients will undergo baseline neurocognitive testing in the form of a Montreal Cognitive Assessment (MoCA) and National Institutes of Health (NIH) Toolbox. MoCA testing only will be conducted on postoperative day 1 in the hospital. The full neurocognitive testing battery will again be conducted at 1-month follow-up in the office. Changes from baseline will be compared between arms at the follow-up time points. Assuming no drop-ins or dropouts and a 10% loss to follow-up, we would need a sample size of 43 patients for 80% power per treatment arm. The primary endpoint, change in MoCA scores, will be analyzed using a random effects model, and secondary outcomes will be analyzed using either linear or logistic regression where appropriate. Conclusions RIPC, if shown to be effective in protecting patients from neurocognitive decline after CEA, represents a safe, inexpensive, and easily implementable method of neuroprotection.

Introduction Abdominal aortic aneurysm (AAA) repair may be performed through open or endovascular approaches, but the factors influencing a patient's repair-type preference are not well characterized. Here we performed a qualitative analysis to better understand factors influencing patient preference within the Preference for Open Versus Endovascular Repair of AAA Trial. Methods Open-ended responses regarding primary (n = 21) and secondary (n = 47) factors influencing patient preference underwent qualitative analysis using the constant comparative method with iterative reviews. Codes were used to generate themes and themes grouped into categories, with each step conducted via consensus agreement between three researchers. Relative prevalence of themes were compared to ascertain trends in patient preference. Results Patient responses regarding both primary and secondary factors fell into four categories: Short-term concerns, long-term concerns, advice & experience, and other. Patients most frequently described short-term concerns (23) as their primary influence, with themes including post-op complications, hospitalization & recovery, and intraoperative concerns. Long-term concerns were more prevalent (20) as secondary factors, which included themes such as survival, and chronic management. The average age of patients voicing only long-term concerns as a primary factor was 11 years younger than those listing only short-term concerns. Conclusion Short-term concerns relating to the procedure and recovery are more often the primary factor influencing patient preference, while long term concerns play a more secondary role. Long-term concerns are more often a primary factor in younger patients. Vascular surgeons should consider this information in shared decision making to reach an optimal outcome.

Venous leg ulceration occurs secondary to venous hypertension and dysregulation of the normal wound healing process. These wounds have a significant socioeconomic impact, as they cause substantial patient morbidity and cost the health-care system billions of dollars. Despite a wide array of therapeutic options, current wound care strategies have met with limited success. Stem cells offer the potential to restore the conditions of physiologic wound healing due to their pluripotent capabilities and the release of trophic factors that can modulate key events in the wound healing cascade. Multiple preclinical and clinical trials have demonstrated that stem cell therapy can accelerate wound healing in diabetic wounds, but little is really known about their benefit in venous wounds. Further research is also necessary to better define the ideal stem cell type, source, and method of delivery in order to maximize their therapeutic potential. Larger clinical trials will be necessary to prove their efficacy and safety in all types of wound repair.

Objective Vascular surgeon scientists shape the future of our specialty through rigorous scientific investigation and innovation in clinical care as well as train the next generation of surgeon-scientists. The Society for Vascular Surgery Foundation (SVSF) supports the development of surgeon-scientists through the Mentored Research Career Development Award (SVSF-CDA) Program, providing supplemental funds to recipients of NIH K08/K23 grants. The ongoing success of this mission was evaluated. Methods Curriculum vitae of the 41 recipients of the SVSF supplemental funding between 1999-2021 were collected and reviewed to evaluate academic achievements to define the programmatic accomplishments, return on investment, and to identify areas for strategic improvement. Results For nearly 22 years, the SVSF awarded supplemental funds for 31 K08 and 10 K23 grants to SVS members from 32 institutions. Thirty-four have completed K-funding while 7 are still being supported. Eleven (27%) awardees have been female including 6 (75%) of the current awardees. However, there has only been little ethnic/racial diversity in the program. Awardees obtained K-funding approximately 4 years after becoming faculty. Eleven awardees (27%) were supported by Howard Hughes, NIH F32, or T32 grants during training. To date, the SVSF has committed $12 million to the SVSF-CDA Program. Among the 34 who have completed their K-funding, 21 (62%) successfully obtained NIH R01, Veterans Affairs, or Department of Defense funding. The awardees have secured over $114M in federal funding, representing a 9.5-fold financial return on investment for the SVSF. In addition to research endeavors, 11 awardees (27%) hold endowed professorships and 19 (46%) have secured tenure at their institutions. Many of the awardees hold or have held leadership positions including 18 (44%) Division Chiefs, 11 (27%) Program Directors, 5 (12%) Chairs of Departments of Surgery, and one (2%) Dean. Eleven (27%) have served as president of a regional or national society and 24 (59%) participate in NIH study sections. Of the 34 who have completed their K-funding, 15 (44%) continue to maintain active independent research funding. Conclusion The SVSF-CDA Program is highly effective in the development of vascular surgeon-scientists who contribute to the leadership and growth of academic vascular surgery with a 9.5-fold return on investment. The number of female awardees has increased in recent years but ethnic/racial diversity remains poor. Despite the fact that 62% successfully transitioned to federal funding, less than half remained funded over time. Retention in research and increasing diversity for the awardees are major concerns and are important areas of strategic focus for the SVSF.

OBJECTIVE: We have previously shown that exogenous administration of the nuclear protein High Mobility Group Box 1 (HMGB1) improves angiogenesis following tissue ischemia. Antagonizing HMGB1 prolongs muscle necrosis and deters regeneration. In this study, we evaluated HMGB1 expression in peripheral arterial disease (PAD), and the mechanisms that promote its release in a murine model of hindlimb ischemia Specifically, we investigated how chloroquine (CQ), a commonly employed disease modifying anti-rheumatic drug promotes HMGB1 release from muscle. We hypothesized that CQ could increase HMGB1 locally and systemically, allowing it to mediate recovery from ischemic injury. METHODS: Muscle biopsies were performed on patients undergoing lower extremity surgery for non-PAD related disease as well as for claudication and critical limb ischemia (CLI). Clinical symptoms and ankle brachial indices (ABIs) were recorded for each patient. HMGB1 was detected in muscle sections using immunohistochemical staining. Unilateral femoral artery ligation (FAL) was performed on both wildtype and inducible HMGB1 knockout mice (iHMGBKO). Wild-type mice were administered intraperitoneal CQ two weeks before and after FAL. Laser Doppler perfusion imaging was used to determine perfusion recovery (LDPI). Serum and tissue levels of HMGB1 were measured at designated time points. In vitro, cultured C2C12 myoblasts were treated with increasing doses of CQ. HMGB1, autophagosome formation, p62/SQSTM1 accumulation, caspase-1 expression and activity and LDH levels were measured in supernatants and cell lysates. RESULTS: Nuclear expression of HMGB1 was prominent in patients with claudication and CLI (P

This cohort study uses data from the Veterans Affairs Surgical Quality Improvement Program database to examine the risk of early postoperative mortality among US veterans with a robust physiologic reserve undergoing open or endovascular abdominal aortic aneurysm repair.

Objective: Endovascular aortic repair (EVAR) has advanced the care of patients with ruptured abdominal aortic aneurysms (rAAA) with improved early postoperative morbidity and mortality. However, this comes at the cost of a rigorous postoperative surveillance schedule to monitor for further aneurysmal degeneration. Adherence to surveillance recommendations is known to be poor in the elective setting, but has yet to be studied in the ruptured population. The aim of this study is to investigate predictors of incomplete surveillance after EVAR for rAAA (rEVAR) and examine how adherence impacts outcomes. Methods: This was a retrospective case control study of patients undergoing rEVAR at a multiple hospital single healthcare center (2003-2020). Patients were excluded if they underwent open conversion during their index hospitalization or died within 60 days of surgery. Follow-up was broadly categorized as complete surveillance (60-day postoperative visit and annually thereafter) or incomplete surveillance, comprising both patients with less than recommended surveillance (minimal surveillance) and completely lost to follow-up (LTF). Any follow-up was defined as patients with complete or minimal surveillance. We investigated predictors of complete versus incomplete surveillance by multivariate logistic regression. Secondary outcomes included overall survival and cumulative incidence of reintervention controlling for the competing risk of mortality, generating hazard ratios (HR) and subdistribution hazard ratios (SHR). Results: One-hundred and sixty patients (mean age 74±10.1 years, 81.2% male) out of 673 total rAAA met study inclusion criteria. Complete surveillance was seen in 41.3% of our cohort, with the remainder with minimal surveillance (29.4%) or LTF (29.4%). Incomplete surveillance was associated with male sex (odds ratio [OR] 2.56; 95% CI 1.02-6.43), lack of a primary care provider (PCP; OR 0.20; 95% CI 0.04-0.99), and longer driving distance from home to treating hospital (OR 2.37; 95% CI 1.08-5.20). Survival was not different between complete and incomplete surveillance groups, however any follow-up conferred improved survival over LTF (HR 0.57; 95% CI 0.331-0.997; P=0.049). Reintervention was associated with incomplete surveillance (SHR 0.29; 95% CI 0.11-0.75), and discharge to a facility (SHR 0.25; 95% CI 0.067-0.94). Conclusions: Incomplete surveillance was observed in over 50% of patients who underwent rEVAR and was associated with male sex, lack of PCP, and longer driving distance. Any follow-up conferred a survival benefit, yet incomplete surveillance was associated with a lower risk of reintervention. Targeted strategies to prevent LTF, and less stringent, personalized follow-up plans that may confer similar survival benefit with better patient adherence should be investigated.

BACKGROUND: The durability of endovascular aneurysm repair (EVAR) when compared to open surgical repair (OSR) in younger patients for elective, infrarenal abdominal aortic aneurysms (AAA) remains unclear due to limited follow up. STUDY DESIGN: We identified all patients

Objective Shared medical decision making is most important when there are competing options for repair such as in treatment of abdominal aortic aneurysm (AAA). We sought to understand the sources of patients' pre-existing knowledge about AAA to better inform treating physicians about patients' needs for preoperative counseling. Methods We performed a multicenter survey of patients facing AAA repair at 20 Veterans Affairs hospitals across the United States as part of the Preferences for Open Versus Endovascular Repair of AAA study. A validated survey instrument was administered to examine the sources of information available and commonly used by patients to learn about their repair options. The survey was administered by study personnel before the patient had any interaction with the vascular surgeon because survey data were collected before the vascular clinic visit. Results Preliminary analysis of data from 99 patients showed that our cohort was primarily male (99%) and elderly (mean age 73 years). Patients commonly had a history of hypertension (86%), prior myocardial infarction (32%), diabetes (32%), and were overweight (58%). Patients arrived at their surgeon's office appointment with limited information. A majority of patients (52%) reported that they had not talked to their primary care physician at all about their options for AAA repair, and one-half (50%) reported that their view of the different surgical options had not been influenced by anyone. Slightly less than one-half of patients reported that they did not receive any information about open surgical aneurysm repair and endovascular aortic aneurysm repair (41% and 37%, respectively). Few patients indicated using the internet as their main source of information about open surgical aneurysm repair and endovascular aortic aneurysm repair (10% and 11%, respectively). Conclusions Patients are commonly referred for AAA repair having little to no information regarding AAA pathology or repair options. Fewer than one in five patients searched the internet or had accessed other sources of information on their own. Most vascular surgeons should assume that patients will present to their first vascular surgery appointment with minimal understanding of the treatment options available to them.

The vast accomplishments of the US Department of Veterans Affairs (VA) during the past century have contributed to the advancement of medicine and benefited patients worldwide. This article highlights some of those accomplishments and the advantages in the VA system that promulgated those successes. Through its affiliation with medical schools, its formation of a structured research and development program, its Cooperative Studies Program, and its National Surgical Quality Improvement Program, the VA has led the world in the progress of health care. The exigencies of war led not only to the organization of VA health care but also to groundbreaking, landmark developments in colon surgery; surgical treatments for vascular disease, including vascular grafts, carotid surgery, and arteriovenous dialysis fistulas; cardiac surgery, including implantable cardiac pacemaker and coronary artery bypass surgery; and the surgical management of many conditions, such as hernias. The birth of successful liver transplantation was also seen within the VA, and countless other achievements have benefited patients around the globe. These successes have created an environment where residents and medical students are able to obtain superb education and postgraduate training and where faculty are able to develop their clinical and academic careers.

Arteriogenesis represents the maturation of preformed vascular connections in response to flow changes and shear stress. These collateral vessels can restore up to 60% of the native blood flow. Shear stress and vascular injury can induce the release of nucleotides from vascular smooth muscle cells and platelets that can serve as signaling ligands, suggesting they may be involved in mediating arteriogenesis. The P2Y2 nucleotide receptor (P2Y2R) has also been shown to mediate smooth muscle migration and arterial remodeling. Thus, we hypothesize that P2Y2R mediates arteriogenesis in response to ischemia.Hind limb ischemia was induced by femoral artery ligation (FAL) in C57Bl/6NJ or P2Y2R negative mice (P2Y2(-/-)). Hind limb perfusion was measured with laser Doppler perfusion imaging and compared with the sham-operated contralateral limb immediately and at 3, 7, 14, 21, and 28 days after ligation. Collateral vessel size was measured by Microfil casting. Muscle specimens were harvested and analyzed with immunohistochemistry for Ki67, vascular cell adhesion molecule, macrophages, and muscle viability by hematoxylin and essoin stain.Hind limb ischemia induced by FAL in C57Bl/6NJ mice resulted in significant ischemia as measured by laser Doppler perfusion imaging. There was rapid recovery to nearly normal levels of perfusion by 2 weeks. FAL in P2Y2(-/-) mice resulted in severe ischemia with greater tissue loss. Recovery of perfusion was impaired, achieving only 40% compared with wild-type mice by 28 days. Collateral vessels in the P2Y2(-/-) mice were underdeveloped, with reduced vascular cell proliferation and smaller vessel size. The collaterals were ∼65% the size of wild-type collateral vessels (P = .011). Angiogenesis at 28 days in the ischemic muscle, however, was greater in the P2Y2(-/-) mice (P.001), possibly related to persistent ischemia leading and angiogenic drive. Early macrophage recruitment was reduced by nearly 70% in P2Y2(-/-) despite significantly more myocyte necrosis. However, inflammation was greater at 28 days in the P2Y2(-/-) mice.P2Y2R deficiency does not alter baseline collateral vessel formation but does significantly impair collateral maturation, with resultant persistent limb ischemia despite enhanced angiogenesis. These findings reinforce the importance of arteriogenesis in the recovery of perfusion in ischemic tissues compared with angiogenesis. They also support the role of P2Y2R in mediating this process. The mechanism by which P2Y2R mediates arteriogenesis may involve the recruitment of inflammatory cells to the ischemic tissues, which is essential to arteriogenesis. Approaches to target P2Y2R may yield new therapeutic strategies for the treatment of arterial occlusive disease.

OBJECTIVES: Initial data on drug-eluting stents (DES) shows that they may increase the durability of endovascular treatment of superficial femoral artery disease compared with traditional bare-metal stents (BMS). Observed decreased target lesion revascularization (TLR) rates have potential for cost savings despite an increased initial cost. The purpose of this study was to run a simulation model of progressive transition from BMS to DES over 5 years evaluating the overall cost impact of that transition. METHODS: Florida State Ambulatory Databases were searched for all patients undergoing superficial femoral artery stenting in 2013 using Current Procedural Terminology codes 37226 and 37227. A simulation model was developed to estimate the impact of a progressive transition from BMS to DES over a 5-year horizon in this patient population. Cost estimates were determined from available cost charge ratio data. For the 5-year model, 2013 served as the initial year with each subsequent year based on the expected number of interventions per year. Up to one TLR per patient was assumed for the model. The 5-year TLR rates for DES and other parameter estimates were based on pooled data from the literature. Institutional data were used to estimate that up to 48% of superficial femoral artery lesions would fit the instructions for use for the Zilver PTX (Cook Medical, Bloomington, Ind), which is currently the only DES approved by the U.S. Food and Drug Administration for peripheral interventions. The net budget impact was expressed as the difference in total costs (primary stenting and reinterventions) for a scenario where BMS is progressively replaced by Zilver PTX compared with a scenario of BMS only. Multiple sensitivity analyses were performed on the base scenario. RESULTS: We identified 4107 peripheral interventions in the first year that fit our study. The overall cost for these procedures in Florida database was $51,362,142.00. In the base case scenario, DES was introduced slowly into the population at a rate of 8% per year up to 48% at the end of the model. This strategy resulted in an overall cost savings of $1,688,953.72 compared with the model with BMS alone. Sensitivity analyses including slower adoption of DES up to only 24% at 5 years, a 20% increase in TLR rates per year for the DES, and a 10% reduction in TLR rates per year for BMS still resulted in a net savings. As long as the additional cost of a DES compared with BMS is less than $677, the DES model remains less expensive. CONCLUSIONS: The adoption of DES in lieu of traditional BMS can lead to significant cost savings in a single state model over a short time horizon. (J Vasc Surg 2018;68:1465–72.)

Purinergic signaling plays a role in vessel remodeling among developing collateral arteries. We have previously shown that the P2Y 2 receptor, an ATP/UTP receptor, mediates arteriogenesis in a murine model of hindlimb ischemia. Intra-arterial administration of UTP increases perivascular inflammation and macrophage accumulation, necessary early events of arteriogenesis, an effect which was abolished among P2Y 2 -/- mice. Extracellular nucleotides can be dephosphorylated by ectonuclotidases which are expressed in various tissues, converting ATP/UTP into ADP/UDP. Our objective was to identify whether receptors for these derivative nucleotides would also have a role in arteriogenesis. Muscular branches of the caudal femoral artery were explanted from rat and assessed for purinergic receptor expression by immunofluorescence. Baseline expression of P2Y 2 is found among endothelial cells predominantly. Conversely, the P2Y 6 receptor is highly expressed in the media, with no appreciable staining in the endothelium. CD39, an ectonucleotidase, is expressed among VSMCs in developing collateral arteries (Figure). We then sought to identify whether inhibition of the P2Y 6 receptor, the ligand for which is UDP, would alter perfusion recovery after femoral artery ligation (FAL). Selective P2Y 6 receptor antagonist MRS2578 was intraperitoneally delivered via osmotic minipump at a rate of 20 ng/hr. FAL was performed and perfusion recovery monitored over 14 days using laser Doppler perfusion imaging (LDPI). Perfusion recovery was reduced in animals treated with MRS2578 when compared to vehicle (DMSO) at 7 and 14 days (figure). Two (out of 12) animals receiving MRS2578 were euthanized due to gangrene of the hindlimb. We have demonstrated in this work that the P2Y 6 receptor is another mediator of arteriogenesis. Our results also suggest an interesting spatial localization of purinergic receptors among resident vascular cells which may facilitate their function in arteriogenesis.

Introduction: Nitric oxide (NO) is required for cutaneous wound healing. Impaired diabetic wound healing has been linked to a deficiency in local NO production and can be enhanced with NO delivery. NO is a short-lived, highly reactive molecule and local delivery is complicated by these properties. An alternate source of NO can be achieved through the ability of nitrite reductases to convert the stable NO end-product, nitrite, back to NO. We have previously demonstrated that skin and wound edge express high levels of xanthine oxidoreductase (XOR). XOR is a strong nitrite reductase. We hypothesize that dietary nitrite supplementation will improve wound healing in diabetic mice. Methods: Db/db mice (N>8/group) were pretreated for 1 week with sodium nitrite supplemented drinking water (50 mg/L), nitrite-free chow, or standard chow. Additionally, nitrite supplemented mice were gavaged with nitrite supplemented water (0.2 ml) every other day for the duration of the experiment. A 1 cm 2 excisional wound was created on the back of each mouse. Wounds were photographed every other day until closure and wound areas calculated with ImageJ and compared with ANOVA and Kaplan Meier. Results: Time to complete healing was significantly different between nitrite supplemented (NS), nitrite depleted (ND), and control mice (18.9±0.7, 21.6±3.3, and 23±3.5 days, respectively, P = 0.035). NS mice reached 75% and 100% healed faster than ND or control mice (P = .009 and P = .042, respectively) [Figure]. Initial wound expansion on day 2 was decreased in NS mice compared to ND mice. XOR activity was increased in wounds compared to skin but similar between all treatment groups. Conclusion: Nitric oxide is a critical component of wound healing. Oral systemic nitrite supplementation improves diabetic wound healing in mice. Dietary nitrite may be an inexpensive and safe method of augmenting NO production through wound XOR expression to improve wound repair.

Intimal hyperplasia (IH) contributes to the failure of vascular interventions. While many investigational therapies inhibit the development of IH in animal models, few of these potential therapies can reverse established lesions. Inhaled carbon monoxide (CO) dramatically inhibits IH in both rats and pigs when given perioperatively. It also prevented the development of pulmonary arterial hypertension in rodents. Interestingly, CO could reverse pulmonary artery structural changes and right heart hemodynamic changes when administered after the establishment of pulmonary hypertension. Thus, we hypothesize that inhaled CO may mediate the regression of established neointimal lesions.Rats underwent carotid artery balloon angioplasty injury. Carotid arteries were collected at 2 and 4 weeks after injury for morphometric analysis of the neointima. Another group was treated with inhaled CO (250 parts per million) for 1 hour daily from week 2 until week 4. Additional rats were sacrificed 3 days after initiating CO treatment, and the carotid arteries were examined for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end-labeling, proliferation by Ki67 staining, and autophagy by microtubule-associated protein light chain 3 I/II staining.At 2 weeks following injury, sizable neointimal lesions had developed (intimal/media = 0.92 ± 0.22). By 4 weeks, lesion size remained stable (0.80 ± 0.09). Delayed inhaled CO treatment greatly reduced neointimal lesion size vs the 2- and 4-week control mice (0.38 ± 0.05; P.05). Arteries from the CO-treated rats exhibited significantly reduced apoptosis compared with control vessels (3.18% ± 1.94% vs 16.26% ± 5.91%; P = .036). Proliferation was also dramatically reduced in the CO-treated animals (2.98 ± 1.55 vs 10.37 ± 2.80; P = .036). No difference in autophagy between control and CO-treated rats was detected.Delayed administration of inhaled CO reduced established neointimal lesion size. This effect was mediated by the antiproliferative effect of CO on medial and intimal smooth muscle cells without increases in arterial wall apoptosis or autophagy. Future studies will examine additional time points to determine if there is temporal variation in the rates of apoptosis and autophagy.

Introduction: Millions of Americans are at risk for amputation from severe peripheral arterial disease (PAD) when surgery is not possible. Pro-regenerative and angiogenic agents may improve outcome in that setting. Chloroquine (CQ) promotes wound healing in scleroderma but has not been tested in PAD. CQ promotes healing of ischemic muscle, increases muscle high mobility group box 1 (HMGB1), an inflammatory, pro-angiogenic protein, and activates caspase-1 in myoblasts. We hypothesize that HMGB1 mediates protective effects of CQ and is regulated by caspase-1 in muscle. Controlled rather than indiscriminate release of HMGB1 from damaged muscle may be protective during ischemia. Methods: C2C12 myoblasts in low serum were treated with CQ (0-50μM) ± Ac-YVAD-cmk (10 μg/ml), a caspase-1 inhibitor. HMGB1 release in supernatants was measured using ELISA. Cytotoxicity was assessed by comparing spontaneous lactate dehydrogenase (LDH) activity in culture media from control, treated and maximally lysed cells. CQ (50μg/ml) or placebo treated wild-type and inducible HMGB1 knockout (iHMGB1KO) mice underwent unilateral femoral artery ligation (FAL). Laser Doppler perfusion imaging (LDPI) before and 1,7,14 and 21d after FAL was reported as % improvement over time. ANOVA was used to assess statistical significance among groups. Results: CQ (5-10uM) attenuated spontaneous LDH leak after 12h from serum-depleted myoblasts (p Conclusion: We present the novel finding that in nutrient-depleted myoblasts, caspase-1 mediates the survival benefits of CQ and regulates HMGB1 release. In turn, HMGB1 is critical for CQ’s beneficial effects on perfusion after FAL, another stress condition. Regulated HMGB1 release may be immunomodulatory, regenerative and modifiable with drugs like CQ. Altering survival and inflammatory pathways through CQ may present a novel therapeutic strategy in PAD.

Practice patterns associated with inferior vena cava (IVC) filter placement have seen considerable variation in the last decade. We used a statewide administrative database to examine trends in IVC filter placement and retrieval in the general population.We reviewed Florida state inpatient and ambulatory surgery databases from 2004 to 2014. International Classification of Diseases, Ninth Revision diagnosis and procedure codes and Current Procedural Terminology codes were searched for patients undergoing inpatient or outpatient IVC filter placement, and each patient was longitudinally tracked to the time of inpatient or outpatient filter retrieval. For inpatient filter placements, associated diagnoses were reviewed to identify indications for placement. Univariate and multivariate logistic regression models were constructed to identify factors associated with improved retrieval rates.During the 11-year period, 131,791 IVC filter placements were identified, with a 50% increase from 2004 to 2010 and a 24% decline from 2010 to 2014. Median age at filter placement was 71 years (interquartile range, 57-81 years). Mean follow-up after filter placement was 17.3 ± 25.5 months. Only 8637 filters (6.6%) were retrieved. The annual retrieval rate trended upward, from 3.4% in 2004 to 8.5% in 2013 (P .001). Median filter dwell time was 96.5 days (interquartile range, 44-178 days). Diagnoses associated with filter placement included venous thromboembolism (75.9%), trauma (35.0%), hemorrhage (29.9%), malignant disease (29.4%), and stroke (5.1%). Retrieval rates were highest in younger patients (34.0% in patients younger than 20 years) and lowest in Medicare patients (2.5%). In a multivariate logistic regression model, Medicare was associated with decreased retrieval rates (odds ratio, 0.33; 95% confidence interval, 0.31-0.35; P .001) after adjusting for age and associated diagnoses. Weaker risk factors included increased age, white race, and diagnoses of deep venous thrombosis, pulmonary embolism, and malignant disease. A trauma diagnosis was associated with improved retrieval. To further investigate the Medicare effect, a propensity score-matched model was created to better account for confounding effects. In this model, Medicare persisted as a risk factor for decreased filter retrieval (odds ratio, 0.43; 95% confidence interval, 0.40-0.46; P .001).IVC filter placements, after a substantial increase between 2004 and 2010, have been declining since 2010. Retrieval rates in the general population are steadily improving but continue to lag behind those described in center-specific literature. Increased age and Medicare as the primary payer are the strongest risk factors for lack of filter retrieval. Widespread improvements on a national scale are needed to improve the appropriateness of filter placements and to enhance filter retrieval rates.

The Second Joan L and Julius H Jacobson Promising Investigator Awardee, Edith Tzeng MD, FACS In 2005, the Surgical Research Committee of the American College of Surgeons was tasked with selecting the recipient of a newly established award, "The Joan L and Julius H Jacobson Promising Investigator Award." According to the Jacobsons, the award funded by Dr Jacobson should be given at least once every 2 years to a surgeon investigator at "the tipping point," who can demonstrate that his or her research shows the promise of leading to a significant contribution to the practice of surgery and patient safety. Every year, the Surgical Research Committee receives many excellent nominations and has the difficult task of selecting one awardee. The first awardee was Michael Longaker MD, FACS, who 10 years later reflected on the award and the impact it had on his career. 1 This year, Edith Tzeng, MD, FACS, the second Jacobson awardee, reflects on her 10-year journey after receiving the award. Dr Tzeng is now a national and international figure in the field of vascular surgery and has studied the effect of nitric oxide and carbon monoxide on intimal hyperplasia. Kamal MF Itani, MD, FACS and Leigh Neumayer, MD, FACS, on behalf of the Surgical Research Committee of the American College of Surgeons.

Background: Impaired diabetic wound healing, a source of significant disability, has been attributed to the reduced nitric oxide (NO) bioavailability and increased oxidative stress. We have previously reported that xanthine oxidoreductase (XOR), important in purine metabolism and reactive oxygen species synthesis, is highly expressed in regenerating skin and is required for normal healing. XOR is also expressed in diabetic wounds at similar or greater levels. Recently, XOR has been identified as a strong nitrite reductase and can generate NO from nitrite. Based on these findings, we hypothesize that topical nitrite applied to diabetic wounds may improve healing. Methods: Anesthetized diabetic db/db mice underwent excisional wounding. The wounds were treated with either Aquaphor with 2% sodium nitrite or Aquaphor alone and covered with a bioocclusive dressing. Wounds were photographed every other day for digital planimetry until day 28. Wounds were collected on day 28 and fixed, sectioned, and examined for proliferation (Ki67) and angiogenesis (CD31) by immunostaining. Results: Wounds in db/db mice treated with topical Aquaphor + nitrite displayed significantly accelerated healing compared to those treated with Aquaphor alone (Table). Aquaphor + nitrite also increased wound angiogenesis (29.3 ± 4.7 vs 13.2 ± 4.8, N=6-8/group; p Conclusion: Topical nitrite significantly accelerated wound healing in db/db mice. Nitrite treatment enhanced angiogenesis and keratinocyte proliferation in the wounds. These prohealing effects are likely mediated by XOR mediated conversion of nitrite to NO which can potentially divert XOR away from ROS production. Future studies will define the ability of nitrite to alter wound oxidative stress as well as the translation of this simple and safe therapy for human application.

Vascular disease is a leading cause of morbidity and mortality worldwide. Current vascular therapeutic interventions directed at diseased vessels are restricted in long-term efficacy by the development of intimal hyperplasia and the reformation of flow-limiting disease. The vascular injury and inflammation that ensues from the intervention, especially in the setting of an existing atherosclerotic vascular disease, results in further endothelial dysfunction and subsequent smooth muscle cell proliferation and migration. Although the etiology of intimal hyperplasia is multifactorial, impaired nitric oxide (NO) signaling has been implicated. The vasoprotective properties of NO have been intensely studied, and many investigations have focused on harnessing this biological system for therapeutic benefit. Continued studies investigate the role of impaired NO signaling via the classical arginine/NO synthase (NOS)/NO pathway in the setting of intimal hyperplasia. Furthermore, the possible protective effects of nitrate and nitrite-generated NO via non-NOS-mediated pathways to limit vascular injury have been recently appreciated and will likely prove to be an important vasoregulatory and vasoprotective signaling pathway.

Evidence for benefit of endovascular aneurysm repair (EVAR) over open surgical repair for de novo infrarenal abdominal aortic aneurysms (AAAs) in younger patients remains conflicting because of heterogeneous study populations and small sample sizes. The objective of this study was to compare perioperative and short-term outcomes for EVAR and open surgery in younger patients using a large national disease and procedure-specific data set.We identified patients 65 years of age or younger undergoing first-time elective EVAR or open AAA repair from the Vascular Quality Initiative (2003-2014). We excluded patients with pararenal or thoracoabdominal aneurysms, those medically unfit for open repair, and those undergoing EVAR for isolated iliac aneurysms. Clinical and procedural characteristics were balanced using inverse propensity of treatment weighting. A supplemental analysis extended the study to those younger than 70 years.We identified 2641 patients, 73% (n = 1928) EVAR and 27% (n = 713) open repair. The median age was 62 years (interquartile range, 59-64 years), and 13% were female. The median follow-up time was 401 days (interquartile range, 357-459 days). Unadjusted perioperative survival was 99.6% overall (open repair, 99.1%; EVAR, 99.8%; P .001), with 97.4% 1-year survival overall (open repair, 97.3%; EVAR, 97.4%; P = .9). Unadjusted reintervention rates were five (open repair) and seven (EVAR) reinterventions per 100 person-years (P = .8). After propensity weighting, the absolute incidence of perioperative mortality was 1% in both groups (open repair, 0.9%, EVAR, 0.2%; P .001), and complication rates were low. Propensity-weighted survival (hazard ratio, 0.88; 95% confidence interval, 0.56-1.38; P = .6) and reintervention rates (open repair, 6; EVAR, 8; reinterventions per 100 person-years; P = .8) did not differ between the two interventions. The analysis of those younger than 70 years showed similar results.In this study of younger patients undergoing repair of infrarenal AAA, 30-day morbidity and mortality for both open surgery and EVAR are low, and the absolute mortality difference is small. The prior published perioperative mortality and 1-year survival benefit of EVAR over open AAA repair is not observed in younger patients. Further studies of long-term durability are needed to guide decision-making for open repair vs EVAR in this population.

Arteriogenesis (collateral artery development) is an adaptive pathway critical for salvage of tissue in the setting of arterial occlusion. Rodent models of arteriogenesis typically involve an experimental occlusion (ligation) of a hindlimb artery and then rely on indirect measures such as laser Doppler perfusion imaging to assess blood flow recovery. Unfortunately, the more commonly utilized measures of distal tissue perfusion at rest are unable to account for hemodynamic and vasoactive variables and thus provide an incomplete assessment of collateral network capacity. We provide a detailed description of modifications to the commonly used model of femoral artery ligation. These serve to alter and then directly assess collateral network’s hemodynamic capacity. By incorporating an arteriovenous fistula distal to the arterial ligation, arterial growth is maximized. Hindlimb perfusion may be isolated to measure minimum resistance of flow around the arterial occlusion, which provides a direct measure of collateral network capacity. Our results reinforce that arteriogenesis is driven by hemodynamic variables, and it can be reliably augmented and measured in absolute terms. Using these modifications to a widely used model, functional arteriogenesis may be more directly studied.

In the past decade, carbon monoxide has lost its reputation as a toxic gas and has gained a following of scientists who now appreciate its potential therapeutic utility as an anti-inflammatory and cytoprotective agent. Its vasoprotective properties have also gained a tremendous amount of attention, especially its ability to inhibit intimal hyperplasia. This review will provide a historical perspective of carbon monoxide biology and summarize its function in disease modulation with a special focus on its potential role as a vascular therapeutic.

Objectives: Arteriogenesis is a process by which small resistance vessels mature into large caliber collateral arteries in response to arterial occlusive disease. Inflammatory cells of monocyte/macrophage lineage are necessary to promote vascular remodeling. We have previously reported that mice lacking the P2Y2 receptor, a purinergic receptor whose agonists include ATP and UTP, exhibited impaired collateral development and persistently reduced perfusion when subjected to femoral artery ligation (FAL). Thus, we hypothesize that purinergic signaling via the P2Y2 receptor mediates inflammatory cell recruitment during arteriogenesis. Methods: C57Bl6 mice underwent FAL and intra-arterial infusion of 200uL of PBS or 100uM UTP in PBS (N=3 each group). Hindlimb perfusion was assessed with laser Doppler imaging at day 7. In a separate experiment, P2Y2 -/- mice underwent femoral artery ligation alongside C57Bl/6 (wild type receptor) mice without UTP infusion. Thigh adductor muscles were fixed, sectioned, and immunostaining was performed for inflammatory cells (anti-CD45) and macrophages/monocytes (anti-F4/80). Two collateral vessels were analyzed per animal, and positive cells were quantified per high-power field. Results: UTP treated mice showed a trend toward increased peri-collateral CD45 positive cells at 12hr, and significantly increased F4/80 positive cells at day 3 (18.3+/-3.3 vs 11.5+/-1.3, p=0.044). This corresponded to improved perfusion at 7 days in UTP treated mice (58.5%+/-3.9 vs 36.0%+/-2.8, p=0.026). P2Y2 -/- mice recruited fewer F4/80 positive cells to the collateral vessels at day 3 when compared to the wild type (1.8 ± 1.3 vs 11.8 ± 2.9, p=.012). Conclusion: UTP treatment increases macrophage recruitment to the immature collateral vessel wall and improves perfusion recovery following FAL. In the absence of P2Y2 receptor, macrophage recruitment was significantly inhibited. These findings suggest that purinergic signaling via the P2Y2 receptor is an important mediator of vascular inflammation which is required for collateral growth. Ongoing studies will focus on the cellular mechanisms involved and whether enhanced collateral maturation and limb perfusion can be driven by the pharmacologic administration of nucleotides.

Objective Vascular restenosis remains a major obstacle to long-term success after vascular intervention. Circulating progenitor cells have been implicated in restenosis, and yet it has remained unclear if these cells, particularly nonendothelial progenitors, have an active role in this pathologic process. We hypothesized that circulating CD34 + /c-kit + progenitors would increase after vascular injury, mirrored by changes in the injury signal, stromal cell-derived factor 1α (sdf1α). We further postulated that an antibody-based depletion would mitigate progenitor surge and, in turn, reduce restenosis in a murine model. Methods C57BL6 mice underwent wire injury of the femoral artery and were compared with mice with sham surgery and vessel ligation by flow cytometry as well as by sdf1α enzyme-linked immunosorbent assay of peripheral blood. Next, injured C57BL6 mice treated with a depleting antibody toward the progenitor marker sca-1 or with an isotype control were compared in terms of sdf1α as well as enumeration of progenitors. At 28 days, restenosis was quantified between sca-1- and isotype-treated animals. Results Wire injury generated an increase in sdf1α as well as a surge of CD34 + /c-kit + progenitors relative to nonsurgical controls ( P = .005). Treatment with sca-1 antibody ablated the peripheral surge compared with isotype-treated, injured animals ( P = .02), and sca progenitor depletion reduced the 28-day intima to media ratio in a statistically significant fashion compared with either nontreated ( P = .04) or isotype-treated ( P = .036) animals. Conclusions Our study has demonstrated that sca-1 antibody reduces both progenitor surge and vascular restenosis after endoluminal vascular injury in a murine model. This suggests that circulating progenitors play an active role in restenotic disease.

ObjectiveCarbon monoxide (CO) and nitric oxide (NO) have both been shown to possess vasoprotective properties. NO has successfully inhibited intimal hyperplasia in both small-animal and large-animal experimental models, whereas CO has only been studied in rodents. Evidence suggests that these two molecules may exert their vascular effects through common as well as unique signaling pathways. The purpose of this study was to determine the effect of a low concentration of inhaled CO on intimal hyperplasia in a large-animal model and if CO and NO treatment could exert a synergistic effect to inhibit this process.MethodsBalloon angioplasty was performed in a porcine model. Animals received inhaled CO (250 ppm) delivered preoperatively for 60 minutes or preoperatively and intraoperatively. Blood was collected for carboxyhemoglobin (COHgb) measurements at the start of the operation and every 30 minutes during the operation. Heart rate, respiratory rate, and oxygen saturation were monitored throughout. To study the effect of combined CO and NO treatment, another group of pigs received inducible NO synthase (iNOS) gene transfer in one iliac artery and control gene transfer (AdlacZ) in the contralateral iliac artery, with or without preoperative and intraoperative inhaled CO. Adenoviral infection was performed immediately after balloon injury. All animals were euthanized at 3 weeks, and iliac arteries were collected for histologic and morphometric analysis.ResultsOne hour of pretreatment with CO was associated with modest and transient elevations in COHgb levels, resulting in a 25.6% reduction in neointimal area and a 10% reduction in intimal area/medial area ratio (I/M) 3 weeks after injury (NS). In contrast, preoperative followed by intraoperative CO administration increased COHgb in a sustained fashion and inhibited neointima formation by 51.7% and I/M by 31% (P < .001). There was no evidence of toxicity associated with this administration of CO. The treatment of injured iliac arteries with the control adenoviral vector AdlacZ did not further increase the inhibitory effect of CO on intimal hyperplasia. The combination of inhaled CO and iNOS gene transfer resulted in greater protection, however, with a 64% reduction in neointimal area and a 48% reduction in I/M (P < .001).ConclusionsCO is an effective means of reducing intimal hyperplasia in large animals after vascular injury when delivered during the operative procedure. No toxicity was associated with the increase in COHgb. The combination of CO and NO provided additional protection against the vascular injury response, with a greater reduction in neointima formation. These data suggest that these agents may prove to be clinically beneficial in prolonging vascular patency after interventions.Clinical RelevanceIntimal hyperplasia is a significant clinical problem that greatly reduces the long-term patency of surgical bypasses and angioplasty/stent procedures. Carbon monoxide (CO) is an emerging tool in the prevention of intimal hyperplasia after vascular injury. We demonstrated in a porcine model of vascular injury that the preoperative and intraoperative administration of inhaled CO significantly reduced neointima formation. In addition, CO showed synergistic effects when combined with nitric oxide (NO) synthase gene therapy, a treatment that had been previously shown to significantly reduce intimal hyperplasia. The main clinical relevance of this study is that, to our knowledge, it is the first large-animal model study to show the benefit of inhaled carbon monoxide on vascular injury. Further studies are required to further hone the optimal dosing regimen of CO and the potential of combined CO/NO therapy.

Type 2 diabetes mellitus (DM) and the metabolic syndrome, both characterized by insulin resistance, are associated with an accelerated form of atherosclerotic vascular disease and poor outcomes following vascular interventions. These vascular effects are thought to stem from a heightened inflammatory environment and reduced bioavailability of nitric oxide (NO). To better understand this process, we characterized the vascular injury response in the obese Zucker rat by examining the expression of adhesion molecules, the recruitment of inflammatory cells, and the development of intimal hyperplasia. We also evaluated the ability of exogenous NO to inhibit the sequela of vascular injury in the metabolic syndrome. Obese and lean Zucker rats underwent carotid artery balloon injury. ICAM-1 and P-selectin expression were increased following injury in the obese animals compared with the lean rats. The obese rats also responded with increased macrophage infiltration of the vascular wall as well as increased neointima formation compared with their lean counterparts (intima/media = 0.91 vs. 0.52, P = 0.001). After adenovirus-mediated inducible NO synthase (iNOS) gene transfer, ICAM-1, P-selectin, inflammatory cell influx, and oxidized low-density lipoprotein (LDL) receptor expression were all markedly reduced versus injury alone. iNOS gene transfer also significantly inhibited proliferative activity (54% and 73%; P < 0.05) and neointima formation (53% and 67%; P < 0.05) in lean and obese animals, respectively. The vascular injury response in the face of obesity and the metabolic syndrome is associated with increased adhesion molecule expression, inflammatory cell infiltration, oxidized LDL receptor expression, and proliferation. iNOS gene transfer is able to effectively inhibit this heightened injury response and reduce neointima formation in this proinflammatory environment.

We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS overexpression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter-regulated human iNOS gene (Adeno-iNOS), or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS-transduced rats demonstrated human iNOS mRNA and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS-treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS-transduced rats, the expression of iNOS protein and exhaled nitric oxide concentrations were increased on days 1–4 and 7 but returned to baseline values by day 14. The administration of the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine dihydrochloride (l-NIL) decreased exhaled nitric oxide concentrations to levels found in Adeno-Control-transduced lungs. In a second group of rats, the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days after transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial, and venous vasoconstrictor responses to U-46619 compared with sham, Adeno-Control, and control groups. In a third set of experiments, the response to 400 nM U-46619 in the presence of 10 μM l-NIL was not different in the isolated lungs from Adeno-Control- and Adeno-iNOS-transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression, which attenuates the vascular constrictor responses to the thromboxane mimetic U-46619.

Background— The 42/44-kD mitogen-activated protein kinases (extracellular signal–regulated kinases, ERKs) regulate smooth muscle cell (SMC) cell-cycle progression and can either promote or inhibit proliferation depending on the activation status of the small GTPase RhoA. RhoA is involved in the regulation of the actin cytoskeleton and converges on multiple signaling pathways. However, the mechanism by which RhoA modulates ERK signaling is not well defined. The purpose of this investigation was to examine whether RhoA regulates ERK downstream signaling and cellular proliferation through its effects on the cytoskeleton and the nuclear localization of ERK. Methods and Results— Treatment of SMCs with Clostridia botulinum C3 exoenzyme, which inhibits RhoA activation, decreased SMC proliferation to 24±7% of that of controls and increased p21 Waf1/Cip1 transcription and protein levels. These effects of RhoA were reversed by inhibition of ERK phosphorylation. However, inactivation of RhoA did not alter levels of ERK phosphorylation but did increase nuclear localization of phosphorylated ERK. In addition, immunostaining demonstrated that phosphorylated ERK associated with the actin cytoskeleton, which was disrupted by C3 exoenzyme. Leptomycin B, an inhibitor of Crm1 that results in ERK nuclear accumulation, similarly increased p21 Waf1/Cip1 . Conclusions— RhoA inhibition increased levels of phosphorylated ERK in the cell nucleus. Inhibition of RhoA or pharmacological inhibition of nuclear export resulted in increased p21 Waf1/Cip1 expression and decreased SMC proliferation, effects that were partially dependent on ERK. RhoA regulation of the actin cytoskeleton may determine ERK subcellular localization and its subsequent effects on SMC proliferation.

OBJECTIVE To determine whether over-expression of nitric oxide synthase (NOS) in the corpus cavernosum of the penis improves erectile function, as NO is an important transmitter for genitourinary tract function, mediating smooth muscle relaxation and being essential for penile erection. MATERIALS AND METHODS The inducible form of the enzyme NOS (iNOS) was introduced into the corpus cavernosum of adult Sprague-Dawley rats (250–300 g) by injecting a solution of plasmid, adenovirus or adenovirus-transduced myoblast cells (adeno-myoblasts). Plasmid, adenovirus and adeno-myoblasts encoding the expression of the β-galactosidase reporter gene were also injected into rats. RESULTS Throughout the corpora cavernosum there was expression of β-galactosidase after injecting each of the three solutions. Maximum staining was greatest for adeno-myoblast, then adenovirus and then plasmid. The mean (sd) basal intracavernosal pressure (ICP) of iNOS-treated animals (adenovirus and adeno-myoblast) increased to 55 (23) cmH2O, compared with naive animals with a basal ICP of 5 (6) cmH2O (P = 0.001). Stimulating the cavernosal nerve (15 Hz, 1.5 ms, 10–40 V, 1 min) resulted in a doubling of the ICP (adenovirus and adeno-myoblast) from the basal level of the iNOS-treated animals. Direct in situ measurement of NO showed the release of 1–1.3 µmol/L in the adeno-myoblast penis. CONCLUSION Myoblast-mediated gene therapy was more successful for delivering iNOS into the corpus cavernosum than direct adenovirus injection or plasmid transfection. Surprisingly, implanting muscle cells into the penis is not only feasible but also beneficial. Gene therapy for NOS may open new avenues of treatment for erectile dysfunction. Control of NOS expression would be necessary to prevent priapism.

Objective: This study was undertaken to determine the effect of nitric oxide (NO) on tissue factor (TF) expression in vascular smooth muscle cells. Study Design: Rat aortic smooth muscle cells (RASMCs) were exposed to NO delivered exogenously with the NO donor S -nitroso- N -acetylpenicillamine (SNAP) or produced endogenously after infection with an adenoviral vector carrying human inducible NO synthase (AdiNOS). Functional TF activity was assessed with chromogenic TF assay. TF antigen was determined with immunohistochemistry. Northern blot analysis was used to determine steady- state TF messenger RNA (mRNA). Electrophoretic mobility gel shift assay was performed to determine the nuclear binding activity of nuclear factor κ-B (NFκB). NFκB activity was inhibited by either prior transduction of RASMCs with mutant IκB or treatment with pyrrolidine dithiocarbamate. Results: RASMCs exposed to SNAP or infected with AdiNOS exhibited increased functional TF activity and antigen. Regardless of the source of NO, a time-dependent and concentration-dependent increase in TF activity was observed. Steady-state TF mRNA levels were also increased by NO delivered via either method. NFκB nuclear binding activity was also increased by NO. Inhibition of NFκB activity by either pyrrolidine dithiocarbamate treatment or mutant IκB transduction abrogated NO-induced enhancement of TF mRNA and functional activity. Conclusion: In RASMC, NO exposure results in upregulation of TF functional activity, antigen, and mRNA. This effect appears to be mediated by an NFκB-dependent pathway. (J Vasc Surg 2003;37:650-9.)

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

Cardiovascular disease is the number one source of morbidity and mortality in the United States. Therapies directed at a variety of cardiovascular diseases have blossomed over the last several decades. The advent of gene therapy, first as an intriguing tool, and subsequently with the early successes of gene trials involving the treatment of SCID, led to the development of gene therapy as a potentially exciting and viable therapy in cardiovascular diseases. A variety of novel vector technologies and delivery systems have been developed to more efficiently deliver the gene product to the desired organ or tissue bed. Early clinical trials focused on stimulating angiogenesis. Subsequently, a number of other aspects of cardiovascular disease have been identified as potential targets for gene therapy, including the prevention of restenosis, the prevention and treatment of thrombosis, and the prevention of transplant vasculopathy. With over forty clinical human trials either completed or currently enrolling, cardiovascular gene therapy has proven to be safe and initial results suggest its efficacy.