Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

OBJECTIVE: This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies. METHODS: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×109 VG to 2×1011 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed. RESULTS: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2×1011 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. CONCLUSION: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×1011 VG aliquots injected IM.

Transforming the Future of Surgeon-Scientists.

OBJECTIVE: To create a blueprint for surgical department leaders, academic institutions, and funding agencies to optimally support surgeon-scientists. BACKGROUND: Scientific contributions by surgeons have been transformative across many medical disciplines. Surgeon-scientists provide a distinct approach and mindset toward key scientific questions. However, lack of institutional support, pressure for increased clinical productivity, and growing administrative burden are major challenges for the surgeon-scientist, as is the time-consuming nature of surgical training and practice. METHODS: An American Surgical Association Research Sustainability Task Force was created to outline a blueprint for sustainable science in surgery. Leaders from top NIH-sponsored departments of surgery engaged in video and in-person meetings between January and April 2023. A strength, weakness, opportunities, threats analysis was performed, and workgroups focused on the roles of surgeons, the department and institutions, and funding agencies. RESULTS: Taskforce recommendations: (1) SURGEONS: Growth mindset : identifying research focus, long-term planning, patience/tenacity, team science, collaborations with disparate experts; Skill set : align skills and research, fill critical skill gaps, develop team leadership skills; DEPARTMENT OF SURGERY (DOS): (2) MENTORSHIP: Chair : mentor-mentee matching/regular meetings/accountability, review of junior faculty progress, mentorship training requirement, recognition of mentorship (eg, relative value unit equivalent, awards; Mentor: dedicated time, relevant scientific expertise, extramural funding, experience and/or trained as mentor, trusted advisor; Mentee : enthusiastic/eager, proactive, open to feedback, clear about goals; (3) FINANCIAL SUSTAINABILITY: diversification of research portfolio, identification of matching funding sources, departmental resource awards (eg, T-/P-grants), leveraging of institutional resources, negotiation of formalized/formulaic funds flow investment from academic medical center toward science, philanthropy; (4) STRUCTURAL/STRATEGIC SUPPORT: Structural: grants administrative support, biostats/bioinformatics support, clinical trial and research support, regulatory support, shared departmental laboratory space/equipment; Strategic: hiring diverse surgeon-scientist/scientists faculty across DOS, strategic faculty retention/ recruitment, philanthropy, career development support, progress tracking, grant writing support, DOS-wide research meetings, regular DOS strategic research planning; (5) COMMUNITY AND CULTURE: Community: right mix of faculty, connection surgeon with broad scientific community; Culture: building research infrastructure, financial support for research, projecting importance of research (awards, grand rounds, shoutouts); (6) THE ROLE OF INSTITUTIONS: Foundation: research space co-location, flexible start-up packages, courses/mock study section, awards, diverse institutional mentorship teams; Nurture: institutional infrastructure, funding (eg, endowed chairs), promotion friendly toward surgeon-scientists, surgeon-scientists in institutional leadership positions; Expectations: RVU target relief, salary gap funding, competitive starting salaries, longitudinal salary strategy; (7) THE ROLE OF FUNDING AGENCIES: change surgeon research training paradigm, offer alternate awards to K-awards, increasing salary cap to reflect market reality, time extension for surgeon early-stage investigator status, surgeon representation on study section, focused award strategies for professional societies/foundations. CONCLUSIONS: Authentic recommitment from surgeon leaders with intentional and ambitious actions from institutions, corporations, funders, and society is essential in order to reap the essential benefits of surgeon-scientists toward advancements of science.

Fibroblasts in Diabetic Foot Ulcers.

Fibroblasts are stromal cells ubiquitously distributed in the body of nearly every organ tissue. These cells were previously considered to be "passive cells", solely responsible for ensuring the turnover of the extracellular matrix (ECM). However, their versatility, including their ability to switch phenotypes in response to tissue injury and dynamic activity in the maintenance of tissue specific homeostasis and integrity have been recently revealed by the innovation of technological tools such as genetically modified mouse models and single cell analysis. These highly plastic and heterogeneous cells equipped with multifaceted functions including the regulation of angiogenesis, inflammation as well as their innate stemness characteristics, play a central role in the delicately regulated process of wound healing. Fibroblast dysregulation underlies many chronic conditions, including cardiovascular diseases, cancer, inflammatory diseases, and diabetes mellitus (DM), which represent the current major causes of morbidity and mortality worldwide. Diabetic foot ulcer (DFU), one of the most severe complications of DM affects 40 to 60 million people. Chronic non-healing DFU wounds expose patients to substantial sequelae including infections, gangrene, amputation, and death. A complete understanding of the pathophysiology of DFU and targeting pathways involved in the dysregulation of fibroblasts are required for the development of innovative new therapeutic treatments, critically needed for these patients.

Novel Gene-Modified Mesenchymal Stem Cell Therapy Reverses Impaired Wound Healing in Ischemic Limbs.

OBJECTIVE: Here, we report a new method to increase the therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biological effects of MSCs modified with E-selectin, a cell adhesion molecule capable of inducing postnatal neovascularization, on a translational murine model. BACKGROUND: Tissue loss significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia. MSC-based therapeutics hold major promise for wound healing and therapeutic angiogenesis, but unmodified MSCs demonstrate only modest benefits. METHODS: Bone marrow cells harvested from FVB/ROSA26Sor mTmG donor mice were transduced with E-selectin-green fluorescent protein (GFP)/AAV-DJ or GFP/AAV-DJ (control). Ischemic wounds were created via a 4 mm punch biopsy in the ipsilateral limb after femoral artery ligation in recipient FVB mice and subsequently injected with phosphate-buffered saline or 1×10 6 donor MSC GFP or MSC E-selectin-GFP . Wound closure was monitored daily for 7 postoperative days, and tissues were harvested for molecular and histologic analysis and immunofluorescence. Whole-body DiI perfusion and confocal microscopy were utilized to evaluate wound angiogenesis. RESULTS: Unmodified MSCs do not express E-selectin, and MSC E-selectin-GFP gain stronger MSC phenotype yet maintain trilineage differentiation and colony-forming capability. MSC E-selectin-GFP therapy accelerates wound healing compared with MSC GFP and phosphate-buffered saline treatment. Engrafted MSC E-selectin-GFP manifest stronger survival and viability in wounds at postoperative day 7. Ischemic wounds treated with MSC E-selectin-GFP exhibit more abundant collagen deposition and enhanced angiogenic response. CONCLUSIONS: We establish a novel method to potentiate regenerative and proangiogenic capability of MSCs by modification with E-selectin/adeno-associated virus. This innovative therapy carries the potential as a platform worthy of future clinical studies.

Racial disparities in presentation and outcomes for endovascular abdominal aortic aneurysm repair.

OBJECTIVE: In the present study, we used a national database to identify racial differences in the presentation and outcomes for patients undergoing endovascular abdominal aortic aneurysm (AAA) repair (EVAR) and identified areas for improving their care. METHODS: We queried the EVAR-targeted National Surgical Quality Improvement Program database (2016-2019) to identify patients who had undergone EVAR for both ruptured and nonruptured AAAs. The patients were categorized according to race (White, Black, and Asian). Patients with a history of abdominal aortic surgery or an indication other than AAAs were excluded. The data was analyzed using the χ2 and Kruskal-Wallis tests, presented as frequencies and percentages or median and interquartile range (IQR) for categorical and continuous variables, respectively. RESULTS: We identified 3629 patients (16.6% female), including 3312 White (91.3%), 248 Black (6.8%), and 69 Asian (1.9%) patients. Black patients were more frequently women (27.0%) compared with White patients (15.9%) and were younger (median age, 71 years; IQR, 64-77 years) than White (median age, 73 years; IQR, 67-79 years) and Asian (median age, 76 years; IQR, 67-81 years) patients (P < .001 for both). The incidence of smoking, congestive heart failure, and dialysis dependency was highest for Black patients, and the incidence of obesity was lowest for Asian patients. The AAAs in Black patients extended more frequently beyond the aortic bifurcation (P = .047). In Asian patients, the internal iliac arteries were more involved (P = .040). For Black patients, 29.8% of the EVARs were performed in a nonelective setting compared with 20.2% for the White and 15.9% for the Asian patients (P < .001). The aneurysm diameter, nonruptured symptomatic rate, and rupture rate were similar across the groups (P = .807). The operative time was prolonged for Black (median, 128 minutes; IQR, 96-177 minutes) compared with White (median, 114 minutes; IQR, 84-162 minutes) patients (P < .001). Postoperatively, Black patients were more likely to require blood transfusion (16.5%) and had prolonged length of hospital stay (median, 2 days; IQR, 1-4 days) compared with White (10.0%; median, 1 day; IQR, 1-3 days) and Asian (4.3%; median, 1 day; IQR, 1-3 days) patients (P = .001 and P < .001, respectively). Black patients also had a higher 30-day readmission rate (P = .038). On multivariate analysis, Black race was an independent factor for length of stay >1 day after both elective and nonelective EVAR and 30-day readmission for elective EVAR, but not 30-day mortality after elective and nonelective EVAR. CONCLUSIONS: In the present nationwide sample of EVAR cases, Black patients were more often women and younger. Despite similar rates of symptomatic and ruptured AAAs at presentation and 30-day mortality, Black patients more often presented and were treated during the same nonelective admission; they also had associated increased length of hospital stay and readmission. These findings signal a missed opportunity to diagnose, optimize, and treat this particular group of patients in an elective setting.

Race-based outcomes of thoracic aortic aneurysms and dissections in the Global Registry for Endovascular Aortic Treatment.

OBJECTIVE: This study characterizes racial differences in presentation, as well as short- and long-term outcomes after endovascular treatment of thoracic aortic aneurysm (TAA) and type B aortic dissection (TBAD). METHODS: We queried the Gore Global Registry for Endovascular Aortic Treatment for thoracic endovascular aortic repairs (TEVARs) performed between 2010 and 2016 and followed through 2022. Pathologies represented were descending TAA, complicated TBAD, and uncomplicated TBAD. Using standard statistical tests, we compared overall and pathology-specific demographics, procedural factors, and outcomes among Black and White patients undergoing TEVAR. RESULTS: We identified 438 TEVAR cases, including 236 descending TAA, 121 complicated TBAD, and 74 uncomplicated TBAD. Overall, Black patients were younger and had a higher incidence of renal insufficiency (P = .001), whereas White patients had more chronic obstructive pulmonary disease (P = .003) and cardiac arrhythmias (P = .037). In patients treated for descending TAA, Black patients had increased device- and procedure-related complications (34.3% vs 17.4%; P = .014), conversion to open repair (2.9% vs 0%; P = .011) and type II endoleak (5.7% vs 1.0%; P = .040), but no differences in mortality, length of hospital stay, or major adverse cardiovascular events. Whereas outcomes of TEVAR for uncomplicated TBAD were comparable, Black patients more frequently presented with complicated TBAD than White patients (Black, 40.5% vs White, 24.8%; P = .008) and had subsequently greater reintervention rates (28.1% vs 12.4%; P = .012), all-cause mortality (hazard ratio, 4.28; 95% confidence interval, 1.74-10.5; P = .002) and aortic-related mortality (hazard ratio, 16.7; 95% confidence interval, 1.49-186; P = .022). CONCLUSIONS: Despite increased device- and procedure-related complications, similar short- and long-term outcomes are achieved in Black and White patients undergoing TEVAR for descending TAA and uncomplicated TBAD. However, Black patients are more likely to present with, require reintervention for, and suffer mortality from complicated TBAD.

Ischemic-Trained Monocytes Improve Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

OBJECTIVE: Monocytes, which play an important role in arteriogenesis, can build immunologic memory by a functional reprogramming that modifies their response to a second challenge. This process, called trained immunity, is evoked by insults that shift monocyte metabolism, increasing HIF (hypoxia-inducible factor)-1α levels. Since ischemia enhances HIF-1α, we evaluate whether ischemia can lead to a functional reprogramming of monocytes, which would contribute to arteriogenesis after hindlimb ischemia. METHODS AND RESULTS: Mice exposed to ischemia by 24 hours (24h) of femoral artery occlusion (24h trained) or sham were subjected to hindlimb ischemia one week later; the 24h trained mice showed significant improvement in blood flow recovery and arteriogenesis after hindlimb ischemia. Adoptive transfer using bone marrow-derived monocytes (BM-Mono) from 24h trained or sham donor mice, demonstrated that recipients subjected to hindlimb ischemia who received 24h ischemic-trained monocytes had remarkable blood flow recovery and arteriogenesis. Further, ischemic-trained BM-Mono had increased HIF-1α and GLUT-1 (glucose transporter-1) gene expression during femoral artery occlusion. Circulating cytokines and GLUT-1 were also upregulated during femoral artery occlusion.Transcriptomic analysis and confirmatory qPCR performed in 24h trained and sham BM-Mono revealed that among the 15 top differentially expressed genes, 4 were involved in lipid metabolism in the ischemic-trained monocytes. Lipidomic analysis confirmed that ischemia training altered the cholesterol metabolism of these monocytes. Further, several histone-modifying epigenetic enzymes measured by qPCR were altered in mouse BM-Mono exposed to 24h hypoxia. CONCLUSIONS: Ischemia training in BM-Mono leads to a unique gene profile and improves blood flow and arteriogenesis after hindlimb ischemia.

Effect of Body Mass Index on Early Outcomes of Endovascular Abdominal Aortic Aneurysm Repair.

BACKGROUND: This study compares the presentation, management, and outcomes of patients undergoing endovascular abdominal aortic aneurysm repair (EVAR), based on their weight status as defined by their body mass index (BMI). METHODS: Patients with primary EVAR for ruptured and intact abdominal aortic aneurysm (AAA) were identified in the National Surgical Quality Improvement Program database (2016-2019). Patients were categorized by weight status (underweight: BMI < 18.5 kg/m2, normal weight: 18.5-24.9 kg/m2, overweight: 25-29.9 kg/m2, Obese I: 30-34.9 kg/m2, Obese II: 35-39.9 kg/m2, Obese III: > 40 kg/m2). Preoperative characteristics and 30-day outcomes were compared. RESULTS: Of 3,941 patients, 4.8% were underweight, 24.1% normal weight, 37.6% overweight, and 22.5% with Obese I, 7.8% Obese II, and 3.3% Obese III status. Underweight patients presented with larger (6.0 [5.4-7.2] cm) and more frequently ruptured (25.0%) aneurysms than normal weight patients (5.5 [5.1-6.2] cm and 4.3%, P < 0.001 for both). Pooled 30-day mortality was worse for underweight (8.5%) compared to all other weight status (1.1-3.0%, P < 0.001), but risk-adjusted analysis demonstrated that aneurysm rupture (odds ratio [OR] 15.9, 95% confidence interval [CI] 8.98-28.0) and not underweight status (OR 1.75, 95% CI 0.73-4.18) accounted for increased mortality in this population. Obese III status was associated with prolonged operative time and respiratory complications after ruptured AAA, but not 30-day mortality (OR 0.82, 95% CI 0.25-2.62). CONCLUSIONS: Patients at either extreme of the BMI range had the worst outcomes after EVAR. Underweight patients represented only 4.8% of all EVARs, but 21% of mortalities, largely attributed to higher incidence of ruptured AAA at presentation. Severe obesity, on the other hand, was associated with prolonged operative time and respiratory complications after EVAR for ruptured AAA. BMI, as an independent factor, was however not predictive of mortality for EVAR.

Utility of Closed Suction Drains in Groin Incisions after Femoral Artery Exposure.

BACKGROUND: Surgical groin wounds are at risk of delayed healing and infection, leading to costly and prolonged postoperative recoveries. This study assesses the use of closed suction drains (CSDs) as a wound care adjunct in groin incisions to prevent surgical site infections (SSI). METHODS: A single-center retrospective review was performed on 210 consecutive patients after vascular surgery with common femoral artery exposure from 2016 to 2021. The cohort was divided into 2 groups, groins with and without CSD, looking for surgical site complications. A subgroup analysis comparing postoperative outcomes between complicated and uncomplicated groin incisions within both groups was also performed. RESULTS: Of 293 surgical groins, 20% (n = 59) had drains. Overall, the CSD group had higher SSI rates (14% vs. 5.6%), but also had higher proportion of smokers (92% vs. 83%; P = 0.019), diabetes (56% vs. 36%; P = 0.005), coronary artery disease (69% vs. 46%; P = 0.001), hyperlipidemia (69% vs. 51%; P = 0.01), and previous groin surgery (54% vs. 17%; P < 0.001). The higher risk of SSI was not significant after adjustment of these confounders. A separate analysis within each group showed SSI groins with CSD had lower reintervention rates (37.5%) than those without CSD (69%), as well as shorter length of hospital stay (7 [5-11] vs. 22 [7-25] days). CONCLUSIONS: Our study suggests that CSDs can be a beneficial adjunct for groin wounds after common femoral artery exposure in patients with comorbidities cited above. CSDs decrease the risk of reintervention and length of hospital stay.

Outcomes and role of shunting during carotid endarterectomy for symptomatic patients.

OBJECTIVE: Shunt placement during carotid endarterectomy (CEA) has often been advocated to protect the ischemic penumbra in patients with symptomatic carotid stenosis. In the present study, we assessed the effect of shunt placement during CEA on postoperative stroke risk in symptomatic patients. METHODS: We queried the American College of Surgeons National Surgical Quality Improvement Program database (2016-2019) for CEA cases with complete CEA procedure-targeted data available. Symptomatic patients were identified as those with a preoperative diagnosis of stroke on presentation (DS), transient ischemic attack, amaurosis fugax, or temporary monocular blindness. The DS patients were further analyzed according to the severity of their stroke using the modified Rankin scale scores. To better assess the effect of shunt placement on the stroke rate, we compared cases of CEA with the patch angioplasty technique stratified by the use of an intraoperative shunt. Patients who had undergone carotid eversion or primary closure were excluded. The baseline demographics and perioperative outcomes were compared using the χ2 and Mann-Whitney U tests. Multivariate analysis was performed to identify the independent risk factors for postoperative stroke and cranial nerve injury. RESULTS: We identified 4652 cases of CEA with patch angioplasty in symptomatic patients, including 1889 with (40.6%) and 2763 without (59.4%) shunt placement. The distribution of age, race, and sex was similar for both procedures. Compared with patients without a shunt, those with a shunt had significantly higher rates of emergency surgery (9.1% vs 7.0%; P = .010), nonelective surgery (40.3% vs 37.2%; P = .035), general anesthesia (97.0% vs 86.3%; P < .001), and bleeding disorders (27.2% vs 22.7%; P < .001). The 30-day incidence of postoperative stroke was similar between the patients with (3.2%) and without (2.6%) shunt placement (P = .219). Additionally, a subgroup analysis failed to show any benefit from shunt placement on the incidence of postoperative stroke, regardless of the preoperative symptoms or neurologic disability. In contrast, shunt placement was associated with an increased rate of cranial nerve injury (4.1% vs 2.4%; P = .001). Multivariate analysis revealed that nonelective surgery (odds ratio [OR], 1.99; 95% confidence interval [CI], 1.36-2.91; P < .001) and DS (vs transient ischemic attack, amaurosis fugax, or temporary monocular blindness; OR, 1.64; 95% CI, 1.12-2.41; P = .012) were predictive of 30-day postoperative stroke. After adjusting for confounders, shunt placement had no effect on stroke risk at 30 days but remained an independent risk factor for cranial nerve injury (adjusted OR, 1.87; 95% CI, 1.32-2.64; P < .001). CONCLUSIONS: For symptomatic patients undergoing CEA with patch angioplasty, shunt placement was associated with an increased risk of cranial nerve injury without a reduction in postoperative stroke risk.

COVID-19-related thrombotic complications experience before and during delta wave.

OBJECTIVE: Hypercoagulability and thrombotic complications seen in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the associated pathophysiology, have been reported extensively. However, there is limited information regarding the factors related to this phenomenon and its association with the Coronavirus disease 2019 (COVID-19) Delta variant. METHODS: A retrospective review including patients admitted to a tertiary center with a COVID-19 positive test and at least one acute thrombotic event confirmed by imaging between June 2020 and August 2021 was performed. We compared the rates of thrombotic events in patients with COVID-19 before and during the Delta peak. We also analyzed the association of the thrombotic complications with demographic characteristics, comorbidities, anticoagulation strategies, and prothrombotic markers while describing other complications secondary to COVID-19 infection. RESULTS: Of 964 patients admitted with COVID-19 diagnosis, 26.5% (n = 256) had a thrombotic event evidenced by ultrasound or computed tomography scan. Venous thromboembolism was found in 60% (n = 153), arterial thrombosis in 23% (n = 60), and both venous and arterial thromboses in 17% (n = 17) of the study cohort. Of all patients, 94% were not vaccinated. Delta variant wave (DW) patients had thrombotic episodes in 34.7% (n = 50/144) of cases compared with 25% (n = 206/820) of non-Delta wave (NDW) patients, posing an estimated risk 1.36 times higher in patients infected with COVID-19 during the DW than NDW. Overall, DW subjects were significantly younger (P < .001) with lower body mass index (P = .021) compared with NDW patients. Statistical analyses showed African American patients were more likely to have arterial thrombosis compared with the other groups when testing positive for COVID-19 (odds ratio [OR], 1.78; 95% confidence interval [CI], 1.04-3.05; P = .035, whereas immunosuppressed patients had less risk of arterial thrombosis (OR, 0.38; 95% CI, 0.15-0.96; P = .042). Female gender (OR, 2.15; 95% CI, 1.20-3.85; P = .009) and patients with active malignancy (OR, 5.99; 95% CI, 2.14-16.78; P = .001) had an increased risk of having multiple thrombotic events at different locations secondary to COVID-19. CONCLUSIONS: COVID-19 infection is associated with elevated rates of thrombotic complications and an especially higher risk in patients infected during the Delta variant peak. We highlight the importance of vaccination and the development of new anticoagulation strategies for patients with COVID-19 with additional hypercoagulable risk factors to prevent thrombotic complications caused by this disease.

A Genetic Model of Constitutively Active Integrin CD11b/CD18.

Pharmacological activation of integrin CD11b/CD18 (αMß2, Mac-1, and CR3) shows anti-inflammatory benefits in a variety of animal models of human disease, and it is a novel therapeutic strategy. Reasoning that genetic models can provide an orthogonal and direct system for the mechanistic study of CD11b agonism, we present in this study, to our knowledge, a novel knock-in model of constitutive active CD11b in mice. We genetically targeted the Itgam gene (which codes for CD11b) to introduce a point mutation that results in the I332G substitution in the protein. The I332G mutation in CD11b promotes an active, higher-affinity conformation of the ligand-binding I/A-domain (CD11b αA-domain). In vitro, this mutation increased adhesion of knock-in neutrophils to fibrinogen and decreased neutrophil chemotaxis to a formyl-Met-Leu-Phe gradient. In vivo, CD11bI332G animals showed a reduction in recruitment of neutrophils and macrophages in a model of sterile peritonitis. This genetic activation of CD11b also protected against development of atherosclerosis in the setting of hyperlipidemia via reduction of macrophage recruitment into atherosclerotic lesions. Thus, our animal model of constitutive genetic activation of CD11b can be a useful tool for the study of integrin activation and its potential contribution to modulating leukocyte recruitment and alleviating different inflammatory diseases.

Hidden Readmissions after Carotid Endarterectomy and Stenting.

BACKGROUND: Historically, carotid procedures incur a readmission rate of approximately 6%; however, these studies are not nationally representative and are limited to tracking only the index hospitals. We sought to evaluate a nationally representative database for readmission rates (including different hospitals) after both carotid endarterectomy (CEA) and carotid artery stenting (CAS) and determine risk factors for poor outcomes including postoperative mortality and myocardial infarction. METHODS: This study was a retrospective analysis utilizing the 2010-2014 Nationwide Readmissions Database to query patients aged >18 years undergoing CEA or CAS. Outcomes included initial admission mortality, and 30-day readmission, including mortality and myocardial infarction (MI). Univariable analysis of 39 demographic, clinical, and hospital variables was conducted with significance set at P < 0.05. Significant variables were included in a multivariable logistic regression to identify independent risk factors for readmission. Results were weighted for national estimates. RESULTS: There were 527,622 patients undergoing carotid procedures and 13% (n = 69,187) underwent CAS. The 30-day readmission rate was 7% (n = 35,782), and of those, 25% (n = 8,862) were readmitted to a different hospital. When controlling for other factors, CAS was a risk factor for mortality at both index admission (odds ratio [OR] 2.29 [2.11-2.49]) and 30-day readmission (OR 1.48 [1.3-1.69]) and 30-day readmissions at both index hospital (OR 1.11 [1.07-1.14]) and different hospital (OR 1.38 [1.29-1.48]). Readmission to a different hospital increased mortality risk (OR 1.45 [1.29-1.63]) but did not have an effect on MI. Postoperative infections comprised 15% of readmissions while 6% of all readmissions were for stroke. CONCLUSIONS: Previously unreported, one in 4 readmissions after carotid procedures occur at a different hospital and this fragmentation of care could increase mortality risk after carotid procedures particularly for CAS which was also an independent risk factor for postoperative mortality and readmissions. Further validation is required to decrease unnecessary hospital after carotid procedures.

c-Kit suppresses atherosclerosis in hyperlipidemic mice.

Atherosclerosis is the most common underlying cause of cardiovascular morbidity and mortality worldwide. c-Kit (CD117) is a member of the receptor tyrosine kinase family, which regulates differentiation, proliferation, and survival of multiple cell types. Recent studies have shown that c-Kit and its ligand stem cell factor (SCF) are present in arterial endothelial cells and smooth muscle cells (SMCs). The role of c-Kit in cardiovascular disease remains unclear. The aim of the current study is to determine the role of c-Kit in atherogenesis. For this purpose, atherosclerotic plaques were quantified in c-Kit-deficient mice (KitMut) after they were fed a high-fat diet (HFD) for 16 wk. KitMut mice demonstrated substantially greater atherosclerosis compared with control (KitWT) littermates (P < 0.01). Transplantation of c-Kit-positive bone marrow cells into KitMut mice failed to rescue the atherogenic phenotype, an indication that increased atherosclerosis was associated with reduced arterial c-Kit. To investigate the mechanism, SMC organization and morphology were analyzed in the aorta by histopathology and electron microscopy. SMCs were more abundant, disorganized, and vacuolated in aortas of c-Kit mutant mice compared with controls (P < 0.05). Markers of the "contractile" SMC phenotype (calponin, SM22α) were downregulated with pharmacological and genetic c-Kit inhibition (P < 0.05). The absence of c-Kit increased lipid accumulation and significantly reduced the expression of the ATP-binding cassette transporter G1 (ABCG1) necessary for lipid efflux in SMCs. Reconstitution of c-Kit in cultured KitMut SMCs resulted in increased spindle-shaped morphology, reduced proliferation, and elevated levels of contractile markers, all indicators of their restored contractile phenotype (P < 0.05).NEW & NOTEWORTHY This study describes the novel vasculoprotective role of c-Kit against atherosclerosis and its function in the preservation of the SMC contractile phenotype.

Transcriptomics of Human Arteriovenous Fistula Failure: Genes Associated With Nonmaturation.

RATIONALE & OBJECTIVE: Improving arteriovenous fistula (AVF) outcomes requires better understanding of the biology underlying maturation or failure. Our current knowledge of maturation relies on extrapolation from other vascular pathologies, which does not incorporate unique aspects of AVF remodeling. This study compares the RNA expression of pre-access (native) veins and AVFs with distinct maturation outcomes. STUDY DESIGN: Case-control study. SETTING & PARTICIPANTS: 64 patients undergoing 2-stage AVF surgeries at a single center. 19 native veins and 19 AVF samples were analyzed using RNA sequencing (RNA-seq). 58 native veins were studied using real-time polymerase chain reaction; 45, using immunohistochemistry; and 19, using Western blot analysis. PREDICTOR: RNA expression in native veins and AVFs. OUTCOME: Anatomic nonmaturation, defined as an AVF that never achieved an internal diameter ≥ 6mm. ANALYTICAL APPROACH: Pre-access native veins and AVF samples were obtained from patients undergoing 2-stage AVF creation. Veins that subsequently matured or failed after access creation were analyzed using RNA-seq to search for genes associated with maturation failure. Genes associated with nonmaturation were confirmed using real-time polymerase chain reaction, immunohistochemistry, and Western blot analysis. In addition, the association between pre-access gene expression and postoperative morphology was evaluated. RNA-seq was also performed on AVFs to search for transcriptional differences between AVFs that matured and those that failed at the time of transposition. RESULTS: Pro-inflammatory genes (CSF3R, FPR1, S100A8, S100A9, and VNN2) were upregulated in pre-access veins that failed (false discovery rate < 0.05), and their expression colocalized to smooth muscle cells. Expression of S100A8 and S100A9 correlated with postoperative intimal hyperplasia and the product of medial fibrosis and intimal hyperplasia (r=0.32-0.38; P < 0.05). AVFs that matured or failed were transcriptionally similar at the time of transposition. LIMITATIONS: Small sample size, analysis of only upper-arm veins and transposed fistulas. CONCLUSIONS: Increased expression of proinflammatory genes in pre-access veins appears to be associated with greater risk for AVF nonmaturation.

Fibrotic Venous Remodeling and Nonmaturation of Arteriovenous Fistulas.

The frequency of primary failure in arteriovenous fistulas (AVFs) remains unacceptably high. This lack of improvement is due in part to a poor understanding of the pathobiology underlying AVF nonmaturation. This observational study quantified the progression of three vascular features, medial fibrosis, intimal hyperplasia (IH), and collagen fiber organization, during early AVF remodeling and evaluated the associations thereof with AVF nonmaturation. We obtained venous samples from patients undergoing two-stage upper-arm AVF surgeries at a single center, including intraoperative veins at the first-stage access creation surgery and AVFs at the second-stage transposition procedure. Paired venous samples from both stages were used to evaluate change in these vascular features after anastomosis. Anatomic nonmaturation (AVF diameter never ≥6 mm) occurred in 39 of 161 (24%) patients. Neither preexisting fibrosis nor IH predicted AVF outcomes. Postoperative medial fibrosis associated with nonmaturation (odds ratio [OR], 1.55; 95% confidence interval [95% CI], 1.05 to 2.30; P=0.03, per 10% absolute increase in fibrosis), whereas postoperative IH only associated with failure in those individuals with medial fibrosis over the population's median value (OR, 2.63; 95% CI, 1.07 to 6.46; P=0.04, per increase of 1 in the intima/media ratio). Analysis of postoperative medial collagen organization revealed that circumferential alignment of fibers around the lumen associated with AVF nonmaturation (OR, 1.38; 95% CI, 1.03 to 1.84; P=0.03, per 10° increase in angle). This study demonstrates that excessive fibrotic remodeling of the vein after AVF creation is an important risk factor for nonmaturation and that high medial fibrosis determines the stenotic potential of IH.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells.

OBJECTIVE: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)-derived EPC and MSC function using a murine diabetic wound healing model. METHODS: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony-forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34- and vascular endothelial growth factor receptor 2-positive cells in the vessel wall. Data were analyzed by analysis of variance. RESULTS: Topical estrogen significantly increased colony-forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen-treated compared with placebo-treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen-treated group, reflecting increased MSC activity and differentiation. CONCLUSIONS: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM-derived EPCs and MSCs.

Intramuscular E-selectin/adeno-associated virus gene therapy promotes wound healing in an ischemic mouse model.

BACKGROUND: Poor wound healing in critical limb ischemia (CLI) is attributed to impaired neovascularization and reperfusion. Optimizing the ischemic wound with adhesion molecules that enhance stem cell homing may revolutionize treatment. The purpose of this study is to test the efficacy of adhesion molecule E-selectin on wound healing in an ischemic mouse wound. METHODS: Adult FVB/NJ mice underwent unilateral femoral artery and vein ligation to induce CLI. A 4-mm punch biopsy wound was created on the anterior thigh to simulate ischemic wounds. Intramuscular injection of adeno-associated virus (AAV) carrying either E-selectin (E-selectin/AAV, n = 11) or LacZ as control (LacZ/AAV, n = 10) was performed. Gross wound size was measured for 10 d postoperatively. Ischemic hindlimb reperfusion was quantified using laser Doppler imaging. Wound tissue neovascularization was visualized using DiI perfusion and confocal microscopy. E-selectin expression in wounds was verified by immunofluorescence. RESULTS: Immunofluorescence confirmed E-selectin/AAV delivery in treatment versus control limbs. Wounds from E-selectin/AAV mice versus controls revealed surface area healing of 54% versus 20% (P < 0.01) on postoperative day (POD) 1, 78% versus 51% on POD 4 (P < 0.01), and 97% versus 84% on POD 10 (P < 0.01). Laser Doppler imaging revealed greater reperfusion in E-selectin/AAV mice versus controls by POD 10 (0.49 versus 0.27, P < 0.05). DiI perfused ligated hindlimb in E-selectin/AAV versus control mice revealed mean neovascularization intensity score of 30 versus 18 (P < 0.05) on POD 10. CONCLUSIONS: Intramuscularly injected E-selectin/AAV gene therapy in mice with CLI significantly increases wound angiogenesis and limb reperfusion, expediting overall wound healing.

A Reliable Mouse Model of Hind limb Gangrene.

BACKGROUND: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. METHODS: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. RESULTS: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. CONCLUSIONS: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

Endovascular treatment of TransAtlantic Inter-Society Consensus D aortoiliac occlusive disease using unibody bifurcated endografts.

OBJECTIVE: Aortobifemoral bypass has been the gold standard treatment for extensive aortoiliac occlusive disease. Endovascular therapy and stenting of aortic and iliac occlusive lesions has proven to be efficacious, especially when dealing with short segment lesions. Endovascular treatment of TransAtlantic Inter-Society Consensus II (TASC) D aortoiliac occlusive lesions remains a challenge, but a valuable treatment option in poor surgical candidates. We present our operative technique and midterm results in treating TASC D aortoiliac occlusive disease using unibody bifurcated endografts. METHODS: We performed a retrospective review of patients with TASC D aortoiliac occlusive disease who underwent transfemoral endovascular revascularization with the Endologix Powerlink unibody bifurcated endograft (Endologix, Irvine, Calif). Demographic data, operative details, and outcomes were collected. Paired t-tests were performed to compare preoperative and postoperative ankle brachial indexes. RESULTS: Between March 2009 and July 2011, 10 high-risk patients (8 male and 2 female) for a traditional aortobifemoral bypass were treated using this endovascular technique. The mean age was 59 ± 6 years (range, 50-69 years). All patients presented with rest pain, and four with tissue loss. Technical success was 100%, with two patients requiring brachial access and eight patients requiring additional stent placement. Postoperatively, all patients reported clinical improvement with resolution of ischemic symptoms. Mean improvement ankle brachial index was 0.50 ± 0.08 (P = .028) and 0.50 ± 0.01 (P = .034) in the left and right legs, respectively. Mean follow-up time was 40 ± 24 months (range, 4-81 months). The primary and secondary patency rates were 80% and 100%, respectively. Complications requiring early reintervention occurred in two patients and included one expanding hematoma from the percutaneous access site and one acute iliac artery thrombosis. Additionally, one patient underwent repeat angioplasty/stenting for threatened endograft limbs at 4 months. One patient expired during follow-up from an unrelated cardiac cause 19 weeks postoperatively. CONCLUSIONS: This series demonstrates that endovascular repair using a unibody bifurcated endograft for TASC D aortoiliac occlusive disease is feasible, effective, and has excellent midterm patency. It should be considered an effective treatment option when the disease process involves the aorta, in particular if the patient is surgically unfit for a traditional aortobifemoral bypass. The unibody configuration preserves the anatomic aortic bifurcation, which is particularly important in patients with peripheral occlusive disease who are deemed to undergo subsequent endovascular interventions.