Peripheral artery disease (PAD) pathophysiology extends beyond hemodynamics to include other operating mechanisms, including endothelial dysfunction. Oxidative stress may be linked to endothelial dysfunction by reducing nitric oxide (NO) bioavailability. We aimed to investigate whether the NO system and its regulators are altered in the setting of PAD and to assess the relationship between NO bioavailability and oxidative stress. Sera from 35 patients with intermittent claudication (IC), 26 patients with critical limb ischemia (CLI), and 35 non-PAD controls were analyzed to determine levels of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), nitrate/nitrite (nitric oxides, or NOx), arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and the oxidative stress markers 8-Oxo-2′-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), advanced glycation end products (AGEs), and protein carbonyls. NOx was significantly lower in IC and CLI patients compared to controls in association with elevated oxidative stress, with the greatest NOx reductions observed in CLI. Compared with controls, IC and CLI patients had reduced BH4, elevated BH2, and a reduced BH4/BH2 ratio. SDMA, the arginine/SDMA ratio, and the arginine/ADMA ratio were significantly higher in CLI patients. The NO system and its regulators are significantly compromised in PAD. This dysregulation appears to be driven by increased oxidative stress and worsens as the disease progresses from claudication to CLI.

BACKGROUND: Peripheral arterial disease (PAD) is estimated to affect 7% of the adult population in the United States; however, there is currently little understanding of the key cellular and molecular pathways at play. With PAD characterized by vascular inflammation and associated calcification, the current study set out to elucidate the role of NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome activation in the current cohort. METHODS AND RESULTS: Global proteomics of human vessels with and without PAD from a total of 14 donors revealed an increase of proinflammatory associated ontologies, specifically acute phase and innate immunity. Targeted mass spectrometry showed a significant increase in NLRP3, confirmed by NLRP3 ELISA. Histological analysis from the same patients demonstrated expression of NLRP3, colocalizing in immunoreactive CD68 (cluster of differentiation 68) and CD209 (cluster of differentiation 209) macrophages. Moreover, transmission electron microscopy showed the locality of macrophage-like cells in the presence of calcification, with confocal microscopy further validating the localization of CD68, NLRP3, and calcification via near-infrared calcium tracer. Systemic inflammation and the presence of the NLRP3 inflammasome was assessed via flow cytometry and ELISA, respectively. Compared with patients without PAD, NLRP3 expression was significantly increased in serum. In addition, proinflammatory cytokine presence was significantly increased in disease versus control, with IL (interleukin)-1β, TNF-α (tumor necrosis factor α), and IL-33 demonstrating the greatest disparity, correlating with NLRP3 activation. CONCLUSIONS: The current findings demonstrate a link between NLRP3, macrophage accumulation, and calcification in arteries of patients with PAD, suggesting an association or possible driver of PAD in these patients.

BACKGROUND: Peripheral arterial disease (PAD) causes leg muscle damage due to inadequate perfusion and increases cardiovascular events and mortality 2- to 3-fold. It is unclear if PAD is a biomarker for high-risk cardiovascular disease or if skeletal muscle injury harms arterial health. The objective of this work is to test if serum myoglobin levels (myoglobinemia) are a marker of PAD, and if so, whether myoglobin impairs vascular health. STUDY DESIGN: Patient blood samples were collected from PAD and control (no PAD) patients and interrogated for myoglobin concentrations and nitric oxide bioavailability. Patient mortality over time was captured from the medical record. Myoglobin activity was tested on endothelial cells and arterial function. RESULTS: Myoglobin is a biomarker for symptomatic PAD and was inversely related to nitric oxide bioavailability; 200 ng/mL myoglobin in vitro increased endothelial cell permeability in vitro and decreased nitrate bioavailability. Ex vivo, 100 ng/mL myoglobin increased vascular tone in naive murine aortas approximately 1.5 times, impairing absolute vessel relaxation. In vivo, we demonstrated that myoglobinemia caused impaired flow-mediated dilation in a porcine model. Patients presenting with myoglobin levels of 100 ng/mL or greater had significantly more deaths than those with myoglobin levels of less than 100 ng/mL. CONCLUSIONS: Using a combination of patient data, in vitro, ex vivo, and in vivo testing, we found that myoglobin is a biomarker for symptomatic PAD and a potent regulator of arterial health that can increase vascular tone, increase vascular permeability, and cause endothelial dysfunction, all of which may contribute to the vulnerability of PAD patients to cardiovascular events and death.

BACKGROUND: Cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythroid 2-related factor 2 is responsible for the expression of antioxidant response element-regulated genes and is known to be upregulated by H2S. We examined the levels of H2S, H2S-producing enzymes, and nuclear factor-erythroid 2-related factor 2 activation status in skeletal muscle obtained from critical limb ischemia (CLI) patients. METHODS AND RESULTS: Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor-erythroid 2-related factor 2 activation as well as antioxidant proteins, such as Cu, Zn-superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. CONCLUSIONS: The data demonstrate that H2S bioavailability and nuclear factor-erythroid 2-related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S-producing enzymes may contribute to the pathogenesis of CLI.

Patients with coronavirus disease 2019 (COVID-19) seem to be at high risk for venous thromboembolism (VTE) development, but there is a paucity of data exploring both the natural history of COVID-19–associated VTE and the risk for poor outcomes after VTE development. This investigation aims to explore the relationship between COVID-19–associated VTE development and mortality. A prospectively maintained registry of patients older than 18 years admitted for COVID-19–related illnesses within an academic health care network between March and September 2020 was reviewed. Codes from the tenth revision of the International Classification of Diseases for VTE were collected. The charts of those patients with a code for VTE were manually reviewed to confirm VTE diagnosis. There were 2,552 patients admitted with COVID-19–related illnesses. One hundred and twenty-six patients (4.9%) developed a VTE. A disproportionate percentage of patients of Black race developed a VTE (70.9% VTE v 57.8% non-VTE; P = .012). A higher proportion of patients with VTE expired during their index hospitalization (22.8% VTE v 8.4% non-VTE; P < .001). On multivariable logistic regression analysis, VTE was independently associated with mortality (odds ratio = 3.17; 95% confidence interval, 1.9–5.2; P < .001). Hispanic/Latinx ethnicity was associated with decreased mortality (odds ratio = 0.45; 95% confidence interval, 0.21–1.00; P = .049). Hospitalized patients of Black race with COVID-19 were more prone to VTE development, and patients with COVID-19 who developed in-hospital VTE had roughly nearly threefold higher odds of mortality. Further emphasis should be placed on optimizing COVID-19 anticoagulation protocols to reduce mortality in this high-risk cohort.

Black race has been shown to be a risk factor for amputation in peripheral artery disease (PAD); however, race has been argued to be a marker for socioeconomic status (SES) rather than true disparity. The aim of this study is to study the impact of race and SES on amputation risk in PAD patients. Methods and Results--Patients with incident PAD in the national Veterans Affairs Corporate Data Warehouse were identified from 2003 to 2014 (N=155 647). The exposures were race and SES (measured by median income in residential ZIP codes). The outcome was incident major amputation. Black veterans were significantly more likely to live in low-SES neighborhoods and to present with advanced PAD. Black patients had a higher amputation risk in each SES stratum compared with white patients. In Cox models (adjusting for covariates), black race was associated with a 37% higher amputation risk compared with white race (hazard ratio: 1.37; 95% confidence interval, 1.30-1.45), whereas low SES was independently predictive of increased risk of amputation (hazard ratio: 1.12; 95% confidence interval, 1.06-1.17) and showed no evidence of interaction with race. In predicted amputation risk analysis, black race and low SES continued to be significant risk factors for amputation regardless of PAD presentation. Conclusions--Black race significantly increases the risk of amputation within the same SES stratum compared with white race and has an independent effect on limb loss after controlling for comorbidities, severity of PAD at presentation, and use of medications.

Background Critical limb ischemia (CLI) is a life- and limb-threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow-derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors. Methods MSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population-doubling calculations. Results MSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL-stimulated compared with FBS-stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs. Conclusions PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI.

Carotid artery intima-media thickness (IMT) is a biomarker for cardiovascular disease that also predicts the risk of cardiovascular mortality. Angiotensin-converting enzyme (ACE) inhibition is a unique therapeutic modality because it both treats hypertension and improves arterial health and cardiovascular disease outcomes. Controversy exists regarding the role of ACE inhibitors and angiotensin receptor blockers (ARBs) in IMT regression. Our article provides an update on how ACE inhibitors and ARBs could play a role in decreasing IMT.

Vascularized composite allotransplantation (VCA) has emerged as a viable limb replacement strategy for selected patients with upper limb amputation. However, allograft rejection has been seen in essentially all reported VCA recipients indicating a requirement for substantial immunosuppressive therapy. Calcineurin inhibitors have served as the centerpiece agent in all reported cases, and CNI-associated complications associated with the broad therapeutic effects and side effects of calcineurin inhibitors have been similarly common. Recently, belatacept has been approved as a calcineurin inhibitor replacement in kidney transplantation, but to date, its use in VCA has not been reported. Herein, we report on the case of a hand transplant recipient who developed recurrent acute rejection with alloantibody formation and concomitant calcineurin inhibitor nephrotoxicity, all of which resolved upon conversion from a maintenance regimen of tacrolimus, mycophenolate mofetil and steroids to belatacept and sirolimus. This case indicates that belatacept may be a reasonable maintenance immunosuppressive alternative for use in VCA, providing sufficient prophylaxis from rejection with a reduced side effect profile, the latter being particularly relevant for nonlife threatening conditions typically treated by VCA.

BACKGROUND: Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries. METHODS: D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-weekold mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model. RESULTS: D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model. CONCLUSIONS: TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.