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Background: Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF 165 a and antiangiogenic VEGF 165 b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF 165 a) to SLTRKD (in VEGF 165 b). We have recently shown that VEGF 165 b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF 165 b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear., Methods and Results: Hypoxia serum starvation was used as an in vitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF 165 b. A synthetic anti-angiogenic VEGF 165 b splice variant in which the KD residues were switched to RR (VEGF 165 b KD→RR ) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity in vitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF 165 a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF 165 b KD→RR over VEGF 165 a is due to its ability to induce P38-activation in ischemic endothelial cells., Conclusions: Our data shows that the KD residues regulate VEGF 165 b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF 165 b KD→RR isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.

Reversal of ischemia is mediated by neo-angiogenesis requiring endothelial cell (EC) and pericyte interactions to form stable microvascular networks. We describe an unrecognized role for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in potentiating neo-angiogenesis and vessel stabilization. We show that the endothelium is a major source of TRAIL in the healthy circulation compromised in peripheral artery disease (PAD). EC deletion of TRAIL in vivo or in vitro inhibited neo-angiogenesis, pericyte recruitment, and vessel stabilization, resulting in reduced lower-limb blood perfusion with ischemia. Activation of the TRAIL receptor (TRAIL-R) restored blood perfusion and stable blood vessel networks in mice. Proof-of-concept studies showed that Conatumumab, an agonistic TRAIL-R2 antibody, promoted vascular sprouts from explanted patient arteries. Single-cell RNA sequencing revealed heparin-binding EGF-like growth factor in mediating EC-pericyte communications dependent on TRAIL. These studies highlight unique TRAIL-dependent mechanisms mediating neo-angiogenesis and vessel stabilization and the potential of repurposing TRAIL-R2 agonists to stimulate stable and functional microvessel networks to treat ischemia in PAD.

Background: Peripheral artery disease (PAD) is an ischemic disease with a rising incidence worldwide. The lncRNA H19 (H19) is enriched in endothelial progenitor cells (EPCs), and transplantation of pyroptosis-resistant H19-overexpressed EPCs (oe-H19-EPCs) may promote vasculogenesis and blood flow recovery in PAD, especially with critical limb ischemia (CLI)., Methods: EPCs isolated from human peripheral blood was characterized using immunofluorescence and flow cytometry. Cell proliferation was determined with CCK8 and EdU assays. Cell migration was assessed by Transwell and wound healing assays. The angiogenic potential was evaluated using tube formation assay. The pyroptosis pathway-related protein in EPCs was detected by western blot. The binding sites of H19 and FADD on miR-107 were analyzed using Luciferase assays. In vivo, oe-H19-EPCs were transplanted into a mouse ischemic limb model, and blood flow was detected by laser Doppler imaging. The transcriptional landscape behind the therapeutic effects of oe-H19-EPCs on ischemic limbs were examined with whole transcriptome sequencing., Results: Overexpression of H19 in EPCs led to an increase in proliferation, migration, and tube formation abilities. These effects were mediated through pyroptosis pathway, which is regulated by the H19/miR-107/FADD axis. Transplantation of oe-H19-EPCs in a mouse ischemic limb model promoted vasculogenesis and blood flow recovery. Whole transcriptome sequencing indicated significant activation of vasculogenesis pathway in the ischemic limbs following treatment with oe-H19-EPCs., Conclusions: Overexpression of H19 increases FADD level by competitively binding to miR-107, leading to enhanced proliferation, migration, vasculogenesis, and inhibition of pyroptosis in EPCs. These effects ultimately promote the recovery of blood flow in CLI., Competing Interests: Declaration of competing interest The authors affirm that there are no commercial or financial relationships that could be interpreted as a possible conflict of interest during the course of the research., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Peripheral artery disease (PAD) is the manifestation of atherosclerosis characterized by the accumulation of plaques in the arteries of the lower limbs. Interestingly, growing evidence suggests that the pathology of PAD is multifaceted and encompasses both vascular and skeletal muscle dysfunctions, which contributes to blunted physical capabilities and diminished quality of life. Importantly, it has been suggested that many of these pathological impairments may stem from blunted reduction-oxidation (redox) handling. Of note, in those with PAD, excessive production of reactive oxygen species (ROS) outweighs antioxidant capabilities resulting in oxidative damage, which may have systemic consequences. It has been suggested that antioxidant supplementation may be able to assist in handling ROS. However, the activation of various ROS production sites makes it difficult to determine the efficacy of these antioxidant supplements. Therefore, this review focuses on the common cellular mechanisms that facilitate ROS production and discusses how excessive ROS may impair vascular and skeletal muscle function in PAD. Furthermore, we provide insight for current and potential antioxidant therapies, specifically highlighting activation of the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear Factor Erythroid 2-related factor 2 (Nrf2) pathway as a potential pharmacological therapy to combat ROS accumulation and aid in vascular function, and physical performance in patients with PAD. Altogether, this review provides a better understanding of excessive ROS in the pathophysiology of PAD and enhances our perception of potential therapeutic targets that may improve vascular function, skeletal muscle function, walking capacity, and quality of life in patients with PAD., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle., Methods: Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model., Results: Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis)., Conclusions: Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD., Competing Interests: None.

Diabetes mellitus (DM) is a metabolic disease that heightens the risks of many vascular complications, including peripheral arterial disease (PAD). Various types of cells, including but not limited to endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages (MΦs), play crucial roles in the pathogenesis of DM-PAD. Long non-coding RNAs (lncRNAs) are epigenetic regulators that play important roles in cellular function, and their dysregulation in DM can contribute to PAD. This review focuses on the developing field of lncRNAs and their emerging roles in linking DM and PAD. We review the studies investigating the role of lncRNAs in crucial cellular processes contributing to DM-PAD, including those in ECs, VSMCs, and MΦ. By examining the intricate molecular landscape governed by lncRNAs in these relevant cell types, we hope to shed light on the roles of lncRNAs in EC dysfunction, inflammatory responses, and vascular remodeling contributing to DM-PAD. Additionally, we provide an overview of the research approach and methodologies, from identifying disease-relevant lncRNAs to characterizing their molecular and cellular functions in the context of DM-PAD. We also discuss the potential of leveraging lncRNAs in the diagnosis and therapeutics for DM-PAD. Collectively, this review provides a summary of lncRNA-regulated cell functions contributing to DM-PAD and highlights the translational potential of leveraging lncRNA biology to tackle this increasingly prevalent and complex disease., (© 2024. The Author(s).)

Although peripheral artery disease (PAD) primarily affects large arteries outside the brain, PAD is also associated with elevated cerebral vulnerabilities, including greater risks for brain injury (such as stroke), cognitive decline and dementia. In the present review, we aim to evaluate recent literature and extract information on potential mechanisms linking PAD and consequences on the brain. Furthermore, we suggest novel therapeutic avenues to mitigate cognitive decline and reduce risk of brain injury in patients with PAD. Various interventions, notably exercise, directly or indirectly improve systemic blood flow and oxygen supply and are effective strategies in patients with PAD or cognitive decline. Moreover, triggering protective cellular and systemic mechanisms by modulating inspired oxygen concentrations are emerging as potential novel treatment strategies. While several genetic and pharmacological approaches to modulate adaptations to hypoxia showed promising results in preclinical models of PAD, no clear benefits have yet been clinically demonstrated. We argue that genetic/pharmacological regulation of the involved adaptive systems remains challenging but that therapeutic variation of inspired oxygen levels (e.g., hypoxia conditioning) are promising future interventions to mitigate associated cognitive decline in patients with PAD., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Peripheral artery disease (PAD) is characterized by varying severity of arterial stenosis, exercise induced claudication, malperfused tissue precluding normal healing and skeletal muscle dysfunction. Revascularization interventions improve circulation, but post-reperfusion changes within the skeletal muscle are not well characterized. This study investigates if revascularization enhanced hemodynamics increases walking performance with concurrent improvement of mitochondrial function and reverses abnormal skeletal muscle morphological features that develop with PAD. Fifty-eight patients completed walking performance testing and muscle biopsy before and 6 months after revascularization procedures. Muscle fiber morphology, desmin structure, and mitochondria respiration assessments before and after the revascularization were evaluated. Revascularization improved limb hemodynamics, walking function, and muscle morphology. Qualitatively not all participants recovered normal structural architecture of desmin in the myopathic myofibers after revascularization. Heterogenous responses in the recovery of desmin structure following revascularization may be caused by other underlying factors not reversed with hemodynamic improvements. Revascularization interventions clinically improve patient walking ability and can reverse the multiple subcellular functional and structural abnormalities in muscle cells. Further study is needed to characterize desmin structural remodeling with improvements in skeletal muscle morphology and function., (© 2024. The Author(s).)

Oxidative stress plays a crucial role in the pathogenesis of peripheral artery disease (PAD). This study aimed to investigate the effect of clopidogrel on oxidative stress in PAD patients. Seventy subjects were divided into three groups: PAD patients before treatment (B-PAD), PAD patients after treatment with clopidogrel (A-PAD), and healthy controls. Serum levels of superoxide dismutase (SOD), copper (Cu), zinc (Zn), manganese (Mn), and oxidized protein were measured. SOD activities were also determined. The results showed that SOD activities, and SOD specific activities were significantly decreased in PAD patients compared to healthy individuals. After treatment with clopidogrel, SOD activities, and SOD specific activities were continuously decrease in PAD patients. The SOD and oxidized protein concentrations were significantly increased in PAD patients compared to healthy individuals. After treatment with clopidogrel, the oxidized protein concentration was significantly decreased, while SOD concentration was significantly increased in PAD patients. These findings suggest that the treatment by clopidogrel stimulated the production of the enzyme but the ratio of active enzyme remained low. The decrease in oxidized protein can be explained by the treatment having antioxidant efficacy that may have compensated for the deficiency in enzyme activity and led to a decrease in oxidized protein. Additionally, the results of this study provide promising evidence that oxidative stress biomarkers including SOD concentration, T-SOD activity, Mn-SOD activity, and oxidized protein levels have potential utility in the diagnosis and management of PAD., (© 2024. Pleiades Publishing, Ltd.)

We investigated the role played by ATP-sensitive purinergic 2 (P2) receptors in evoking the pressor response to treadmill exercise in male and female rats with and without femoral arteries that were ligated for ∼72 h to induce simulated peripheral artery disease (PAD). We hypothesized that PPADS (P2 receptor antagonist, 10 mg iv) would reduce the pressor response to 4 min of treadmill exercise (15 m·min -1 , 1° incline) and steady-state exercise plasma norepinephrine (NE) values in male and female rats, and that the magnitude of effect of PPADS would be greater in rats with simulated PAD ("ligated") than in sham-operated rats. In males, PPADS significantly reduced the difference between steady-state exercise and baseline mean arterial pressure (ΔMAP) response to treadmill exercise in sham ( n = 8; pre-PPADS: 12 ± 2, post-PPADS: 1 ± 5 mmHg; P = 0.037) and ligated ( n = 4; pre-PPADS: 20 ± 2, post-PPADS: 11 ± 3 mmHg; P = 0.028) rats with a similar magnitude of effect observed between groups ( P = 0.720). In females, PPADS had no effect on the ΔMAP response to treadmill exercise in sham ( n = 6; pre-PPADS: 9 ± 2, post-PPADS: 7 ± 2 mmHg; P = 0.448) or ligated ( n = 6; pre-PPADS: 15 ± 2, post-PPADS: 16 ± 3 mmHg; P = 0.684) rats. When NE values were grouped by sex independent of ligation/sham status, PPADS significantly reduced plasma NE in male ( P = 0.016) and female ( P = 0.027) rats. The data indicate that P2 receptors contribute to the sympathetic response to exercise in both male and female rats but that the sympathoexcitatory role for P2 receptors translates into an obligatory role in the blood pressure response to exercise in male but not in female rats. NEW & NOTEWORTHY Here, we demonstrate that purinergic 2 (P2) receptors contribute significantly to the blood pressure response to treadmill exercise in male rats both with and without simulated PAD induced by femoral artery ligation. We found no role for P2 receptors in the blood pressure response to treadmill exercise in female rats, thus revealing clear sex differences in P2 receptor-mediated blood pressure control during exercise.

Peripheral artery disease (PAD) is a prevalent cardiovascular disease with risks of limb loss. Our objective is to establish an autologous cell source for vascular regeneration to achieve limb salvage in PAD. Six PAD patients (age 50-80) were enrolled with their peripheral blood collected to derive vascular endothelial cells (ECs) with two different approaches: (1) endothelial progenitor cell (EPC) approach and (2) induced pluripotent stem cell (iPSC) approach. The iPSC approach successfully generated patient-specific ECs for all PAD patients, while the EPC approach did not yield any colony-forming ECs in any of the patients. The patient-derived iPSC-ECs expressed endothelial markers and exhibited endothelial functions. However, elevated inflammatory status with VCAM-1 expression was observed in the patient-derived cells. Pharmacological treatment with resveratrol resulted in patient-specific responses in cell viability and VCAM-1 expression. Our study demonstrates the potential of iPSC-ECs for autologous regenerative therapy in PAD, offering promise for personalized treatments for ischemic PAD., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fibroblast growth factor 23 (FGF23) levels are often elevated in chronic kidney disease (CKD). FGF23 and inflammation are common characteristics in CKD, and both are associated with worse disease progression and the occurrence of complications. The existence of an interaction between FGF23 and inflammation has been suggested, each of which influences the expression and activity of the other, leading to a vicious feedback loop with adverse outcomes, including cardiovascular disease and mortality. In this work, we determined circulating FGF23 levels in a group of patients with CKD stages 3 and 4 subjected to elective femoral endarterectomy due to established peripheral artery disease (PAD), a condition resulting from an athero-inflammatory process, and we studied its associations with different inflammatory markers and mediators. We evaluated its association with serum tumor necrosis factor (TNF)α, interleukin (IL) 6, and IL10, as well as with the gene expression levels of these parameters and A disintegrin and metalloproteinase domain-containing protein (ADAM) 17 in femoral vascular tissue and peripheral blood circulating cells (PBCCs). We also analyzed its association with serum concentrations of C-reactive protein (CRP), the systemic immune inflammation index (SII), and the neutrophil-to-lymphocyte ratio (NLR). Finally, we determined the vascular immunoreactivity of protein TNFα in a subgroup of patients. FGF23 concentrations were independently associated with circulating and PBCC mRNA levels of TNFα. Worst kidney function and diabetes were also found to be contributing to FGF23 levels. Patients with higher levels of FGF23 also had greater vascular immunoreactivity for TNFα.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of βIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Ethylene diamine tetra-acetic acid (EDTA) is a chelating agent used to dissolve calcium deposits but evidence in decalcifying atherosclerotic lesions is limited., Aims: We assessed the feasibility and efficacy of EDTA delivered via porous balloon to target calcified lesions in cadaveric below-the-knee (BTK) arteries., Methods: Using porcine carotid arteries, EDTA concentration was measured in the arterial wall and outside the artery at the 0-, 0.5-, 4-, and 24-h circulation after the injection through a porous balloon. In cadaver BTK samples, the proximal and distal anterior tibial artery (ATA) and distal posterior tibial artery (PTA) were studied. EDTA-2Na/H2O or EDTA-3Na/H2O were administrated using a porous balloon, then circulated for 6 h for EDTA-3Na/H2O and 24 h for EDTA-2Na/H2O and EDTA-3Na/H2O. Micro-CT imaging of the artery segments before and after the circulation and cross-sectional analyses were performed to evaluate calcium burden., Results: In the porcine carotid study, EDTA was delivered through a porous balloon present in the arterial wall and was retained there for 24 h. In BTK arteries, cross-sectional analyses of micro-CT revealed a significant decrease in the calcium area in the distal ATA segment under 24-h circulation with EDTA-2Na/H2O and in the distal ATA segment under 24-h circulation with EDTA-3Na/H2O. The proximal ATA segment under 6-h circulation with EDTA-3Na/H2O showed no significant change in any parameters of calcium CONCLUSION: EDTA-3Na/H2O or EDTA-2Na/H2O with longer circulation times resulted in greater calcium reduction in atherosclerotic lesion. EDTA may have a potential therapeutic option for the treatment of atherosclerotic calcified lesions., (© 2024 Wiley Periodicals LLC.)

Functional cell treatment for critical limb ischemia is limited by cell viability loss and dysfunction resulting from a harmful ischemic microenvironment. Metal-polyphenol networks have emerged as novel cell delivery vehicles for protecting cells from the detrimental ischemic microenvironment and prolonging the survival rate of cells in the ischemic microenvironment. M2 macrophages are closely related to tissue repair, and they secrete anti-inflammatory factors that contribute to lesion repair. However, these cells are easily metabolized in the body with low efficiency. Herein, M2 macrophages were decorated with a metal‒polyphenol network that contains copper ions and epigallocatechin gallate (Cu-EGCG@M2) to increase cell survival and therapeutic potential. Cu-EGCG@M2 synergistically promoted angiogenesis through the inherent angiogenesis effect of M2 macrophages and copper ions. We found that Cu-EGCG@M2 increased in vitro viability and strengthened the in vivo therapeutic effect on the ischemic hindlimbs of mice, which promoted the recovery of blood and muscle regeneration, resulting in superior limb salvage. These therapeutic effects were ascribed to the increased survival rate and therapeutic period of M2 macrophages, as well as the ameliorated microenvironment at the ischemic site. Additionally, Cu-EGCG exhibited antioxidant, anti-inflammatory, and proangiogenic effects. Our findings provide a feasible option for cell-based treatment of CLI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The pathophysiology of muscle damage in peripheral artery disease (PAD) includes increased oxidant production and impaired antioxidant defenses. Epicatechin (EPI), a naturally occurring flavanol, has antioxidant properties that may mediate the beneficial effects of natural products such as cocoa. In a phase II randomized trial, a cocoa-flavanol-rich beverage significantly improved walking performance compared with a placebo in people with PAD. In the present work, the molecular mechanisms underlying the therapeutic effect of cocoa flavanols were investigated by analyzing baseline and follow-up muscle biopsies from participants. Increases in nuclear factor erythroid 2-related factor 2 (Nrf2) target antioxidants heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) in the cocoa group were significantly associated with reduced accumulation of central nuclei, a myopathy indicator, in type II muscle fibers ( P = 0.017 and P = 0.023, respectively). Protein levels of the mitochondrial respiratory complex III subunit, cytochrome b-c1 complex subunit 2 (UQCRC2), were significantly higher in the cocoa group than in the placebo group ( P = 0.032), and increases in UQCRC2 were significantly associated with increased levels of Nrf2 target antioxidants HO-1 and NQO1 ( P = 0.001 and P = 0.035, respectively). Exposure of non-PAD human myotubes to ex vivo serum from patients with PAD reduced Nrf2 phosphorylation, an indicator of activation, increased hydrogen peroxide production and oxidative stress, and reduced mitochondrial respiration. Treatment of myotubes with EPI in the presence of serum from patients with PAD increased Nrf2 phosphorylation and protected against PAD serum-induced oxidative stress and mitochondrial dysfunction. Overall, these findings suggest that cocoa flavanols may enhance antioxidant capacity in PAD via Nrf2 activation. NEW & NOTEWORTHY The current study supports the hypothesis that in people with PAD, cocoa flavanols activate Nrf2, thereby increasing antioxidant protein levels, protecting against skeletal muscle damage, and increasing mitochondrial protein abundance. These results suggest that Nrf2 activation may be an important therapeutic target for improving walking performance in people with PAD.

Background: Restenosis (RS) poses a significant concern, leading to recurrent ischemia and the potential for amputation following intraluminal angioplasty in the treatment of Peripheral Artery Disease (PAD). Through microRNA microarray analysis, the study detected a significant downregulation of miR-199a-5p within arterial smooth muscle cells (ASMCs) associated with RS., Objective: This research aims to explore the possible function and the underlying mechanisms of miR-199a-5p in the context of RS., Methods: Primary ASMCs were extracted from the femoral arteries of both healthy individuals and patients with PAD or RS. The expression levels of miR-199a-5p were assessed using both qRT-PCR and in situ hybridization techniques. To examine the impacts of miR-199a-5p, a series of experiments were performed, including flow cytometry, TUNEL assay, EdU assay, CCK8 assay, Transwell assay, and wound closure assay. A rat carotid balloon injury model was employed to elucidate the mechanism through which miR-199a-5p mitigated neointimal hyperplasia., Results: MiR-199a-5p exhibited downregulation in RS patients and was predominantly expressed within ASMCs. Elevated the expression of miR-199a-5p resulted in an inhibitory effect of proliferation and migration in ASMCs. Immunohistochemistry and a dual-luciferase reporter assay uncovered that RS exhibited elevated expression levels of both HIF-1α and E2F3, and they were identified as target genes regulated by miR-199a-5p. The co-transfection of lentiviruses carrying HIF-1α and E2F3 alongside miR-199a-5p further elucidated their role in the cellular responses mediated by miR-199a-5p. In vivo , the delivery of miR-199a-5p via lentivirus led to the mitigation of neointimal formation following angioplasty, achieved by targeting HIF-1α and E2F3., Conclusion: MiR-199a-5p exhibits promise as a prospective therapeutic target for RS since it alleviates the condition by inhibiting the proliferation and migration of ASMCs via its regulation of HIF-1α and E2F3., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cushing syndrome (CS), characterised by endogenous or exogenous glucocorticoid hormone excess, is associated with several systemic complications, including impaired glucose metabolism, which often becomes clinically manifest as diabetes mellitus (DM). In addition, CS can harm the arterial wall because of hyperglycaemia, dyslipidaemia, hepatic steatosis, and central obesity. These metabolic disorders promote atherosclerosis by synthesising adipokines, leptin, and proinflammatory cytokines. Lower limb arterial complications in CS are common and significantly impact morbidity and mortality. Furthermore, CS, in combination with DM, is likely to cause more diffuse vascular disease that predominantly affects distal arterial beds. In conclusion, CS promotes atherosclerosis, including peripheral artery disease, by causing functional and morphological deterioration of the arterial vessel wall and increasing the presence of classical risk factors of atherosclerosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: A noninvasive and reliable approach to quantitatively measure muscle perfusion of lower extremity is needed to aid the diagnosis and treatment of peripheral artery disease (PAD)., Purpose: To verify the reproductivity of using blood oxygen level-dependent (BOLD) imaging to evaluate perfusion in lower extremities, and explore its correlation with walking performance in patients with PAD., Study Type: Prospective observational study., Subjects: Seventeen patients with lower extremity PAD (mean age: 67 ± 6 years, 15 males) and eight older adults (controls)., Field Strength/sequence: Dynamic multi-echo gradient echo T2* weighted imaging at 3T., Assessment: Perfusion was analyzed in regions of interest according to muscle groups. Perfusion parameters were measured, such as minimum ischemia value (MIV), time to peak (TTP), and gradient during reactive hyperemia (Grad) by two independent users. Walking performance experiments including short physical performance battery (SPPB) and 6-minute walk were tested in patients., Statistical Tests: BOLD parameters were compared using Mann-Whitney U test and Kruskal-Wallis test. Relations between parameters and walking performance were assessed by Mann-Whitney U test and Spearman's correlation coefficient., Results: Good to perfect agreement was demonstrated for all perfusion parameters of interuser reproducibility, and the interscan reproducibility of MIV, TTP, and Grad was good. The TTP of the patients was longer than that of the controls (87.85 ± 38.85 s vs. 36.54 ± 7.27 s), while the Grad of patients was smaller (0.16 ± 0.12 msec/s vs. 0.24 ± 0.11 msec/s). Among PAD patients, the MIV was significantly lower in the low SPPB subgroup (score 6-8) than in the high SPPB group (score 9-12), and the TTP was negatively correlated with 6-minute walk distance (ρ = -0.549)., Data Conclusion: BOLD imaging method had overall good reproducibility for the perfusion assessment of calf muscles. The perfusion parameters were different between PAD patients and controls, and were correlated with lower extremity function., Evidence Level: 2 TECHNICAL EFFICACY: Stage 2., (© 2023 International Society for Magnetic Resonance in Medicine.)

Chronic kidney disease (CKD) is a strong risk factor for peripheral artery disease (PAD) that is associated with worsened clinical outcomes. CKD leads to the accumulation of tryptophan metabolites that are associated with adverse limb events in PAD and are ligands of the aryl hydrocarbon receptor (AHR), which may regulate ischemic angiogenesis. To test if endothelial cell-specific deletion of the AHR (AHR ecKO ) alters ischemic angiogenesis and limb function in mice with CKD subjected to femoral artery ligation. Male AHR ecKO mice with CKD displayed better limb perfusion recovery and enhanced ischemic angiogenesis compared with wild-type mice with CKD. However, the improved limb perfusion did not result in better muscle performance. In contrast to male mice, deletion of the AHR in female mice with CKD had no impact on perfusion recovery or angiogenesis. With the use of primary endothelial cells from male and female mice, treatment with indoxyl sulfate uncovered sex-dependent differences in AHR activating potential and RNA sequencing revealed wide-ranging sex differences in angiogenic signaling pathways. Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. There are sex-dependent differences in Ahr activating potential within endothelial cells that are independent of sex hormones. NEW & NOTEWORTHY This study provides novel insights into the mechanisms by which chronic kidney disease worsens ischemic limb outcomes in an experimental model of peripheral artery disease. Deletion of the aryl hydrocarbon receptor (AHR) in the endothelium improved ischemic angiogenesis suggesting that AHR inhibition could be a viable therapeutic target; however, this effect was only observed in male mice. Subsequent analysis in primary endothelial cells reveals sex differences in Ahr activating potential independent of sex hormones.

Critical limb ischemia incidence and prevalence have increased over the years. However, there are no successful treatments to improve quality of life and to reduce the risk of cardiovascular and limb events in these patients. Advanced regenerative therapies have focused their interest on the generation of new blood vessels to repair tissue damage through the use of stem cells. One of the most promising sources of stem cells with high potential in cell-based therapy is adipose-derived stem cells (ASCs). ASCs are adult mesenchymal stem cells that are relatively abundant and ubiquitous and are characterized by a multilineage capacity and low immunogenicity. The proangiogenic benefits of ASCs may be ascribed to: (a) paracrine secretion of proangiogenic molecules that may stimulate angiogenesis; (b) secretion of microvesicles/exosomes that are also considered as a novel therapeutic prospect for treating ischemic diseases; and (c) their differentiation capability toward endothelial cells (ECs). Although we know the proangiogenic effects of ASCs, the therapeutic efficacy of ASCs after transplantation in peripheral artery diseases patients is still relatively low. In this review, we evidence the potential therapeutic use of ASCs in ischemic regenerative medicine. We also highlight the main challenges in the differentiation of these cells into functional ECs. However, significant efforts are still needed to ascertain relevant transcription factors, intracellular signaling and interlinking pathways in endothelial differentiation.

Background: Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle., Methods: We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas., Results: Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD., Conclusions: This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition., Competing Interests: Disclosures None.

Peripheral artery disease (PAD) causes an ischemic myopathy contributing to patient disability and mortality. Most preclinical models to date use young, healthy rodents with limited translatability to human disease. Although PAD incidence increases with age, and obesity is a common comorbidity, the pathophysiologic association between these risk factors and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of age, diet-induced obesity and chronic hindlimb ischemia (HLI) on (1) mobility, (2) muscle contractility, and markers of muscle (3) mitochondrial content and function, (4) oxidative stress and inflammation, (5) proteolysis, and (6) cytoskeletal damage and fibrosis. Following 16-weeks of high-fat, high-sucrose, or low-fat, low-sucrose feeding, HLI was induced in 18-month-old C57BL/6J mice via the surgical ligation of the left femoral artery at 2 locations. Animals were euthanized 4-weeks post-ligation. Results indicate mice with and without obesity shared certain myopathic changes in response to chronic HLI, including impaired muscle contractility, altered mitochondrial electron transport chain complex content and function, and compromised antioxidant defense mechanisms. However, the extent of mitochondrial dysfunction and oxidative stress was significantly greater in obese ischemic muscle compared to non-obese ischemic muscle. Moreover, functional impediments, such as delayed post-surgical recovery of limb function and reduced 6-minute walking distance, as well as accelerated intramuscular protein breakdown, inflammation, cytoskeletal damage, and fibrosis were only evident in mice with obesity. As these features are consistent with human PAD myopathy, our model could be a valuable tool to test new therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Peripheral arterial disease (PAD) is a prevalent cardiovascular complication of limb vascular insufficiency, causing ischemic injury, mitochondrial metabolic damage and functional impairment in the skeletal muscle, and ultimately leading to immobility and mortality. While potential therapies have been mostly focussed on revascularization, none of the currently available pharmacological treatments are fully effective in PAD, often leading to amputations, particularly in chronic metabolic diseases. One major limitation of focussed angiogenesis and revascularization as a therapeutic strategy is a limited effect on metabolic restoration and muscle regeneration in the affected limb. Therefore, additional preclinical investigations are needed to discover novel treatment options for PAD preferably targeting multiple aspects of muscle recovery. In this review, we propose nuclear receptors expressed in the skeletal muscle as potential candidates for ischemic muscle repair in PAD. We review classic steroid and orphan receptors that have been reported to be involved in the regulation of paracrine muscle angiogenesis, oxidative metabolism, mitochondrial biogenesis and muscle regeneration, and discuss how these receptors could be critical for recovery from ischemic muscle damage. Furthermore, we identify existing gaps in our understanding of nuclear receptor signalling in the skeletal muscle and propose future areas of research that could be instrumental in exploring nuclear receptors as therapeutic candidates for treating PAD., (© 2022 Federation of European Biochemical Societies.)

Tissular hypoxia stimulates vascular morphogenesis. Vascular morphogenesis shapes the cell and, consecutively, tissue growth. The development of new blood vessels is intermediated substantially through the tyrosine kinase pathway. There are several types of receptors inferred to be located in the blood vessel structures. Vascular endothelial growth factor A (VEGF-A) is the leading protagonist of angiogenesis. VEGF-A's interactions with its receptors VEGFR1, VEGFR2, and VEGFR3, together with disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), connective tissue growth factor (CTGF), and neuropilin-1 (NRP1), independently, are studied computationally. Peripheral artery disease (PAD), which results in tissue ischemia, is more prevalent in the senior population. Presently, medical curatives used to treat cases of PAD-antiplatelet and antithrombotic agents, statins, antihypertensive remedies with ACE (angiotensin-converting enzyme) impediments, angiotensin receptor blockers (ARB) or β- blockers, blood glucose control, and smoking cessation-are not effective. These curatives were largely established from the treatment of complaint cases of coronary disease. However, these medical curatives do not ameliorate lower limb perfusion in cases of PAD. Likewise, surgical or endovascular procedures may be ineffective in relieving symptoms. Eventually, after successful large vessel revascularization, the residual microvascular circulation may well limit the effectiveness of curatives in cases of PAD. It would thus feel rational to attempt to ameliorate perfusion in PAD by enhancing vascular rejuvenescence and function. Likewise, stimulating specific angiogenesis in these cases (PAD) can ameliorate the patient's symptomatology. Also, the quality of life of PAD patients can be improved by developing new vasodilative and angiogenetic molecules that stimulate the tyrosine kinase pathway. In this respect, the VEGFA angiogenetic pathway was explored computationally. Docking methodologies, molecular dynamics, and computational molecular design methodologies were used. VEGFA's interaction with its target was primarily studied. Common motifs in the vascular morphogenesis pathway are suggested using conformational energy and Riemann spaces. The results show that interaction with VEGFR2 and ADAMTS1 is pivotal in the angiogenetic process. Also, the informational content of two VEGFA complexes, VEGFR2 and ADAMTS1, is crucial in the angiogenesis process.

Background: Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD., Methods: AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHR mKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD., Results: PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes ( Cyp1a1 , Cyp1b1 , and Aldh3a1 ) when compared with either muscle from the PAD condition with normal renal function ( P <0.05 for all 3 genes) or nonischemic controls. AHR mKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function., Conclusions: These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions., Competing Interests: Disclosures None.

Saphenous vein bypass grafting is an effective technique used to treat peripheral arterial disease (PAD). However, restenosis is the major clinical challenge for the graft vessel among people with PAD postoperation. We hypothesize that there is a common culprit behind arterial occlusion and graft restenosis. To investigate this hypothesis, we found TGF-β, a gene specifically upregulated in PAD arteries, by bioinformatics analysis. TGF-β has a wide range of biological activities and plays an important role in vascular remodeling. We discuss the molecular pathway of TGF-β and elucidate its mechanism in vascular remodeling and intimal hyperplasia, including EMT, extracellular matrix deposition, and fibrosis, which are the important pathways contributing to stenosis. Additionally, we present a case report of a patient with graft restenosis linked to the TGF-β pathway. Finally, we discuss the potential applications of targeting the TGF-β pathway in the clinic to improve the long-term patency of vein grafts.

Peripheral artery disease (PAD) is a vascular disorder caused by occlusive atherosclerosis, which commonly impairs blood flow to the lower extremities. The prevalence of PAD is increasing globally with >200 million people affected. PAD remains a growing global health problem as the population continues to age and diabetes incidence grows. Many patients with PAD, most notably those with critical limb ischemia, fail attempts at surgical and percutaneous intervention to improve blood flow and are at risk of amputation. Gene therapy provides an opportunity to change the clinical course of PAD in these patients via strategies that increase vascular supply through angiogenesis and arteriogenesis improving muscle perfusion and function in ischemic legs. This article discusses gene therapy approaches in the context of PAD, both intermittent claudication and critical limb ischemia, and the promise of adeno-associated virus-based strategies delivering not just VEGFs (vascular endothelial growth factors) but a range of other mediators as potential new therapeutics. We also highlight challenges and failures in the clinical translation of gene therapy for PAD and how at least some of these obstacles may be overcome using adeno-associated virus., Competing Interests: Disclosures None.

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, isoform-3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF 165 a (vascular endothelial growth factor 165 a) and microRNA-93 both promote angiogenesis. Methods and Results Mice following hind-limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA-93 and VEGF 165 a. Post-HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post-HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA-93-treated Balb/CJ mice post-HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF 165 a-treated Balb/CJ mice post-HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF 165 a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF 165 a upregulated glucose-6-phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA-93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF 165 a significantly increased and miR-93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.

Atherogenesis involves a complex multifactorial process including chronic inflammation that requires the participation of several cell types and molecules. In addition to their role in vascular homeostasis, extracellular vesicles also appear to play an important role in atherogenesis, including monocyte transmigration and foam cell formation, SMC proliferation and migration, leukocyte transmigration, and thrombosis. Peripheral arterial disease, a major form of peripheral vascular disease, is characterized by structural or functional impairment of peripheral arterial supply, often secondary to atherosclerosis. Elevated levels of extracellular vesicles have been demonstrated in patients with peripheral arterial disease and implicated in the development of atherosclerosis within peripheral vascular beds. However, extracellular vesicles also appear capable of delivering cargo with atheroprotective effects. This capability has been exploited in vesicles engineered to carry content capable of neovascularization, suggesting potential for therapeutic angiogenesis. This dual capacity holds substantial promise for diagnosis and therapy, including possibly limb- and life-saving options for peripheral arterial disease management., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Diabetes is a major risk factor for peripheral arterial disease. Clinical and preclinical studies suggest an impaired collateral remodeling and angiogenesis in response to atherosclerotic arterial occlusion in diabetic conditions, although the underlying mechanisms are poorly understood., Objective: To clarify the cellular and molecular mechanisms underlying impaired postischemic adaptive vascular responses and to evaluate rHDL (reconstituted HDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic mouse model of hindlimb ischemia., Methods and Results: Hindlimb ischemia was induced by unilateral femoral artery ligation. Collateral and capillary parameters together with blood flow recovery were analyzed from normoxic adductor and ischemic gastrocnemius muscles, respectively, at day 3 and 7 post-ligation. In response to femoral artery ligation, collateral lumen area was significantly reduced in normoxic adductor muscles. Distally, ischemic gastrocnemius muscles displayed impaired perfusion recovery and angiogenesis paralleled with persistent inflammation. Muscle-specific mRNA sequencing revealed differential expression of genes critical for smooth muscle proliferation and sprouting angiogenesis in normoxic adductor and ischemic gastrocnemius, respectively, at day 7 post-ligation. Genes typical for macrophage (Mϕ) subsets were differentially expressed across both muscle types. Cell-specific gene expression, flow cytometry, and immunohistochemistry revealed persistent IFN-I response gene upregulation in arterial endothelial cells, ECs and Mϕs from T2DM mice associated with impaired collateral remodeling, angiogenesis and perfusion recovery. Furthermore, rHDL nanotherapy rescued impaired collateral remodeling and angiogenesis through dampening EC and Mϕ inflammation in T2DM mice., Conclusions: Our results suggest that an impaired collateral remodeling and sprouting angiogenesis in T2DM mice is associated with persistent IFN-I response in ECs and Mϕs. Dampening persistent inflammation and skewing ECs and Mϕ phenotype toward less inflammatory ones using rHDL nanotherapy may serve as a potential therapeutic target for T2DM peripheral arterial disease.

Patients with peripheral artery disease (PAD) have reduced muscle capillary density. Walking training (WT) is recommended for PAD patients. The goal of the study was to verify whether WT promotes angiogenesis in PAD-affected muscle and to investigate the possible role of miRNA-126 and the vascular endothelium growth factor (VEGF) angiogenic pathways on this adaptation. Thirty-two men with PAD were randomly allocated to two groups: WT ( n = 16, 2 sessions/week) and control (CO, n = 16). Maximal treadmill tests and gastrocnemius biopsies were performed at baseline and after 12 weeks. Histological and molecular analyses were performed by blinded researchers. Maximal walking capacity increased by 65% with WT. WT increased the gastrocnemius capillary-fiber ratio (WT = 109 ± 13 vs. 164 ± 21 and CO = 100 ± 8 vs. 106 ± 6%, p < 0.001). Muscular expression of miRNA-126 and VEGF increased with WT (WT = 101 ± 13 vs. 130 ± 5 and CO = 100 ± 14 vs. 77 ± 20%, p < 0.001; WT = 103 ± 28 vs. 153 ± 59 and CO = 100 ± 36 vs. 84 ± 41%, p = 0.001, respectively), while expression of PI3KR2 decreased (WT = 97 ± 23 vs. 75 ± 21 and CO = 100 ± 29 vs. 105 ± 39%, p = 0.021). WT promoted angiogenesis in the muscle affected by PAD, and miRNA-126 may have a role in this adaptation by inhibiting PI3KR2, enabling the progression of the VEGF signaling pathway.

Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in peripheral artery diseases (PAD). However, the precise signaling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. Our previous study suggests that A-type voltage-gated K + (K V 4) channels regulate the excitability in muscle dorsal root ganglion (DRG) neurons of PAD rats; however, it is still lacking regarding the effects of PAD on characteristics of K V 4 currents and engagement of bradykinin (BK) subtype receptors. Thus, we examined K V 4 currents in two distinct muscle DRG neurons, namely isolectin B 4 -positive and B 4 -negative (IB4+ and IB4-) DRG neurons. IB4+ neurons express receptors for glial cell line-derived neurotrophic factor (GDNF), whereas IB4- DRG neurons are depending on nerve growth factors for survival. Our data showed that current density in muscle DRG neurons of PAD rats was decreased and this particularly appeared in IB4+ DRG neurons as compared with IB4- DRG neurons. We also showed that stimulation of BK B1 and B2 receptors led to a greater inhibitory effect on K V 4 currents in IB4+ muscle DRG neurons and siRNA knockdown of K V 4 subunit K V 4.3 decreased the activity of K V 4 currents in IB4+ DRG neurons. In conclusion, our data suggest that limb ischemia and/or ischemia-induced BK inhibit activity of K V 4 channels in a subpopulation of the thin fiber muscle afferent neurons depending on GDNF, which is likely a part of signaling pathways involved in the exaggerated blood pressure response during activation of muscle afferent nerves in PAD.

Background Peripheral artery disease (PAD) is associated with gastrocnemius muscle abnormalities. However, the biological pathways associated with gastrocnemius muscle dysfunction and their associations with progression of PAD are largely unknown. This study characterized differential gene and microRNA (miRNA) expression in gastrocnemius biopsies from people without PAD compared with those with PAD. Participants with PAD included those with and without PAD progression. Methods and Results mRNA and miRNA sequencing were performed to identify differentially expressed genes, differentially expressed miRNAs, mRNA-miRNA interactions, and associated biological pathways for 3 sets of comparisons: (1) PAD progression (n=7) versus non-PAD (n=7); (2) PAD no progression (n=6) versus non-PAD; and (3) PAD progression versus PAD no progression. Immunohistochemistry was performed to determine gastrocnemius muscle fiber types and muscle fiber size. Differentially expressed genes and differentially expressed miRNAs were more abundant in the comparison of PAD progression versus non-PAD compared with PAD with versus without progression. Among the top significant cellular pathways in subjects with PAD progression were muscle contraction or development, transforming growth factor-beta, growth/differentiation factor, and activin signaling, inflammation, cellular senescence, and notch signaling. Subjects with PAD progression had increased frequency of smaller Type 2a gastrocnemius muscle fibers in exploratory analyses. Conclusions Humans with PAD progression exhibited greater differences in the number of gene and miRNA expression, biological pathways, and Type 2a muscle fiber size compared with those without PAD. Fewer differences were observed between people with PAD without progression and control patients without PAD. Further study is needed to confirm whether the identified transcripts may serve as potential biomarkers for diagnosis and progression of PAD.

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.

Nitric oxide (NO) is the critical regulator of VEGFR2-induced angiogenesis. Neither VEGF-A over-expression nor L-Arginine (NO-precursor) supplementation has been effective in helping patients with Peripheral Artery Disease (PAD) in clinical trials. One incompletely studied reason may be due to the presence of the less characterized anti-angiogenic VEGF-A (VEGF 165 b) isoform. We have recently shown that VEGF 165 b inhibits ischemic angiogenesis by blocking VEGFR1, not VEGFR2 activation. Here we wanted to determine whether VEGF 165 b inhibition using a monoclonal isoform-specific antibody against VEGF 165 b vs. control, improved perfusion recovery in preclinical PAD models that have impaired VEGFR2-NO signaling, including (1) type-2 diabetic model, (2) endothelial Nitric oxide synthase-knock out mice, and (3) Myoglobin transgenic mice that have impaired NO bioavailability. In all PAD models, VEGF 165 b inhibition vs. control enhanced perfusion recovery, increased microvascular density in the ischemic limb, and activated VEGFR1-STAT3 signaling. In vitro, VEGF 165 b inhibition vs. control enhanced a VEGFR1-dependent endothelial survival/proliferation and angiogenic capacity. These data demonstrate that VEGF 165 b inhibition induces VEGFR1-STAT3 activation, which does not require increased NO to induce therapeutic angiogenesis in PAD. These results may have implications for advancing therapies for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired.

Previous studies have demonstrated that circulating microRNA (miR)-210 levels are elevated in peripheral artery disease (PAD) patients. MiR-210 is known to be a negative regulator of mitochondrial respiration; however, the relationship between miR-210 and mitochondrial function has yet to be studied in PAD. We aimed to compare skeletal muscle miR-210 expression of PAD patients to non-PAD controls (CON) and to examine the relationship between miR-210 expression and mitochondrial function. Skeletal muscle biopsies from CON (n = 20), intermittent claudication (IC) patients (n = 20), and critical limb ischemia (CLI) patients (n = 20) were analyzed by high-resolution respirometry to measure mitochondrial respiration of permeabilized fibers. Samples were also analyzed for miR-210 expression by real-time PCR. MiR-210 expression was significantly elevated in IC and CLI muscle compared to CON (P = 0.008 and P < 0.001, respectively). Mitochondrial respiration of electron transport chain (ETC) Complexes II (P = 0.001) and IV (P < 0.001) were significantly reduced in IC patients. Further, CLI patients demonstrated significant reductions in respiration during Complexes I (state 2: P = 0.04, state 3: P = 0.003), combined I and II (P < 0.001), II (P < 0.001), and IV (P < 0.001). The expression of the miR-210 targets, cytochrome c oxidase assembly factor heme A: farnesyltransferase (COX10), and iron-sulfur cluster assembly enzyme (ISCU) were down-regulated in PAD muscle. MiR-210 may play a role in the cellular adaptation to hypoxia and may be involved in the metabolic myopathy associated with PAD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Crohn's disease (CD) and peripheral arterial disease (PAD) are closely related. The pathophysiological mechanisms underlying the coexistence of CD and PAD are unknown. The aim of this study was to investigate the key molecules and pathways mediating the co-occurrence of CD and PAD through quantitative bioinformatic analysis of a public RNA sequencing database., Methods: Datasets of CD (GSE111889) and PAD (GSE120642) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were analyzed using the 'edgeR' and 'limma' packages of R. Gene Ontology and Kyoto Encyclopedia analyses of common DEGs were performed to explore the functions of DEGs. Protein-protein interaction (PPI) networks were established by the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized by Cytoscape. Hub genes were selected using the plugin cytoHubba. Hub gene validation was performed in GSE95095 for CD and GSE134431 for PAD. Receiver operating characteristic curves were used to evaluate the predictive values of the hub genes. Gene set enrichment analysis and immune infiltration of the hub genes were performed., Results: A total of 54 common DEGs (2 downregulated and 52 upregulated) were identified. Pathways of neutrophil chemotaxis, neutrophil migration and cytokine and cytokine receptors were enriched in CD and PAD. S100A8, S100A9, S100A12 and CXCR2 were identified as hub genes after validation, with all area under the curve > 0.7 for both CD and PAD. Neutrophil infiltration was associated with upregulation of the hub genes. Pathways of immune processes, including neutrophil activation, neutrophil chemotaxis, neutrophil migration were significantly correlated with high expression of S100A8, S100A9, S100A12 and CXCR2 in both CD and PAD., Conclusions: This bioinformatic study elucidates S100A8, S100A9, S100A12 and CXCR2 as hub genes for the co-occurrence of Crohn's disease and peripheral artery disease. Inflammation and immune regulation modulated by neutrophil infiltration play a central role in the development of CD and PAD and may be potential targets for diagnosis and treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yao, Zhang, Niu, Yan, Tong, Zou, Li and Yang.)

Background: Previous studies on the relationship between the visceral adiposity index (VAI) and peripheral arterial disease (PAD) are limited. Therefore, this study explored the relationship between VAI and PAD in normal-weight patients with hypertension., Methods: A total of 6615 normal-weight patients with hypertension were included in the current study. The VAI, a simple index calculated using blood lipid and waist circumference (WC), can be used as a simple biomarker of body fat distribution. The outcome was PAD, which was defined as present when each side's ankle-brachial index (ABI) was ≤ 0.90., Results: A significant positive association was observed between VAI and PAD prevalence. For per unit increment in LnVAI, the adjusted odds ratios (ORs) of PAD for the total participants and males were 1.55 (95% CI 1.15-2.10) and 2.12 (95% CI 1.46-3.07), respectively. However, the VAI was not associated with PAD in female patients with hypertension (OR 1.28; 95% confidence interval [CI] 0.85-1.95). There was no interaction between sex and VAI (P for interaction = 0.128). Accordingly, in total participants, when VAI was assessed in quartiles and compared with quartile 1 (< 0.84), the PAD prevalence was higher than that of quartiles 2 (0.84 to < 1.36: OR 1.49; 95% CI 0.92-2.44), 3 (1.36 to < 2.25: OR 1.95; 95% CI 1.14-3.32), and 4 (≥ 2.25: OR 1.93; 95% CI 1.04-3.57). There were no significant interactions with the other confounders., Conclusion: This study showed a positive association between VAI and PAD in normal-weight adults with hypertension among men but not among women., (© 2022. The Author(s).)

Peripheral artery disease (PAD) is an atherosclerotic disease that impairs blood flow and muscle function in the lower limbs. A skeletal muscle myopathy characterized by mitochondrial dysfunction and oxidative damage is present in PAD; however, the underlying mechanisms are not well established. We investigated the impact of chronic ischemia on skeletal muscle microcirculatory function and its association with leg skeletal muscle mitochondrial function and oxygen delivery and utilization capacity in PAD. Gastrocnemius samples and arterioles were harvested from patients with PAD ( n = 10) and age-matched controls (Con, n = 11). Endothelium-dependent and independent vasodilation was assessed in response to flow (30 μL·min -1 ), acetylcholine, and sodium nitroprusside (SNP). Skeletal muscle mitochondrial respiration was quantified by high-resolution respirometry, microvascular oxygen delivery, and utilization capacity (tissue oxygenation index, TOI) were assessed by near-infrared spectroscopy. Vasodilation was attenuated in PAD ( P < 0.05) in response to acetylcholine (Con: 71.1 ± 11.1%, PAD: 45.7 ± 18.1%) and flow (Con: 46.6 ± 20.1%, PAD: 29.3 ± 10.5%) but not SNP ( P = 0.30). Complex I + II state 3 respiration ( P < 0.01) and TOI recovery rate were impaired in PAD ( P < 0.05). Both flow and acetylcholine-mediated vasodilation were positively associated with complex I + II state 3 respiration ( r = 0.5 and r = 0.5, respectively, P < 0.05). Flow-mediated vasodilation and complex I + II state 3 respiration were positively associated with TOI recovery rate ( r = 0.8 and r = 0.7, respectively, P < 0.05). These findings suggest that chronic ischemia attenuates skeletal muscle arteriole endothelial function, which may be a key mediator for mitochondrial and microcirculatory dysfunction in the PAD leg skeletal muscle. Targeting microvascular dysfunction may be an effective strategy to prevent and/or reverse disease progression in PAD. NEW & NOTEWORTHY Ex vivo skeletal muscle arteriole endothelial function is impaired in claudicating patients with PAD, and this is associated with attenuated skeletal muscle mitochondrial respiration. In vivo skeletal muscle oxygen delivery and utilization capacity is compromised in PAD, and this may be due to microcirculatory and mitochondrial dysfunction. These results suggest that targeting skeletal muscle arteriole function may lead to improvements in skeletal muscle mitochondrial respiration and oxygen delivery and utilization capacity in claudicating patients with PAD.

Peripheral arterial disease (PAD) is caused by atherosclerotic plaque accumulation, which results in ischemia in lower extremity ischemia. Cell-based therapy using endothelial progenitor cells (EPCs) or endothelial cells (ECs) has been challenging due to an insufficient number and replicative senescence of primary cells isolated from patients. To overcome this limitation, we generated induced pluripotent stem cells (iPSCs) from a patient with PAD for the first time. The patient-specific iPSCs have unlimited proliferation and can be used to generate a clinically relevant number of functional ECs. Here we developed a strategy to efficiently generate high EC yields within 5 days of differentiation. The generated iPSC-derived ECs from a PAD patient were phenotypically and functionally similar to the primary blood outgrowth endothelial cells (BOECs) and iPSC-ECs derived from healthy donors as evidenced by expression of EC-specific markers, capillary-like tube-forming potential, and the ability to uptake acetylated low-density lipoprotein (Ac-LDL). Our approach may provide an alternative renewable source of large-scale ECs for regenerative therapy. This study represents the first step toward the development of an autologous cell-based strategy for the treatment of PAD in the future., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD., Methods: Paired EAT and SAT samples were collected from 82 patients undergoing elective cardiac surgery either for coronary artery bypass grafting (CAD group, N = 66) or valve surgery (NCAD group, N = 16). ABCA1 and ABCG1 mRNA levels in EAT and SAT samples were analyzed using real time polymerase chain reaction, ABCA1 protein levels in EAT samples were assessed by western blotting. ABCA1 and ABCG1 DNA methylation analysis was performed in 24 samples from the CAD group and 9 samples from the NCAD group via pyrosequencing., Results: DNA methylation levels in the ABCA1 promoter and ABCG1 cg27243685 and cg06500161 CpG sites were higher in EAT samples from patients with CAD compared with NCAD (21.92% vs 10.81%, p = 0.003; 71.51% vs 68.42%, p = 0.024; 46.11% vs 37.79%, p = 0.016, respectively). In patients with CAD, ABCA1 and ABCG1 DNA methylation levels were higher in EAT than in SAT samples (p < 0.05). ABCA1 mRNA levels in EAT samples were reduced in the subgroup of patients with CAD and concomitant carotid artery disease or peripheral artery disease compared with the NCAD group (p = 0.024). ABCA1 protein levels in EAT samples tended to be lower in CAD patients than in the NCAD group (p = 0.053). DNA methylation levels at the ABCG1 cg27243685 site positively correlated with plasma triglyceride concentration (r = 0.510, p = 0.008), body mass index (r = 0.556, p = 0.013) and waist-to-hip ratio (r = 0.504, p = 0.012) in SAT samples., Conclusion: CAD is associated with ABCA1 and ABCG1 DNA hypermethylation in EAT. CAD with concomitant carotid artery disease or peripheral artery disease is accompanied by decreased ABCA1 gene expression in EAT. DNA methylation levels at the ABCG1 cg27243685 locus in SAT are associated with hypertriglyceridemia and obesity., (© 2021. The Author(s).)

The mechanoreflex is exaggerated in patients with peripheral artery disease (PAD) and in a rat model of simulated PAD in which a femoral artery is chronically (∼72 h) ligated. We found recently that, in rats with a ligated femoral artery, blockade of thromboxane A 2 (TxA 2 ) receptors on the sensory endings of thin fiber muscle afferents reduced the pressor response to 1 Hz repetitive/dynamic hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). Conversely, we found no effect of TxA 2 receptor blockade in rats with freely perfused femoral arteries. Here, we extended the isolated mechanoreflex findings in "ligated" rats to experiments evoking dynamic hindlimb skeletal muscle contractions. We also investigated the role played by inositol 1,4,5-trisphosphate (IP 3 ) receptors, receptors associated with intracellular signaling linked to TxA 2 receptors, in the exaggerated response to dynamic mechanoreflex and exercise pressor reflex activation in ligated rats. Injection of the TxA 2 receptor antagonist daltroban into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic contraction in ligated but not "freely perfused" rats. Moreover, injection of the IP 3 receptor antagonist xestospongin C into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic stretch and contraction in ligated but not freely perfused rats. These findings demonstrate that, in rats with a ligated femoral artery, sensory neuron TxA 2 receptor and IP 3 receptor-mediated signaling contributes to a chronic sensitization of the mechanically activated channels associated with the mechanoreflex and the exercise pressor reflex.

Introduction: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC., Methods: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique., Results: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening., Conclusion: R/NIR vasodilation requires indirect activation of the BKca channel., Competing Interests: The authors have declared that no competing interests exist.

Recent evidence demonstrates an involvement of impaired mitochondrial function in peripheral arterial disease (PAD) development. Specific impairments have been assessed by different methodological in-vivo (near-infrared spectroscopy, 31 P magnetic resonance spectroscopy), as well as in-vitro approaches (Western blotting of mitochondrial proteins and enzymes, assays of mitochondrial function and content). While effects differ with regard to disease severity, chronic malperfusion impacts subcellular energy homeostasis, and repeating cycles of ischemia and reperfusion contribute to PAD disease progression by increasing mitochondrial reactive oxygen species production and impairing mitochondrial function. With the leading clinical symptom of decreased walking capacity due to intermittent claudication, PAD patients suffer from a subsequent reduction of quality of life. Different treatment modalities, such as physical activity and revascularization procedures, can aid mitochondrial recovery. While the relevance of these modalities for mitochondrial functional recovery is still a matter of debate, recent research indicates the importance of revascularization procedures, with increased physical activity levels being a subordinate contributor, at least during mild stages of PAD. With an additional focus on the role of revascularization procedures on mitochondria and the identification of suitable mitochondrial markers in PAD, this review aims to critically evaluate the relevance of mitochondrial function in PAD development and progression.

Background: Lipoprotein-related traits have been consistently identified as risk factors for atherosclerotic cardiovascular disease, largely on the basis of studies of coronary artery disease (CAD). The relative contributions of specific lipoproteins to the risk of peripheral artery disease (PAD) have not been well defined. We leveraged large-scale genetic association data to investigate the effects of circulating lipoprotein-related traits on PAD risk., Methods: Genome-wide association study summary statistics for circulating lipoprotein-related traits were used in the mendelian randomization bayesian model averaging framework to prioritize the most likely causal major lipoprotein and subfraction risk factors for PAD and CAD. Mendelian randomization was used to estimate the effect of apolipoprotein B (ApoB) lowering on PAD risk using gene regions proxying lipid-lowering drug targets. Genes relevant to prioritized lipoprotein subfractions were identified with transcriptome-wide association studies., Results: ApoB was identified as the most likely causal lipoprotein-related risk factor for both PAD (marginal inclusion probability, 0.86; P =0.003) and CAD (marginal inclusion probability, 0.92; P =0.005). Genetic proxies for ApoB-lowering medications were associated with reduced risk of both PAD (odds ratio,0.87 per 1-SD decrease in ApoB [95% CI, 0.84-0.91]; P =9×10 -10 ) and CAD (odds ratio,0.66 [95% CI, 0.63-0.69]; P =4×10 -73 ), with a stronger predicted effect of ApoB lowering on CAD (ratio of effects, 3.09 [95% CI, 2.29-4.60]; P <1×10 -6 ). Extra-small very-low-density lipoprotein particle concentration was identified as the most likely subfraction associated with PAD risk (marginal inclusion probability, 0.91; P =2.3×10 -4 ), whereas large low-density lipoprotein particle concentration was the most likely subfraction associated with CAD risk (marginal inclusion probability, 0.95; P =0.011). Genes associated with extra-small very-low-density lipoprotein particle and large low-density lipoprotein particle concentration included canonical ApoB pathway components, although gene-specific effects were variable. Lipoprotein(a) was associated with increased risk of PAD independently of ApoB (odds ratio, 1.04 [95% CI, 1.03-1.04]; P =1.0×10 -33 )., Conclusions: ApoB was prioritized as the major lipoprotein fraction causally responsible for both PAD and CAD risk. However, ApoB-lowering drug targets and ApoB-containing lipoprotein subfractions had diverse associations with atherosclerotic cardiovascular disease, and distinct subfraction-associated genes suggest possible differences in the role of lipoproteins in the pathogenesis of PAD and CAD.

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.

Background: Peripheral arterial disease (PAD) is accompanied by myopathy characterized by mitochondrial dysfunction. The aim of this experimental study was to investigate the effect of revascularization procedures on mitochondrial function in ischemic and non-ischemic muscle., Methods: Muscle biopsies from patients with symptomatic stage IIB/III PAD caused by isolated pathologies of the superficial femoral artery were obtained from muscle regions within the chronic ischemic muscle (gastrocnemius) and from non-ischemic muscle (vastus lateralis) before and 6 weeks after invasive revascularization. High-resolution respirometry was used to investigate mitochondrial function and results were normalized to citrate synthase activity (CSA). Results are given in absolute values and fold over basal (FOB)., Results: Respiratory states (OXPHOS (P) and electron transfer (E) capacity) normalized to CSA decreased while CSA was increased in chronic ischemic muscle after revascularization. There were no changes in in non-ischemic muscle. The FOB of chronic ischemic muscle was significantly higher for CSA (chronic ischemic 1.37 (IQR 1.10-1.64) vs. non-ischemic 0.93 (IQR 0.69-1.16) p = 0.020) and significantly lower for respiratory states normalized to CSA when compared to the non-ischemic muscle (P per CSA chronic ischemic 0.64 (IQR 0.46-0.82) vs non-ischemic 1.16 (IQR 0.77-1.54) p = 0.011; E per CSA chronic ischemic 0.61 (IQR 0.47-0.76) vs. non-ischemic 1.02 (IQR 0.64-1.40) p = 0.010)., Conclusions: Regeneration of mitochondrial content and function following revascularization procedures only occur in muscle regions affected by malperfusion. This indicates that the restoration of blood and oxygen supply are important mediators aiding mitochondrial recovery.

Ruan Jian Qing Mai formula (RJQM), a multicomponent herbal formula, has been widely used to treat peripheral arterial disease (PAD) in China. However, its active compounds and mechanisms of action are still unknown. First, RNA sequencing analysis of 15 healthy and 16 PAD samples showed that 524 PAD differential genes were significantly enriched in Go Ontology (ribonucleotide metabolic process, oxidoreductase complex, and electron transfer activity), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA pathways (OXPHOS and TCA cycle), miRNA (MIR183), and kinase (PAK6). Fifty-three active ingredients in RJQM had similar structures to the seven drug molecules in CLUE. Then, network topology analysis of the 53 components-target-pathway-disease network yielded 10 active ingredients. Finally, computational toxicity estimations showed that the median lethal dose (LD50) of the 10 active ingredients was above 1000 mg/kg, and eight of them did not cause hepatotoxicity, mutagenicity, carcinogenicity, cytotoxicity, and immunotoxicity nor activate 12 toxic pathways. In conclusion, RJQM has a protection effect on PAD by regulating a complex molecular network. Part of the mechanism is associated with the regulation of OXPHOS by 10 active components, which may alleviate mitochondrial dysfunction and pathological metabolic programming., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.