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BACKGROUND: Systemic cytokines are elevated in patients with chronic kidney disease (CKD) and on hemodialysis compared with the general population. However, whether cytokine levels interfere with vascular remodeling, increasing the risk of arteriovenous fistula (AVF) failure, remains unknown. METHODS: This is a case-control study of 64 patients who underwent surgery for AVF creation (32 with AVF maturation failure and 32 matching controls with successful maturation). A total of 74 cytokines, including chemokines, interferons, interleukins, and growth factors, were measured in preoperative plasma samples using multiplex assays. Sixty-two patients were included in the statistical analyses. Associations with AVF failure were assessed using paired comparisons and conditional logistic regressions accounting for paired strata. RESULTS: Seven cytokines were significantly higher in patients with AVF maturation failure than in matching controls (G-CSF, IL-6, MDC, RANTES, SDF-1α/β, TGFα, and TPO). Of these, G-CSF (odds ratio [OR]=1.71; 95% confidence interval [95% CI], 1.05 to 2.79 per 10 pg/ml), MDC (OR=1.60, 95% CI, 1.08 to 2.38 per 100 pg/ml), RANTES (OR=1.55, 95% CI, 1.10 to 2.17 per 100 pg/ml), SDF-1α/β (OR=1.18, 95% CI, 1.04 to 1.33 per 1000 pg/ml), and TGFα (OR=1.39, 95% CI 1.003, 1.92 per 1 pg/ml) showed an incremental association by logistic regression. CONCLUSIONS: This study identified a profile of plasma cytokines associated with adverse maturation outcomes in AVFs. These findings may open the doors for future therapeutics and markers for risk stratification.

Endovascular stent fractures are commonly seen in arteries but are rare events in the venous system. Stents deployed in hemodialysis vascular accesses can fracture and migrate to proximal locations. Complications associated with stent fracture include in-stent stenosis and central vein stenosis. In this report, we present a unique case of a hemodialysis access stent fracture that migrated to the left ventricle and manifested with chest pain.

INTRODUCTION: The molecular transformation of the human preaccess vein after arteriovenous fistula (AVF) creation is poorly understood. This limits our ability to design efficacious therapies to improve maturation outcomes. METHODS: Bulk RNA sequencing (RNA-seq) followed by paired bioinformatic analyses and validation assays were performed in 76 longitudinal vascular biopsies (veins and AVFs) from 38 patients with stage 5 chronic kidney disease or end-stage kidney disease undergoing surgeries for 2-stage AVF creation (19 matured, 19 failed). RESULTS: A total of 3637 transcripts were differentially expressed between veins and AVFs independent of maturation outcomes, with 80% upregulated in fistulas. The postoperative transcriptome demonstrated transcriptional activation of basement membrane and interstitial extracellular matrix (ECM) components, including preexisting and novel collagens, proteoglycans, hemostasis factors, and angiogenesis regulators. A postoperative intramural cytokine storm involved >80 chemokines, interleukins, and growth factors. Postoperative changes in ECM expression were differentially distributed in the AVF wall, with proteoglycans and fibrillar collagens predominantly found in the intima and media, respectively. Interestingly, upregulated matrisome genes were enough to make a crude separation of AVFs that failed from those with successful maturation. We identified 102 differentially expressed genes (DEGs) in association with AVF maturation failure, including upregulation of network collagen VIII in medial smooth muscle cells (SMCs) and downregulation of endothelial-predominant transcripts and ECM regulators. CONCLUSION: This work delineates the molecular changes that characterize venous remodeling after AVF creation and those relevant to maturation failure. We provide an essential framework to streamline translational models and our search for antistenotic therapies.

Periadventitial biomaterials have been employed for nearly three decades to promote adaptive venous remodeling following hemodialysis vascular access creation in preclinical models and clinical trials. These systems are predicated on the combination of scaffolds, hydrogels, and/or particles with therapeutics (small molecules, proteins, genes, and cells) to prevent venous stenosis and subsequent maturation failure. Periadventitial biomaterial therapies have evolved from simple drug delivery vehicles for traditional drugs to more thoughtful designs tailored to the pathophysiology of access failure. The emergence of tissue engineering strategies and gene therapies are another exciting new direction. Despite favorable results in experimental and preclinical studies, no periadventitial therapy has been clinically approved to improve vascular access outcomes. After conducting an exhaustive review of the literature, we identify the seminal studies and clinical trials that utilize periadventitial biomaterials and discuss the key features of each biomaterial format and their respective shortcomings as they pertain to access maturation. This review provides a foundation from which clinicians, surgeons, biologists, and engineers can refer to and will hopefully inspire thoughtful, translatable treatments to finally address access failure.

The development of venous intimal hyperplasia (IH) has been historically associated with failure of arteriovenous fistulas (AVF) used for hemodialysis. This long-standing assumption, based on histological observations, has been recently challenged by clinical studies indicating that the size of the intima by itself is not enough to explain stenosis or AVF maturation failure. Irrespective of this lack of association, IH is present in most native veins and fistulas, is prominent in many cases, and suggests a role in the vein that may not be reflected by its dimensions. Therefore, the contribution of IH to AVF dysfunction remains controversial. Using only clinical data and avoiding extrapolations from animal models, we critically discuss the biological significance of IH in vein remodeling, vascular access function, and the response of the venous wall to repeated trauma in hemodialysis patients. We address questions and pose new ones such as: What are the factors that contribute to IH in pre-access veins and AVFs? Do cellular phenotypes and composition of the intima influence AVF function? Are there protective roles of the venous intima? This review explores these possibilities, with hopes of rekindling a critical discussion about venous IH that goes beyond thickness and AVF outcomes.

Pulmonary hypertension (PH) is common in end-stage renal disease (ESRD) patients and is associated with increased all-cause and cardiovascular mortality in this group. There is scarce data on the long-term effect of arteriovenous fistula (AVF) creation on pulmonary hypertension (PH) and the reflected changes in echocardiographic measurements.This is a retrospective study of 54 patients who underwent AVF creation between 2009 and 2014 and with echocardiographic evaluations before and after surgery. We analyzed pairwise changes in right ventricular systolic pressure (RVSP), right atrial pressure (RAP) during systole, left ventricular mass (LVM), tricuspid regurgitation (TR), mitral E/E' ratio, and ejection fraction (EF), as well as the factors that predicted change in RVSP after surgery.The median time for the preoperative echocardiogram was 0.3 years (interquartile range (IQR) 0.2 - 0.7 years) prior to AVF creation, while the follow-up echo was done 1.3 (0.6 - 2.1) years after surgery. 67% of the patients had RVSP gt; 37 mmHg at baseline. There was a significant reduction in RVSP after AVF creation compared to baseline (median 33 (IQR 26 - 43) vs. 46 mmHg, p = 0.0015), with 59% of the patients experiencing a decrease and 19% remaining stable. There were also significant decreases in LVM (201 (143 - 256) vs. 215 (163 - 276), p = 0.045) and RAP systole (10 (10 - 15) vs. 3 (3 - 8); p lt; 0.001) after surgery. Higher preoperative weight (p = 0.038) and RVSP (p = 0.006), and use of loop diuretics (p = 0.015) were significantly associated with improvement in RVSP after AVF creation.Our results suggest that AVF creation is associated with a significant reduction or stable measurements of RVSP in the ESRD population, likely due to an improvement in volume status.

Neointimal cells are an elusive population with ambiguous origins, functions, and states of differentiation. Expansion of the venous intima in arteriovenous fistula (AVF) is one of the most prominent remodeling processes in the wall after access creation. However, most of the current knowledge about neointimal cells in AVFs comes from extrapolations from the arterial neointima in non-AVF systems. Understanding the origin of neointimal cells in fistulas may have important implications for the design and effective delivery of therapies aimed to decrease intimal hyperplasia (IH). In addition, a broader knowledge of cellular dynamics during postoperative remodeling of the AVF may help clarify other transformation processes in the wall that combined with IH determine the successful remodeling or failure of the access. In this review, we discuss the possible anatomical sources of neointimal cells in AVFs and their relative contribution to intimal expansion.

BACKGROUND: The role of hyaluronan (HA) in the development and progression of diabetic kidney disease (DKD), as well as the precise mechanisms and consequences of HA involvement in this pathology are still to be clarified. METHODS: In this study, we assayed the effects of the HA synthesis inhibitor 4-methylumbelliferone (4-MU) on the development of DKD. Diabetic type 2 model mice (eNOS(−/−) C57BLKS/J(db)) were fed artificial diets containing 5% 4-MU or not for 9 weeks. Plasma glucose, glomerular filtration rate (GFR), albumin to creatinine ratio (ACR), and biomarkers of kidney function and systemic inflammation were measured at baseline and after treatment. Diabetic nephropathy was further characterized in treated and control mice by histopathology. RESULTS: Treated animals consumed a daily dose of approximately 6.2 g of 4-MU per kg of body weight. At the end of the experimental period, the 4-MU supplemented diet resulted in a significant decrease in non-fasting plasma glucose (516 [interquartile range 378–1170] vs. 1149 [875.8–1287] mg/dL, P=0.050) and a trend toward lower HA kidney content (5.6 ± 1.5 vs. 8.8 ± 3.1 ng/mg of kidney weight, P=0.070) compared to the control diet, respectively. Diabetic animals treated with 4-MU showed significantly higher GFR and lower urine ACR and plasma cystatin C levels than diabetic controls. Independent histological assessment of DKD also demonstrated a significant decrease in mesangial expansion score and glomerular injury index in 4-MU-treated mice compared to controls. Plasma glucose showed a strong correlation with kidney HA levels (r=0.66, P=0.0098). Both total hyaluronan (r=0.76, P=0.0071) and low-molecular-weight hyaluronan content (r=0.64, P=0.036) in the kidneys correlated with urine ACR in mice. CONCLUSION: These results show that the hyaluronan synthesis inhibitor 4-MU effectively slowed the progression of DKD and constitutes a potential new therapeutic approach to treat DKD.

Background and aims Increased receptor tyrosine kinase (RTK) activity has been historically linked to atherosclerosis. Paradoxically, we recently found that global deficiency in c-Kit function increased atherosclerosis in hyperlipidemic mice. This study aimed to investigate if such unusual atheroprotective phenotype depends upon c-Kit's function in smooth muscle cells (SMC). Methods We studied atherosclerosis in a SMC-specific conditional knockout mice (KitSMC) and control littermate. Tamoxifen (TAM) and vehicle treated mice were fed high fat diet for 16 weeks before atherosclerosis assessment in the whole aorta using oil red staining. Smooth muscle cells were traced within the aortic sinus of conditional c-Kit tracing mice (KitSMC eYFP) and their control littermates (KitWT eYFP) by immunofluorescent confocal microscopy. We then performed RNA sequencing on primary SMC from c-Kit deficient and control mice, and identified significantly altered genes and pathways as a result of c-Kit deficiency in SMC. Results Atherosclerosis significantly increased in KitSMC mice with respect to control groups. In addition, the loss of c-Kit in SMC increased plaque size and necrotic core area in the aortic sinus of hyperlipidemic mice. Smooth muscle cells from KitSMC eYFP mice were more prone to migrate and express foam cell markers (e.g., Mac2 and MCAM) than those from control littermate animals. RNAseq analysis showed a significant upregulation in genes associated with cell proliferation, migration, lipid metabolism, and inflammation secondary to the loss of Kit function in primary SMCs. Conclusions Loss of c-Kit increases SMC migration, proliferation, and expression of foam cell markers in atherosclerotic plaques from hyperlipidemic mice.

The rate of arteriovenous fistula (AVF) maturation failure remains unacceptably high despite continuous efforts on technique improvement and careful pre-surgery planning. In fact, half of all newly created AVFs are unable to be used for hemodialysis (HD) without a salvage procedure. While vascular stenosis in the venous limb of the access is the culprit, the underlying factors leading to vascular narrowing and AVF maturation failure are yet to be determined. We have recently demonstrated that AVF non-maturation is associated with post-operative medial fibrosis and fibrotic stenosis, and post-operative intimal hyperplasia (IH) exacerbates the situation. Multiple pathological processes and signaling pathways are underlying the stenotic remodeling of the AVF. Our group has recently indicated that a pro-inflammatory cytokine platelet factor 4 (PF4/CXCL4) is upregulated in veins that fail to mature after AVF creation. Platelet factor 4 is a fibrosis marker and can be detected in vascular stenosis tissue, suggesting that it may contribute to AVF maturation failure through stimulation of fibrosis and development of fibrotic stenosis. Here, we present an overview of the how PF4-mediated fibrosis determines AVF maturation failure.

Introduction: In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established. Hypothesis: Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice. Methods: qRT-PCR, Western blot, and scRNAseq demonstrated the loss of Lox gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between Lox gene expression and Frizzled-related protein 3 ( Frzb ), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of Bmp2 and Myc , both β-catenin/TCF4 regulated genes, in SMC following gene deletion of Lox and 8 weeks of HFD. LOX knockout SMCs also had lower expression of Frzb and were more resistant to osteoblastic transformation in vitro. Conclusions: Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs.

Introduction: Arteriovenous fistula (AVF) nonmaturation is associated with postoperative fibrosis and an increase in lysyl oxidase (LOX) expression. Objective: The purpose of this study was to determine whether unidirectional periadventitial delivery of β-aminoproprionitrile (BAPN) using bilayer poly lactic-co-glycolic acid nanofiber scaffolds is a feasible approach to reduce AVF fibrosis and encourage adaptive vascular remodeling. Methods: AVFs were created in thirty Sprague Dawley rats (200-350 g) of both sexes by an end-to-side anastomosis of the epigastric vein to the femoral artery. BAPN-loaded (b-BAPN) (n=10) or vehicle (b-VEH) (n=10) scaffolds (5 mm x 5 mm) were wrapped around the juxta-anastomotic zone of the epigastric vein immediately after anastomosis. AVFs without treatment were used as an additional control (n=10). Changes in lumen diameter were followed-up weekly using ultrasound. Flow was calculated using the pulse-wave velocity and lumen diameter. AVFs were collected at 21 days for histomorphometric analysis and to assess fibrosis. Results: b-BAPN significantly increased dilation of AVFs at day 14 (185.9%) compared to b-VEH (135.5%; p=0.0006) and control AVFs (113.6%; p2 vs. 95,580 μm 2 ; p=0.0048). While there was no difference in intimal growth (p=0.5460), b-BAPN significantly reduced luminal occlusion with respect to control AVFs (0.0611 vs. 0.2067; p=0.0002). Finally, b-BAPN (49.48%) significantly reduced the fibrosis percent area compared to b-VEH (72.14%; p=0.0016) and control AVFs (74.5%; p=0.0007). Conclusion: Periadventitial LOX inhibition prevents AVF postoperative fibrosis and promotes adaptive vascular remodeling.

Introduction: The arteriovenous fistula (AVF) is the lifeline for patients with end-stage kidney disease (ESKD) requiring hemodialysis therapy. The mechanism of “arterialization”, by which a vein transforms into an AVF after anastomosis, is one of the most understudied transformative processes in vascular biology despite the urgent need for new interventions to facilitate and accelerate fistula usability to improve the life of these patients. Hypothesis: Dysregulation of type VIII collagen biosynthesis increases the risk for failure in newly created AVFs Methods and Results: We first deciphered the transcriptional transformation of the pre-access vein after arterial anastomosis using paired venous samples from 38 CKD patients undergoing surgeries for two-stage AVF creation. A total of 3,637 transcripts were differentially expressed (DE) between veins and AVFs in pairwise analyses (log2FC ≥ 1 or ≤ -1, FDR Conclusions: Vascular accumulation of type VIII collagen in the AVF wall after anastomosis strongly increases the risk of failure.

The number of people worldwide living with end-stage renal disease is increasing. Arteriovenous fistulas are the preferred method of vascular access in patients who will require hemodialysis. As the number of patients with arteriovenous fistulas grows, the role of physicians who intervene who maintain and salvage these fistulas will grow in importance. This review aims to familiarize practitioners with the rationale for arteriovenous fistula creation, the detection of fistula dysfunction, and the state of the art on fistula maintenance and preservation. Current controversies are briefly reviewed.

Objective Novel therapeutic angiogenic concepts for critical limb ischemia are still needed for limb salvage. E-selectin, a cell-adhesion molecule, is vital for recruitment of the stem/progenitor cells necessary for neovascularization in ischemic tissues. We hypothesized that priming ischemic limb tissue with E-selectin/adeno-associated virus (AAV) gene therapy, in a murine hindlimb ischemia and gangrene model, would increase therapeutic angiogenesis and improve gangrene. Methods FVB/NJ mice were given intramuscular hindlimb injections of either E-selectin/AAV or LacZ/AAV and then underwent induction of gangrene via femoral artery ligation and concomitant systemic injections of the nitric oxide synthesis inhibitor L-NAME (L-NG-Nitro arginine methyl ester; 40 mg/kg). Gangrene was evaluated via the Faber hindlimb appearance score. The rate of ischemic limb reperfusion and ischemic tissue angiogenesis were evaluated using laser Doppler perfusion imaging and DiI perfusion with confocal laser scanning microscopy of the ischemic footpads, respectively. The treadmill exhaustion test was performed on postoperative day (POD) 8 to determine hindlimb functionality. Results The E-selectin/AAV–treated mice (n = 10) had decreased Faber ischemia scores compared with those of the LacZ/AAV–treated mice (n = 7) at both PODs 7 and 14 (P < .05 and P < .01, respectively), improved laser Doppler perfusion imaging reperfusion indexes by POD 14 (P < .01), and greater gangrene footpad capillary density (P < .001). E-selectin/AAV–treated mice also had improved exercise tolerance (P < .05) and lower relative muscular atrophy (P < .01). Conclusion We surmised that E-selectin/AAV gene therapy would significantly promote hindlimb angiogenesis, reperfusion, and limb functionality in mice with hindlimb ischemia and gangrene. Our findings highlight the reported novel gene therapy approach to critical limb ischemia as a potential therapeutic option for future clinical studies., Clinical Relevance In the United States, >150,000 limb amputations are performed annually for critical limb ischemia (CLI) despite the use of medical and surgical therapy. Thus, novel therapeutic angiogenic concepts for CLI are still needed for limb salvage. We used a novel gene therapy approach in a mouse model of gangrene, the most severe form of CLI, to demonstrate the efficacy of our gene therapy with E-selectin. Preclinical studies such as ours are a vital step in the development of new therapies for CLI patients with threatened limbs and no further medical or surgical options., Visual abstract

Pharmacological activation of integrin CD11b/CD18 (αMβ2, Mac-1, and CR3) shows anti-inflammatory benefits in a variety of animal models of human disease, and it is a novel therapeutic strategy. Reasoning that genetic models can provide an orthogonal and direct system for the mechanistic study of CD11b agonism, we present in this study, to our knowledge, a novel knock-in model of constitutive active CD11b in mice. We genetically targeted the Itgam gene (which codes for CD11b) to introduce a point mutation that results in the I332G substitution in the protein. The I332G mutation in CD11b promotes an active, higher-affinity conformation of the ligand-binding I/A-domain (CD11b αA-domain). In vitro, this mutation increased adhesion of knock-in neutrophils to fibrinogen and decreased neutrophil chemotaxis to a formyl–Met–Leu–Phe gradient. In vivo, CD11bI332G animals showed a reduction in recruitment of neutrophils and macrophages in a model of sterile peritonitis. This genetic activation of CD11b also protected against development of atherosclerosis in the setting of hyperlipidemia via reduction of macrophage recruitment into atherosclerotic lesions. Thus, our animal model of constitutive genetic activation of CD11b can be a useful tool for the study of integrin activation and its potential contribution to modulating leukocyte recruitment and alleviating different inflammatory diseases.

Early remodeling of the arteriovenous fistula (AVF) determines maturation outcomes. However, the cellular response of the venous wall early after AVF creation remains largely enigmatic because of the lack of venous biopsies obtained shortly after anastomosis. This report presents a detailed immunohistochemistry analysis of a pre-access cephalic vein and the resulting seven-day-old AVF that required ligation due to steal syndrome. We test for markers of mature and progenitor endothelial cells (CD31, CD34, VWF), contractile smooth muscle cells and myofibroblasts (MYH11, SMA), and immune cell populations (CEACAM8, CD3, CD20, CD11b, CD45, CD68, CD163, tryptase). We demonstrated near complete endothelial coverage of the fistula at 7 days, a high degree of wall neovascularization, pronounced loss of myofibroblasts and smooth muscle cells, and significant infiltration of mast cells, neutrophils, monocytes, and macrophages. Of interest, the presence of CD163+ macrophages in the AVF suggests a reactive response to increased intramural oxygenation. In conclusion, these images provide for the first time a glimpse of early remodeling in a human AVF by immunohistochemistry. This case demonstrates the possibility to obtain additional precious samples of this early stage through future multicenter collaborative efforts.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

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

AimsNovel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile.Methods and ResultsFemoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP+ (MSCGFP) or E-selectin-GFP+ (MSCE−selectin−GFP). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSCE−selectin−GFP-treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSCE−selectin−GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSCE−selectin−GFP-treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation.ConclusionThis innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies.

Background Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration and accumulation, causing venous neointimal hyperplasia (VNH)/venous stenosis (VS) in arteriovenous fistula (AVF). The effect of blocking CX3CR1 using an anti-human variable VHH molecule (hCX3CR1 VHH, BI 655088) on VNH/VS was determined using a humanized mouse in which the human CX3CR1 (hCX3CR1) gene was knocked in (KI). Methods Whole-transcriptomic RNA sequencing with bioinformatics analysis was used on human stenotic AVF samples, C57BL/6J, hCX3CR1 KI mice with AVF and CKD, and in in vitro experiments to identify the pathways involved in preventing VNH/VS formation after hCX3CR1 VHH administration. Results Accumulation of CX3CR1 and CD68 was significantly increased in stenotic human AVFs. In C57BL/6J mice with AVF, there was increased Cx3cr1, Cx3cl1, Cd68, and Tnf-α gene expression, and increased immunostaining of CX3CR1 and CD68. In hCX3CR1-KI mice treated with hCX3CR1 VHH molecule (KI-A), compared with vehicle controls (KI-V), there was increased lumen vessel area and patency, and decreased neointima in the AVF outflow veins. RNA-seq analysis identified TNF-α and NF-κB as potential targets of CX3CR1 inhibition. In KI-A-treated vessels compared with KI-V, there was decreased gene expression of Tnf- α, Mcp-1, and Il-1 β; with reduction of Cx3cl1, NF-κB, and Cd68; decreased M1, Ly6C, smooth muscle cells, fibroblast-activated protein, fibronectin, and proliferation; and increased TUNEL and M2 staining. In cell culture, monocytes stimulated with PMA and treated with hCX3CR1 VHH had decreased TNF- α, CD68, proliferation, and migration. Conclusions CX3CR1 blockade reduces VNH/VS formation by decreasing proinflammatory cues.

Background and Aims Low arteriovenous fistula (AVF) maturation rate remains a bottleneck in creating a long lasting lifeline to hemodialysis. AVF maturation is determined by the intricate balance between outward remodeling (OR) and formation of intimal hyperplasia (IH), during which vascular smooth muscle cell (VSMC) proliferation is essential. Recently, the von Willebrand Factor (VWF) has been shown to induce VSMC proliferation in vitro and in vivo. In this study we investigate the role of VWF on VSMC proliferation and AVF functionality to gain a better understanding of the relationship between the two latter processes. Method AVFs were created in wild-type (WT) and VWF (-/-) deficient (B6.129S2-Vwf tm1Wgr) mice using the external jugular vein and the common carotid artery (CCA). Mean velocity and diameter of the CCA was measured at baseline before surgery, t=0 immediately post-surgery, t=7 and t=14 using ultrasonography to determine flow volume (mL/s). The mice were sacrificed at t=14, AVFs were collected and OR and IH were analyzed using Weigert’s elastin and aSMA staining, using the Mann-Whitney test. To study VSMC proliferation in vitro, VSMCs were isolated from vena cava explants. VSMCs from VWF -/- mice were cultured with 10% VWF -/- plasma with or without addition of VWF (Wilfactin). Proliferation was measured after 40 hours. Humane patient-matched samples were obtained during two stage brachio-basalic AVF surgery: the native veins at creation and venous AVF samples at transposition. Maturation was defined as a luminal AVF diameter of > 6 mm. Samples were stained for VWF and aSMA. Positively stained tissue in the medial layer was quantified using Histoquant and analyzed with the Wilcoxon signed-rank test. Results WT mice showed better outcomes in AVF functionality. Whereas blood flow did not increase in VWF deficient mice over two weeks after AVF-creation, WT mice showed a steady increase in flow rate from t=0 to t=7 (1.97 fold) and t=14 (3.0 fold) . At 14 days, VWF -/- mice showed significantly reduced OR compared to WT mice, with a 1.46 fold smaller venous perimeter (p=0.008). Formation of IH was also significantly reduced in VWF -/- mice (9.58 fold change, p = 0,0001), with a non-significant 1.42 fold difference in luminal area. Lastly, an 8.05 fold decrease was observed in aSMA+ cells in the IH of VWF -/-mice (p = 0.0002). In vitro, addition of VWF caused an 1.5 fold increase in proliferation compared to VWF deficient VSMCs cultured without VWF (p=0.016). As for chronic kidney disease (CKD) patients, our study showed significant wall thickening and increase in aSMA+ tissue only in matured AVFs. There was significant upregulation of VWF+ tissue in the media from native veins to venous AVF samples, with 183% in failed AVFs (p= 0.026) and 425% in matured AVFs (p= 0.013). Matured AVFs had a 4.6 fold increase in VWF+ area in the medial layer compared to failed AVFs (p= 0.033). Conclusion Our study demonstrates VWF as an inducer of VSMC proliferation and thereby both OR and IH. Secondly, we show that VWF is essential for AVF maturation and that enhanced expression in the media coincides with AVF maturation in CKD patients.

Background and objectives:The venous vasa vasorum is the mesh of microvessels that provide oxygen and nutrients to the walls of large veins. Whether changes to the vasa vasorum have any effects on human arteriovenous fistula outcomes remains undetermined. In this study, we challenged the hypothesis that inadequate vascularization of the arteriovenous fistula wall is associated with maturation failure.Design, setting, participants, and measurements:This case–control pilot study includes pre-access veins and arteriovenous fistula venous samples (i.e. tissue pairs) from 30 patients undergoing two-stage arteriovenous fistula creation (15 matured and 15 failed to mature). Using anti-CD31 immunohistochemistry, we quantified vasa vasorum density and luminal area (vasa vasorum area) in the intima, media, and adventitia of pre-access veins and fistulas. We evaluated the association of pre-existing and postoperative arteriovenous fistula vascularization with maturation failure and with postoperative morphometry.Results:Vascularization of veins and arteriovenous fistulas was predominantly observed in the outer media and adventitia. Only the size of the microvasculature (vasa vasorum area), but not the number of vessels (vasa vasorum density), increased after arteriovenous fistula creation in the adventitia (median vasa vasorum area 1366 µm2/mm2(interquartile range 495–2582) in veins versus 3077 µm2/mm2(1812–5323) in arteriovenous fistulas, p Conclusion:The lack of change in intimal and medial vascularization after arteriovenous fistula creation argues against higher oxygen demand in the inner walls of the fistula during the vein to arteriovenous fistula transformation. Postoperative intimal hyperplasia in the arteriovenous fistula wall appears to thrive under hypoxic conditions. Vasa vasorum density and area by themselves are not predictive of maturation outcomes.

Introduction Receptor tyrosine kinases have been implicated in various vascular remodeling processes and cardiovascular disease. However, their role in the regulation of vascular tone is poorly understood. Herein, we evaluate the contribution of c-Kit signaling to vasoactive responses. Methods The vascular reactivity of mesenteric arteries was assessed under isobaric conditions in c-Kit deficient (KitW/W−v) and littermate control mice (Kit+/+) using pressure myography. Protein levels of soluble guanylyl cyclase beta 1 (sGCβ1) were quantified by Western blot. Mean arterial pressure was measured after high salt (8% NaCl) diet treatment using the tail-cuff method. Results Smooth muscle cells (SMCs) from c-Kit deficient mice showed a 5-fold downregulation of sGCβ1 compared to controls. Endothelium-dependent relaxation of mesenteric arteries demonstrated a predominance of prostanoid vs. nitric oxide (NO) signaling in both animal groups. The dependence on prostanoid-induced dilation was higher in c-Kit mutant mice than in controls, as indicated by a significant impairment in vasorelaxation with indomethacin with respect to the latter. Endothelium-independent relaxation showed significant dysfunction of NO signaling in c-Kit deficient SMCs compared to controls. Mesenteric artery dilation was rescued by addition of a cGMP analog, but not with a NO donor, indicating a deficiency in cGMP production in c-Kit deficient SMCs. Finally, c-Kit deficient mice developed higher blood pressure on an 8% NaCl diet compared to their control littermates. Conclusion c-Kit deficiency inhibits NO signaling in SMCs. The existence of this c-Kit/sGC signaling axis may be relevant for vascular reactivity and remodeling.

Background The arteriovenous fistula (AVF) is the preferred hemodialysis access for end-stage renal disease (ESRD) patients. Yet, establishment of a functional AVF presents a challenge, even for the most experienced surgeons, since postoperative stenosis frequently occludes the AVF. Stenosis results from the loss of compliance in fibrotic areas of the fistula which turns intimal hyperplasia into an occlusive feature. Fibrotic remodeling depends on deposition and crosslinking of collagen by lysyl oxidase (LOX), an enzyme that catalyzes the deamination of lysine and hydroxylysine residues, facilitating intra/intermolecular covalent bonds. We postulate that pharmacological inhibition of lysyl oxidase (LOX) increases postoperative venous compliance and prevents stenosis in a rat AVF model. Methods LOX gene expression and vascular localization were assayed in rat AVFs and human pre-access veins, respectively. Collagen crosslinking was measured in humans AVFs that matured or failed, and in rat AVFs treated with β-aminopropionitrile (BAPN), an irreversible LOX inhibitor. BAPN was either injected systemically or delivered locally around rat AVFs using nanofiber scaffolds. The major endpoints were AVF blood flow, wall fibrosis, collagen crosslinking, and vascular distensibility. Results Non-maturation of human AVFs was associated with higher LOX deposition in pre-access veins (N=20, P=0.029), and increased trivalent crosslinks (N=18, P=0.027) in human AVF tissues. Systemic and local inhibition of LOX increased AVF distensibility, while reducing wall fibrosis and collagen crosslinking in rat fistulas. Conclusions Our results demonstrate that BAPN-mediated inhibition of LOX significantly improves vascular remodeling in experimental fistulas.

Introduction: Recent studies have shown that innate response can build immunological memory. This process called “trained immunity” is an epigenetic reprogramming of the monocytes driven by a metabolism shift towards glycolysis. Hypothesis: Since HIF-1a is a major player in monocytes activation and also targets some epigenetic enzymes, we hypothesize that hypoxia/ischemia can lead to “trained immunity” improving arteriogenesis in a mouse model of hindlimb ischemia. Methods: Mice subjected to a unilateral single ischemia insult for 24hours (24h group: femoral artery ligation for 24 h) or by ischemia reperfusion cycles (Cycle group: ischemia-reperfusion in 4 cycles of 5 minutes) or non-ischemia (sham group), were used as donors in the monocyte transfer experiment. Recipient mice underwent to permanent hindlimb ischemia one day prior the tail vein injection of bone marrow (BM) monocytes from the ischemic leg. Laser Doppler assessed blood flow recovery before and after hindlimb ischemia. Arteriogenesis was quantified on recipient mice by assessing the diameter of gracilis collaterals. A 24h group monocytes from ischemia and non-ischemia leg was used as donors to test the systemic vs. local effect of the ischemic-trained monocytes. Lin- cells were used as a control. Cultured human monocytes were subjected to 24h hypoxia and gene expression for epigenetic enzymes was performed by real time rt-PCR Results: Blood flow recovery in recipients who received ischemic-trained monocytes (24h and cycle) improved overtime compared to sham. Arteriogenesis was significant greater in 24h group compared to sham. Surprisingly, the improvement in blood flow recovery was abolished using combined monocytes from ischemic and non-sichemic, suggesting that ischemia training has a local effect in the monocytes. The Lin- experiment confirmed that the ischemia training is monocyte specific. Moreover, hypoxia regulated epigenetic enzymes responsible for histones methylation in human monocytes. Conclusions: Monocytes can be trained by previous ischemia/hypoxia insult and improve arteriogenesis during hindlimb ischemia. The molecular mechanisms that lead the trained immunity by hypoxia still unknown, however epigenetic modifications might play a role in this process.

Introduction: Lysyl oxidase (LOX) is a copper-dependent amino-oxidase that initiates covalent cross-linking of collagen and elastin, modifies growth factor action and controls gene expression. LOX upregulation is associated with cardiovascular diseases. Because embryonic LOX knockout (KO) mice die perinatally of aortic aneurysms and cardiovascular dysfunction, the studies about LOX function in cardiovascular disease has relied mostly on the use of beta-aminopropionitrile. However, this irreversible inhibitor also inhibits other members of LOX family. Hypothesis: To overcome this limitation, we hypothesized that conditional inactivation of lysyl oxidase would improve vascular function in apoE -/- mice. Methods: We developed, for the first time, a global (LOX f/f CAG-CreERT2 ApoE -/- ) and a smooth muscle cell (SMC) specific (LOX f/f Myh11-CreERT2 ApoE -/- ) LOX conditional knockout mice. Results: LOX inactivation in SMC decreased immature collagen crosslinking in the aorta, carotid pulse wave velocity and blood pressure. It led to a significant reduction in atherosclerotic plaque deposition in the aorta after 16 weeks of high-fat diet (HFD). Global inactivation of LOX, on the other hand, also reduced the systolic blood pressure and prevented vascular stiffness after 4 weeks of HFD. In in-vitro studies, we found that LOX is present in the extracellular space and the nucleus of SMC. Bulk RNA-seq revealed that LOX deletion elevated the expression of SMC alpha-actin, transgelin, and filamin A genes, which are typically present in the contractile phenotype. Conclusion: Our data show that conditional deletion of LOX is atheroprotective and improved vascular and hemodynamic function in ApoE KO mice.