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

OBJECTIVE: This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies. METHODS: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed. RESULTS: Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. CONCLUSIONS: E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.

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

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

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

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

A Genetic Model of Constitutively Active Integrin CD11b/CD18.

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

Echocardiographic changes after arteriovenous fistula creation in hemodialysis patients.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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 > 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 < 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. CONCLUSION: 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.

Anti Human CX3CR1 VHH Molecule Attenuates Venous Neointimal Hyperplasia of Arteriovenous Fistula in Mouse Model.

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 ß; 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.

c-Kit suppresses atherosclerosis in hyperlipidemic mice.

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

c-Kit deficiency impairs nitric oxide signaling in smooth muscle cells.

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 (sGCß1) 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 sGCß1 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.

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

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

Fibrotic Venous Remodeling and Nonmaturation of Arteriovenous Fistulas.

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

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

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

A Reliable Mouse Model of Hind limb Gangrene.

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

Dialysis Arteriovenous Fistula Failure and Angioplasty: Intimal Hyperplasia and Other Causes of Access Failure.

The arteriovenous fistula (AVF) is the preferred hemodialysis access type because it has better patency rates and fewer complications than other access types. However, primary failure remains a common problem impeding AVF maturation and adding to patients' morbidity and mortality. Juxta-anastomotic (or inflow) stenosis is the most common reason leading to primary failure, and percutaneous transluminal angioplasty continues to be the gold-standard treatment with excellent success rates. Intimal hyperplasia (IH) has been traditionally blamed as the main pathophysiologic culprit, but new evidence raises doubts regarding the contribution of IH alone to primary failure. We report a 64-year-old man with a 2-stage brachiobasilic AVF that was complicated by failure 4 months after creation. An angiogram showed multiple juxta-anastomotic and midfistula stenotic lesions. Percutaneous transluminal angioplasty was successful in assisting maturation and subsequently cannulating the AVF for hemodialysis treatment. We failed to identify the underlying cause of stenosis because biopsy specimens from fistula tissue obtained at the time of transposition revealed no occlusive IH. This case emphasizes the need for additional research on factors contributing to AVF failure besides IH and highlights the need for more therapeutic options to reduce AVF failure rate.

Pre-existing and Postoperative Intimal Hyperplasia and Arteriovenous Fistula Outcomes.

BACKGROUND: The contribution of intimal hyperplasia (IH) to arteriovenous fistula (AVF) failure is uncertain. This observational study assessed the relationship between pre-existing, postoperative, and change in IH over time and AVF outcomes. STUDY DESIGN: Prospective cohort study with longitudinal assessment of IH at the time of AVF creation (pre-existing) and transposition (postoperative). Patients were followed up for up to 3.3 years. SETTING & PARTICIPANTS: 96 patients from a single center who underwent AVF surgery initially planned as a 2-stage procedure. Veins and AVF samples were collected from 66 and 86 patients, respectively. Matched-pair tissues were available from 56 of these patients. PREDICTORS: Pre-existing, postoperative, and change in IH over time. OUTCOMES: Anatomic maturation failure was defined as an AVF that never reached a diameter > 6mm. Primary unassisted patency was defined as the time elapsed from the second-stage surgery to the first intervention. MEASUREMENTS: Maximal intimal thickness in veins and AVFs and change in intimal thickness over time. RESULTS: Pre-existing IH (>0.05mm) was present in 98% of patients. In this group, the median intimal thickness increased 4.40-fold (IQR, 2.17- to 4.94-fold) between AVF creation and transposition. However, this change was not associated with pre-existing thickness (r(2)=0.002; P=0.7). Ten of 96 (10%) AVFs never achieved maturation, whereas 70% of vascular accesses remained patent at the end of the observational period. Postoperative IH was not associated with anatomic maturation failure using univariate logistic regression. Pre-existing, postoperative, and change in IH over time had no effects on primary unassisted patency. LIMITATIONS: The small number of patients from whom longitudinal tissue samples were available and low incidence of anatomic maturation failure, which decreased the statistical power to find associations between end points and IH. CONCLUSIONS: Pre-existing, postoperative, and change in IH over time were not associated with 2-stage AVF outcomes.

The impact of arteriovenous fistulae on the myocardium: the impact of creation and ligation in the transplant era.

Cardiac hypertrophy is a relatively common complication seen in patients with advanced chronic kidney disease (CKD) and end-stage renal disease (ESRD). Moreover, cardiac hypertrophy is even more frequently seen in patients with ESRD who have an arteriovenous (AV) access. There has been substantial evidence pertaining to the effects of AV access creation on the heart structure and function. Similarly, there is increasing evidence on the effects of AV access closure, flow reduction, transplantation, and immunosuppressive medication on both endpoints. In this review, we present the evidence available in the literature on these topics and open the dialog for further research in this interesting field.

A meta-analysis of randomized clinical trials assessing hemodialysis access thrombosis based on access flow monitoring: where do we stand?

The National Kidney Foundation Kidney Disease Outcomes Quality Initiative recommends the routine use of hemodialysis arteriovenous (AV) access surveillance to detect hemodynamically significant stenoses and appropriately correct them to reduce the incidence of thrombosis and to improve accesses patency rates. Access blood flow monitoring is considered as one of the preferred surveillance method for both AV fistulas (AVF) and AV grafts (AVG); however, published studies have reported conflicting results of its utility that led healthcare professionals to doubt the benefits of this surveillance method. We performed a meta-analysis of the published randomized controlled trials (RCTs) of AV access surveillance using access blood flow monitoring. Our hypothesis was that access blood flow monitoring lowers the risk of AV access thrombosis and that the outcome differs between AVF and AVG. The estimated overall pooled risk ratio (RR) of thrombosis was 0.87 (95% confidence interval [CI], 0.67-1.13) favoring access blood flow monitoring. The pooled RR of thrombosis were 0.64 (95% CI, 0.41-1.01) and 1.06 (95% CI, 0.77-1.46) in the subgroups of only AVF and only AVG, respectively. Our results added to the uncertainty of access blood flow monitoring as a surveillance method of hemodialysis accesses.

Small molecule agonists of integrin CD11b/CD18 do not induce global conformational changes and are significantly better than activating antibodies in reducing vascular injury.

BACKGROUND: CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action. METHODS: We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells. RESULTS: Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective. CONCLUSIONS: Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference. GENERAL SIGNIFICANCE: CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.

c-Kit signaling determines neointimal hyperplasia in arteriovenous fistulae.

Stenosis of arteriovenous (A-V) fistulae secondary to neointimal hyperplasia (NIH) compromises dialysis delivery, which worsens patients' quality of life and increases medical costs associated with the maintenance of vascular accesses. In the present study, we evaluated the role of the receptor tyrosine kinase c-Kit in A-V fistula neointima formation. Initially, c-Kit was found in the neointima and adventitia of human brachiobasilic fistulae, whereas it was barely detectable in control veins harvested at the time of access creation. Using the rat A-V fistula model to study venous vascular remodeling, we analyzed the spatial and temporal pattern of c-Kit expression in the fistula wall. Interestingly, c-Kit immunoreactivity increased with time after anastomosis, which concurred with the accumulation of cells in the venous intima. In addition, c-Kit expression in A-V fistulae was positively altered by chronic kidney failure conditions. Both blockade of c-Kit with imatinib mesylate (Gleevec) and inhibition of stem cell factor production with a specific short hairpin RNA prevented NIH in the outflow vein of experimental fistulae. In agreement with these data, impaired c-Kit activity compromised the development of NIH in A-V fistulae created in c-KitW/Wv mutant mice. These results suggest that targeting of the c-Kit signaling pathway may be an effective approach to prevent postoperative NIH in A-V fistulae.

Myofibroblasts: the ideal target to prevent arteriovenous fistula failure?

The arteriovenous fistula (AVF) failure is a major cause of morbidity in the hemodialysis population. Most AVFs fail due to neointimal hyperplasia (NIH). In this issue, Yang et al. delineated a mechanism responsible for transforming the fistula adventitia into a fertile soil for neointimal precursors. These authors pondered the role of hypoxia-regulated hypoxia-inducible factor-1 (HIF-1α), vascular endothelial growth factor A (VEGF-A), and matrix metalloproteinases (MMPs) in the activation of those adventitial myofibroblasts that may significantly contribute to the formation of the fistula neointima.