Protein-rich foods, sea foods, and gut microbiota amplify immune responses in chronic diseases and cancers - Targeting PERK as a novel therapeutic strategy for chronic inflammatory diseases, neurodegenerative disorders, and cancer.

The endoplasmic reticulum (ER) is a cellular organelle that is physiologically responsible for protein folding, calcium homeostasis, and lipid biosynthesis. Pathological stimuli such as oxidative stress, ischemia, disruptions in calcium homeostasis, and increased production of normal and/or folding-defective proteins all contribute to the accumulation of misfolded proteins in the ER, causing ER stress. The adaptive response to ER stress is the activation of unfolded protein response (UPR), which affect a wide variety of cellular functions to maintain ER homeostasis or lead to apoptosis. Three different ER transmembrane sensors, including PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1), are responsible for initiating UPR. The UPR involves a variety of signal transduction pathways that reduce unfolded protein accumulation by boosting ER-resident chaperones, limiting protein translation, and accelerating unfolded protein degradation. ER is now acknowledged as a critical organelle in sensing dangers and determining cell life and death. On the other hand, UPR plays a critical role in the development and progression of several diseases such as cardiovascular diseases (CVD), metabolic disorders, chronic kidney diseases, neurological disorders, and cancer. Here, we critically analyze the most current knowledge of the master regulatory roles of ER stress particularly the PERK pathway as a conditional danger receptor, an organelle crosstalk regulator, and a regulator of protein translation. We highlighted that PERK is not only ER stress regulator by sensing UPR and ER stress but also a frontier sensor and direct senses for gut microbiota-generated metabolites. Our work also further highlighted the function of PERK as a central hub that leads to metabolic reprogramming and epigenetic modification which further enhanced inflammatory response and promoted trained immunity. Moreover, we highlighted the contribution of ER stress and PERK in the pathogenesis of several diseases such as cancer, CVD, kidney diseases, and neurodegenerative disorders. Finally, we discuss the therapeutic target of ER stress and PERK for cancer treatment and the potential novel therapeutic targets for CVD, metabolic disorders, and neurodegenerative disorders. Inhibition of ER stress, by the development of small molecules that target the PERK and UPR, represents a promising therapeutic strategy.

The intricate cellular ecosystem of human peripheral veins as revealed by single-cell transcriptomic analysis.

The venous system has been historically understudied despite its critical roles in blood distribution, heart function, and systemic immunity. This study dissects the microanatomy of upper arm veins at the single cell level, and how it relates to wall structure, remodeling processes, and inflammatory responses to injury. We applied single-cell RNA sequencing to 4 non-diseased human veins (3 basilic, 1 cephalic) obtained from organ donors, followed by bioinformatic and histological analyses. Unsupervised clustering of 20,006 cells revealed a complex ecosystem of endothelial cell (EC) types, smooth muscle cell (SMCs) and pericytes, various types of fibroblasts, and immune cell populations. The venous endothelium showed significant upregulation of cell adhesion genes, with arteriovenous zonation EC phenotypes highlighting the heterogeneity of vasa vasorum (VV) microvessels. Venous SMCs had atypical contractile phenotypes and showed widespread localization in the intima and media. MYH11+DESlo SMCs were transcriptionally associated with negative regulation of contraction and pro-inflammatory gene expression. MYH11+DEShi SMCs showed significant upregulation of extracellular matrix genes and pro-migratory mediators. Venous fibroblasts ranging from secretory to myofibroblastic phenotypes were 4X more abundant than SMCs and widely distributed throughout the wall. Fibroblast-derived angiopoietin-like factors were identified as versatile signaling hubs to regulate angiogenesis and SMC proliferation. An abundant monocyte/macrophage population was detected and confirmed by histology, including pro-inflammatory and homeostatic phenotypes, with cell counts positively correlated with age. Ligand-receptor interactome networks identified the venous endothelium in the main lumen and the VV as a niche for monocyte recruitment and infiltration. This study underscores the transcriptional uniqueness of venous cells and their relevance for vascular inflammation and remodeling processes. Findings from this study may be relevant for molecular investigations of upper arm veins used for vascular access creation, where single-cell analyses of cell composition and phenotypes are currently lacking.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats.

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-ß1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-ß1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-ß1 levels. However, treatment with TC14012 significantly reduced the TGF-ß1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

Functions for platelet factor 4 (PF4/CXCL4) and its receptors in fibroblast-myofibroblast transition and fibrotic failure of arteriovenous fistulas (AVFs).

BACKGROUND: Over 60% of End Stage Renal Disease (ESRD) patients are relying on hemodialysis (HD) to survive, and the arteriovenous fistula (AVF) is the preferred vascular access method for HD. However approximately half of all newly created AVF fail to mature and cannot be used without a salvage procedure. We have recently demonstrated an association between AVF maturation failure and post-operative fibrosis, while our RNA-seq study also revealed that veins that ultimately failed during AVF maturation had elevated levels of platelet factor 4 (PF4/CXCL4). However, a link between these two findings was yet to be established. METHODS: In this study, we investigated potential mechanisms between PF4 levels and fibrotic remodeling in veins. We compared the local expression of PF4 and fibrosis marker integrin ß6 (ITGB6) in veins that successfully underwent maturation with that in veins that ultimately failed to mature. We also measured the changes of expression level of α-smooth muscle actin (αSMA/ACTA2) and collagen (Col1/COL1A1) in venous fibroblasts upon various treatments, such as PF4 pharmacological treatment, alteration of PF4 expression, and blocking of PF4 receptors. RESULTS: We found that PF4 is expressed in veins and co-localizes with αSMA. In venous fibroblasts, PF4 stimulates expression of αSMA and Col1 via different pathways. The former requires integrins αvß5 and α5ß1, while chemokine receptor CXCR3 is needed for the latter. Interestingly, we also discovered that the expression of PF4 is associated with that of ITGB6, the ß subunit of integrin αvß6. This integrin is critical for the activation of the major fibrosis factor TGFß, and overexpression of PF4 promotes activation of the TGFß pathway. CONCLUSIONS: These results indicate that upregulation of PF4 may cause venous fibrosis both directly by stimulating fibroblast differentiation and expression of extracellular matrix (ECM) molecules and indirectly by facilitating the activation of the TGFß pathway.

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.

Chronic Kidney Disease Transdifferentiates Veins into a Specialized Immune-Endocrine Organ with Increased MYCN-AP1 Signaling.

Most patients with end-stage renal disease (ESRD) and advanced chronic kidney disease (CKD) choose hemodialysis as their treatment of choice. Thus, upper-extremity veins provide a functioning arteriovenous access to reduce dependence on central venous catheters. However, it is unknown whether CKD reprograms the transcriptome of veins and primes them for arteriovenous fistula (AVF) failure. To examine this, we performed transcriptomic analyses of bulk RNA sequencing data of veins isolated from 48 CKD patients and 20 non-CKD controls and made the following findings: (1) CKD converts veins into immune organs by upregulating 13 cytokine and chemokine genes, and over 50 canonical and noncanonical secretome genes; (2) CKD increases innate immune responses by upregulating 12 innate immune response genes and 18 cell membrane protein genes for increased intercellular communication, such as CX3CR1 chemokine signaling; (3) CKD upregulates five endoplasmic reticulum protein-coding genes and three mitochondrial genes, impairing mitochondrial bioenergetics and inducing immunometabolic reprogramming; (4) CKD reprograms fibrogenic processes in veins by upregulating 20 fibroblast genes and 6 fibrogenic factors, priming the vein for AVF failure; (5) CKD reprograms numerous cell death and survival programs; (6) CKD reprograms protein kinase signal transduction pathways and upregulates SRPK3 and CHKB; and (7) CKD reprograms vein transcriptomes and upregulates MYCN, AP1, and 11 other transcription factors for embryonic organ development, positive regulation of developmental growth, and muscle structure development in veins. These results provide novel insights on the roles of veins as immune endocrine organs and the effect of CKD in upregulating secretomes and driving immune and vascular cell differentiation.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

The Transcriptomics of the Human Vein Transformation After Arteriovenous Fistula Anastomosis Uncovers Layer-Specific Remodeling and Hallmarks of Maturation Failure.

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 ß-aminopropionitrile-loaded nanofibers reduce fibrosis and improve arteriovenous fistula remodeling in rats.

Background: Arteriovenous fistula (AVF) postoperative stenosis is a persistent healthcare problem for hemodialysis patients. We have previously demonstrated that fibrotic remodeling contributes to AVF non-maturation and lysyl oxidase (LOX) is upregulated in failed AVFs compared to matured. Herein, we developed a nanofiber scaffold for the periadventitial delivery of ß-aminopropionitrile (BAPN) to determine whether unidirectional periadventitial LOX inhibition is a suitable strategy to promote adaptive AVF remodeling in a rat model of AVF remodeling. Methods: Bilayer poly (lactic acid) ([PLA)-]- poly (lactic-co-glycolic acid) ([PLGA)] scaffolds were fabricated with using a two-step electrospinning process to confer directionality. BAPN-loaded and vehicle control scaffolds were wrapped around the venous limb of a rat femoral-epigastric AVF during surgery. AVF patency and lumen diameter were followed monitored using Doppler ultrasound surveillance and flow was measured before euthanasia. AVFs were harvested after 21 days for histomorphometry and immunohistochemistry. AVF compliance was measured using pressure myography. RNA from AVF veins was sequenced to analyze changes in gene expression due to LOX inhibition. Results: Bilayer periadventitial nanofiber scaffolds extended BAPN release compared to the monolayer design (p < 0.005) and only released BAPN in one direction. Periadventitial LOX inhibition led to significant increases in AVF dilation and flow after 21 days. Histologically, BAPN trended toward increased lumen and significantly reduced fibrosis compared to control scaffolds (p < 0.01). Periadventitial BAPN reduced downregulated markers associated with myofibroblast differentiation including SMA, FSP-1, LOX, and TGF-ß while increasing the contractile marker MYH11. RNA sequencing revealed differential expression of matrisome genes. Conclusion: Periadventitial BAPN treatment reduces fibrosis and promotes AVF compliance. Interestingly, the inhibition of LOX leads to increased accumulation of contractile VSMC while reducing myofibroblast-like cells. Periadventitial LOX inhibition alters the matrisome to improve AVF vascular remodeling.

Aorta- and liver-generated TMAO enhances trained immunity for increased inflammation via ER stress/mitochondrial ROS/glycolysis pathways.

We determined whether gut microbiota-produced trimethylamine (TMA) is oxidized into trimethylamine N-oxide (TMAO) in nonliver tissues and whether TMAO promotes inflammation via trained immunity (TI). We found that endoplasmic reticulum (ER) stress genes were coupregulated with MitoCarta genes in chronic kidney diseases (CKD); TMAO upregulated 190 genes in human aortic endothelial cells (HAECs); TMAO synthesis enzyme flavin-containing monooxygenase 3 (FMO3) was expressed in human and mouse aortas; TMAO transdifferentiated HAECs into innate immune cells; TMAO phosphorylated 12 kinases in cytosol via its receptor PERK and CREB, and integrated with PERK pathways; and PERK inhibitors suppressed TMAO-induced ICAM-1. TMAO upregulated 3 mitochondrial genes, downregulated inflammation inhibitor DARS2, and induced mitoROS, and mitoTEMPO inhibited TMAO-induced ICAM-1. ß-Glucan priming, followed by TMAO restimulation, upregulated TNF-α by inducing metabolic reprogramming, and glycolysis inhibitor suppressed TMAO-induced ICAM-1. Our results have provided potentially novel insights regarding TMAO roles in inducing EC activation and innate immune transdifferentiation and inducing metabolic reprogramming and TI for enhanced vascular inflammation, and they have provided new therapeutic targets for treating cardiovascular diseases (CVD), CKD-promoted CVD, inflammation, transplantation, aging, and cancer.

A snapshot of early venous remodeling in a 7-day-old arteriovenous fistula.

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.

The anatomical sources of neointimal cells in the arteriovenous fistula.

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.

Vein morphometry in end-stage kidney disease: Teasing out the contribution of age, comorbidities, and vintage to chronic wall remodeling.

Background: Chronic kidney disease (CKD) is a highly comorbid condition with significant effects on vascular health and remodeling. Upper extremity veins are important in end-stage kidney disease (ESKD) due to their potential use to create vascular accesses. However, unlike arteries, the contribution of CKD-associated factors to the chronic remodeling of veins has been barely studied. Methods: We measured morphometric parameters in 315 upper extremity veins, 131 (85% basilic) from stage 5 CKD/ESKD patients and 184 (89% basilic) from non-CKD organ donors. Associations of demographic and clinical characteristics with intimal hyperplasia (IH) and medial fibrosis were evaluated using multivariate regression models. Results: The study cohort included 33% females, 30% blacks, 32% Hispanics, and 37% whites. Over 60% had hypertension, and 25% had diabetes independent of CKD status. Among kidney disease participants, 26% had stage 5 CKD, while 22 and 52% had ESKD with and without history of a previous arteriovenous fistula/graft (AVF/AVG), respectively. Intimal hyperplasia was associated with older age (ß = 0.13 per year, confidence interval [CI] = 0.002-0.26), dialysis vintage > 12 months (ß = 0.22, CI = 0.09-0.35), and previous AVF/AVG creation (ß = 0.19, CI = 0.06-0.32). Upper quartile values of IH were significantly associated with diabetes (odds ratio [OR] = 2.02, CI = 1.08-3.80), which demonstrated an additive effect with previous AVF/AVG history and longer vintage in exacerbating IH. Medial fibrosis also increased as a function of age (ß = 0.17, CI = 0.04-0.30) and among patients with diabetes (ß = 0.15, CI = 0.03-0.28). Age was the predominant factor predicting upper quartile values of fibrosis (OR = 1.03 per year, CI = 1.01-1.05) independent of other comorbidities. Conclusion: Age and diabetes are the most important risk factors for chronic development of venous IH and fibrosis independent of CKD status. Among kidney disease patients, longer dialysis vintage, and history of a previous AVF/AVG are strong predictors of IH.

Periadventitial biomaterials to improve arteriovenous fistula and graft outcomes.

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.

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.

von Willebrand Factor: A Central Regulator of Arteriovenous Fistula Maturation Through Smooth Muscle Cell Proliferation and Outward Remodeling.

Background Arteriovenous fistula (AVF) maturation failure is a main limitation of vascular access. Maturation is determined by the intricate balance between outward remodeling and intimal hyperplasia, whereby endothelial cell dysfunction, platelet aggregation, and vascular smooth muscle cell (VSMC) proliferation play a crucial role. von Willebrand Factor (vWF) is an endothelial cell-derived protein involved in platelet aggregation and VSMC proliferation. We investigated AVF vascular remodeling in vWF-deficient mice and vWF expression in failed and matured human AVFs. Methods and Results Jugular-carotid AVFs were created in wild-type and vWF-/- mice. AVF flow was determined longitudinally using ultrasonography, whereupon AVFs were harvested 14 days after surgery. VSMCs were isolated from vena cavae to study the effect of vWF on VSMC proliferation. Patient-matched samples of the basilic vein were obtained before brachio-basilic AVF construction and during superficialization or salvage procedure 6 weeks after AVF creation. vWF deficiency reduced VSMC proliferation and macrophage infiltration in the intimal hyperplasia. vWF-/- mice showed reduced outward remodeling (1.5-fold, P=0.002) and intimal hyperplasia (10.2-fold, P<0.0001). AVF flow in wild-type mice was incremental over 2 weeks, whereas flow in vWF-/- mice did not increase, resulting in a two-fold lower flow at 14 days compared with wild-type mice (P=0.016). Outward remodeling in matured patient AVFs coincided with increased local vWF expression in the media of the venous outflow tract. Absence of vWF in the intimal layer correlated with an increase in the intima-media ratio. Conclusions vWF enhances AVF maturation because its positive effect on outward remodeling outweighs its stimulating effect on intimal hyperplasia.

E-Selectin/AAV2/2 Gene Therapy Alters Angiogenesis and Inflammatory Gene Profiles in Mouse Gangrene Model.

For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia.

Systemic Profile of Cytokines in Arteriovenous Fistula Patients and Their Associations with Maturation Failure.

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/ß, 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/ß (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.

Role of platelet factor 4 in arteriovenous fistula maturation failure: What do we know so far?

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.