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 (IND)-enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-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 three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×109 VG to 2×1011 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 superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2×1011 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's 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. CONCLUSION: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×1011 VG aliquots injected IM.

Loss of c-Kit in Endothelial Cells Protects against Hindlimb Ischemia.

BACKGROUND: Critical limb ischemia (CLI) is the end stage of peripheral artery disease (PAD), and around 30% of CLI patients are ineligible for current treatments. The angiogenic benefits of c-Kit have been reported in the ischemia scenario; however, the present study demonstrates the effects of specific endothelial c-Kit signaling in arteriogenesis during hindlimb ischemia. METHODS: We created conditional knockout mouse models that decrease c-Kit (c-Kit VE-Cadherin CreERT2-c-Kit) or its ligand (SCF VE-Cadherin CreERT2-SCF) specifically in endothelial cells (ECs) after tamoxifen treatment. These mice and a control group (wild-type VE-Cadherin CreERT2-WT) were subjected to hindlimb ischemia or aortic crush to evaluate perfusion/arteriogenesis and endothelial barrier permeability, respectively. RESULTS: Our data confirmed the lower gene expression of c-Kit and SCF in the ECs of c-Kit and SCF mice, respectively. In addition, we confirmed the lower percentage of ECs positive for c-Kit in c-Kit mice. Further, we found that c-Kit and SCF mice had better limb perfusion and arteriogenesis compared to WT mice. We also demonstrated that c-Kit and SCF mice had a preserved endothelial barrier after aortic crush compared to WT. CONCLUSIONS: Our data demonstrate the deleterious effects of endothelial SCF/c-Kit signaling on arteriogenesis and endothelial barrier integrity.

Single-Cell Analyses Offer Insights into the Different Remodeling Programs of Arteries and Veins.

Arteries and veins develop different types of occlusive diseases and respond differently to injury. The biological reasons for this discrepancy are not well understood, which is a limiting factor for the development of vein-targeted therapies. This study contrasts human peripheral arteries and veins at the single-cell level, with a focus on cell populations with remodeling potential. Upper arm arteries (brachial) and veins (basilic/cephalic) from 30 organ donors were compared using a combination of bulk and single-cell RNA sequencing, proteomics, flow cytometry, and histology. The cellular atlases of six arteries and veins demonstrated a 7.8× higher proportion of contractile smooth muscle cells (SMCs) in arteries and a trend toward more modulated SMCs. In contrast, veins showed a higher abundance of endothelial cells, pericytes, and macrophages, as well as an increasing trend in fibroblasts. Activated fibroblasts had similar proportions in both types of vessels but with significant differences in gene expression. Modulated SMCs and activated fibroblasts were characterized by the upregulation of MYH10, FN1, COL8A1, and ITGA10. Activated fibroblasts also expressed F2R, POSTN, and COMP and were confirmed by F2R/CD90 flow cytometry. Activated fibroblasts from veins were the top producers of collagens among all fibroblast populations from both types of vessels. Venous fibroblasts were also highly angiogenic, proinflammatory, and hyper-responders to reactive oxygen species. Differences in wall structure further explain the significant contribution of fibroblast populations to remodeling in veins. Fibroblasts are almost exclusively located outside the external elastic lamina in arteries, while widely distributed throughout the venous wall. In line with the above, ECM-targeted proteomics confirmed a higher abundance of fibrillar collagens in veins vs. more basement ECM components in arteries. The distinct cellular compositions and transcriptional programs of reparative populations in arteries and veins may explain differences in acute and chronic wall remodeling between vessels. This information may be relevant for the development of antistenotic therapies.

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.

Early hyperlipidemia triggers metabolomic reprogramming with increased SAH, increased acetyl-CoA-cholesterol synthesis, and decreased glycolysis.

To identify metabolomic reprogramming in early hyperlipidemia, unbiased metabolome was screened in four tissues from ApoE-/- mice fed with high fat diet (HFD) for 3 weeks. 30, 122, 67, and 97 metabolites in the aorta, heart, liver, and plasma, respectively, were upregulated. 9 upregulated metabolites were uremic toxins, and 13 metabolites, including palmitate, promoted a trained immunity with increased syntheses of acetyl-CoA and cholesterol, increased S-adenosylhomocysteine (SAH) and hypomethylation and decreased glycolysis. The cross-omics analysis found upregulation of 11 metabolite synthetases in ApoE‾/‾ aorta, which promote ROS, cholesterol biosynthesis, and inflammation. Statistical correlation of 12 upregulated metabolites with 37 gene upregulations in ApoE‾/‾ aorta indicated 9 upregulated new metabolites to be proatherogenic. Antioxidant transcription factor NRF2-/- transcriptome analysis indicated that NRF2 suppresses trained immunity-metabolomic reprogramming. Our results have provided novel insights on metabolomic reprogramming in multiple tissues in early hyperlipidemia oriented toward three co-existed new types of trained immunity.

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.

Hyaluronan Inhibition as a Therapeutic Target for Diabetic Kidney Disease: What Is Next?

Diabetic kidney disease (DKD) is the leading cause of CKD and ESKD in the United States and worldwide. Pharmacotherapy and lifestyle modifications for glycemia, dyslipidemia, and BP control have shown success in slowing the progression of DKD. Traditional treatments, such as angiotensin-converting enzyme inhibitors or angiotensin receptor blockers and more recently the use of sodium-glucose cotransporter 2 inhibitors, nonsteroidal selective mineralocorticoid receptor antagonists, such as finerenone, and glucagon-like peptide 1 receptor agonists, have led to added benefits on various outcomes. However, significant residual risk for DKD progression remains despite the current standard-of-care approaches. Arteriolar hyalinosis (AH) is among the key findings seen on kidney biopsies of patients with DKD. It results from the excessive accumulation of hyaluronan (HA) in the arterioles. AH has not been targeted specifically by any of the therapeutic methods currently being used. We discuss in this manuscript the potential use of a selective therapy targeting AH and the increased total renal HA deposits using a HA synthesis inhibitor in DKD.

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.

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.

The outcomes of a novel two-stage proximal brachial artery to proximal basilic/brachial vein arteriovenous graft extension for dialysis access.

BACKGROUND: We describe a technique to mature a basilic/brachial vein in the mid-arm in preparation for a second stage loop proximal brachial artery to basilic/brachial vein arteriovenous graft (BBAVG). This can occur after a failed basilic/brachial vein transposition or a lack of adequate veins in the distal arm. This allows a mature vein to be used in an end-to-end configuration as an outflow to a BBAVG while preserving proximal vessels for the future. METHODS: This single-center retrospective study was performed from 2015 to 2021, including 104 AVG patients divided into three groups: (1) Patients who failed a basilic vein transposition and had an enlarged vein suitable for an AVG outflow; (2) Patients who had a small caliber basilic/brachial vein after the transposition, requiring a mid-arm brachial artery to brachial/basilic arteriovenous fistula (AVF) creation with a subsequent AVG extension; (3) and lastly, patients who had no distal arm veins available and required a primary brachial artery to basilic/brachial AVF with AVG extension. A survival analysis was performed looking at time to loss of primary and secondary patency, calculated with Kaplan-Meier estimates and Cox regression models adjusted for covariates. RESULTS: The median follow-up time was 11 months (IQ = 11-30 months). The survival analysis showed 28% lost primary patency at a median time of 9 months, and 14% lost secondary patency at a median time of 61 months. Overall secondary patency of the vascular access measured at 12 months was 85.6%. Loss of primary (p = 0.008) and secondary patency (p = 0.017), as well as patency during the first 12 months (p = 0.036), were all significantly associated with increased age when adjusting for covariates. CONCLUSIONS: Our results suggest that the graft extension technique using a mature vein from a previous fistula can result in reliable and durable access. This is important for patients with limited access for hemodialysis, as the axillary vein is preserved for future use if needed.

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.

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.

Innate immunity of vascular smooth muscle cells contributes to two-wave inflammation in atherosclerosis, twin-peak inflammation in aortic aneurysms and trans-differentiation potential into 25 cell types.

Introduction: Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in aortic diseases. Innate immunity is the main driving force for cardiovascular diseases. Methods: To determine the roles of innate immunity in VSMC and aortic pathologies, we performed transcriptome analyses on aortas from ApoE-/- angiotensin II (Ang II)-induced aortic aneurysm (AAA) time course, and ApoE-/- atherosclerosis time course, as well as VSMCs stimulated with danger-associated molecular patterns (DAMPs). Results: We made significant findings: 1) 95% and 45% of the upregulated innate immune pathways (UIIPs, based on data of 1226 innate immune genes) in ApoE-/- Ang II-induced AAA at 7 days were different from that of 14 and 28 days, respectively; and AAA showed twin peaks of UIIPs with a major peak at 7 days and a minor peak at 28 days; 2) all the UIIPs in ApoE-/- atherosclerosis at 6 weeks were different from that of 32 and 78 weeks (two waves); 3) analyses of additional 12 lists of innate immune-related genes with 1325 cytokine and chemokine genes, 2022 plasma membrane protein genes, 373 clusters of differentiation (CD) marker genes, 280 nuclear membrane protein genes, 1425 nucleoli protein genes, 6750 nucleoplasm protein genes, 1496 transcription factors (TFs) including 15 pioneer TFs, 164 histone modification enzymes, 102 oxidative cell death genes, 68 necrotic cell death genes, and 47 efferocytosis genes confirmed two-wave inflammation in atherosclerosis and twin-peak inflammation in AAA; 4) DAMPs-stimulated VSMCs were innate immune cells as judged by the upregulation of innate immune genes and genes from 12 additional lists; 5) DAMPs-stimulated VSMCs increased trans-differentiation potential by upregulating not only some of 82 markers of 7 VSMC-plastic cell types, including fibroblast, osteogenic, myofibroblast, macrophage, adipocyte, foam cell, and mesenchymal cell, but also 18 new cell types (out of 79 human cell types with 8065 cell markers); 6) analysis of gene deficient transcriptomes indicated that the antioxidant transcription factor NRF2 suppresses, however, the other five inflammatory transcription factors and master regulators, including AHR, NF-KB, NOX (ROS enzyme), PERK, and SET7 promote the upregulation of twelve lists of innate immune genes in atherosclerosis, AAA, and DAMP-stimulated VSMCs; and 7) both SET7 and trained tolerance-promoting metabolite itaconate contributed to twin-peak upregulation of cytokines in AAA. Discussion: Our findings have provided novel insights on the roles of innate immune responses and nuclear stresses in the development of AAA, atherosclerosis, and VSMC immunology and provided novel therapeutic targets for treating those significant cardiovascular and cerebrovascular diseases.