Yang, Binxia

Introduction: Arteriovenous fistulas (AVFs) used for hemodialysis vascular access will fail because of venous neointimal hyperplasia (VNH) that causes stenosis formation at the venous outflow tract. As a result, only 60% of all AVFs are patent at 1-year. There are several factors which have been hypothesized to contribute to VNH including shear stress, hypoxia, and both local and systemic inflammation. Mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammation while having antiproliferitive properties that aid abrogating vascular injury. Hypothesis: We hypothesized that adventitial transplantation of human adipose derived MSCs to the outflow vein of AVF created in CD1-FoxN1 would attenuate VNH failure by reducing pro-inflammatory mediators including tissue necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-1 beta (IL-1β). Methods: Adventitial transplantation of human MSC was performed in CD1-FoxN1 mice. A carotid artery to ipsilateral jugular AVF was placed and animals were divided into three groups: MSCs (M), CorMatrix ECM scaffold (S, CorMatrix Scaffold, Roswell, GE), or scaffold embedded with 250,000 MSCs (S+M) which were delivered to the adventitia. The mice were then sacrificed at 7 and 28 days post AVF placement and the outflow veins were harvested for histomorphometric and qPCR expression analysis. Results: The average lumen vessel area was significantly increased in the outflow veins removed from the S + M group compared to S or M alone at days 7 and 21 (both, P<0.05). The average area of neointima was significantly decreased in the S+M group when compared to S or M groups (P<0.05) while cell density and Ki-67 index were also significantly decreased at day 7 between S and S+M groups (P<0.05). RNA expression analysis revealed that MCP-1, IL-1β, and TNF-α were all significantly decreased in the outflow vein removed from the S+M group when compared to the S or M group (P<0.05). Conclusion: Adventitial transplantation of MSCs to the outflow vein of the AVF at the time of placement attenuates VNH mediated through a reduction of MCP-1, IL-1β, and TNF-α.

Purpose: The majority of patients with end stage renal disease who require hemodialysis use a arteriovenous fistula (AVF) for their vascular access. However, only 40% of AVFs are clinically usable after 1 year. Arteriovenous fistulas fail because of venous neointimal hyperplasia (VNH) that develops in the proximal outflow vein. The immediate early gene, IEX-1 (IER-3), is increased in response to shear stress and hypoxia and both factors contribute to VNH. IEX-1 expression was increased in stenotic venous stenoses removed from patients with hemodialysis vascular access compared to controls. We hypothesized that reducing the expression of IEX-1 using a knockout mouse model with chronic kidney disease (CKD) and AVFs would have decreased VNH compared to wild type (WT) mice. Methods: CKD was induced by performing a nephrectomy. Four weeks later, an AVF was placed to connect the carotid artery to ipsilateral jugular vein. The mice were sacrificed 3 and 28 days after fistula creation and outflow veins were harvested for gene expression and histomorphometric analyses. Results: By day 3, the average gene expression of vascular endothelial growth factor-A (VEGF-A) was significantly lower in the IEX-1 KO vs. WT mice (average reduction: 40%, P<0.05). At day 28, the average lumen vessel area was increased significantly (average increase: 133%, P<0.05, IEX-1 KO vs. WT, respectively) while the average area of the neointima was significantly decreased (average reduction: 20%, IEX-1 KO vs. WT, respectively). Accompanied with this, there was a significant decrease in α-SMA staining and Ki-67 proliferation while the average TUNEL staining was significantly increased (all P<0.05, IEX-1 KO vs. WT). Finally, there was a significant decrease in the average staining intensity for VEGF-A and matrix metalloproteinase-9 (MMP-9) in the KO vs. WT mice (both, P<0.05). Conclusion: A reduction in IEX-1 gene expression results in positive vascular remodeling accompanied with a reduction in proliferation and α-SMA density mediated through a VEGF-A/MMP-9 pathway. Taken collectively, these findings indicate that reducing IEX-1 expression is protective for VNH formation and provide a rationale for use of anti-IEX-1 therapies aimed at prolonging AVF function.

The antigen specificity of Anti‐Neuronal Nuclear Antibody‐type 3 (ANNA3)‐IgG is unknown. We identified Dachshund‐homolog 1 (DACH1) as the ANNA3 autoantigen and confirmed it by antigen‐specific assays, immunohistochemical colocalization and immune absorption experiments. Patients' median age was 63.5 years (range, 49–88); 67% were female. Neurological manifestations (information available for 30 patients) included one or more of neuropathy, 12; cognitive difficulties, 11; ataxia, 8; dysautonomia, 7. Evidence of a neoplasm was present in 27 of 30 (90%), most of neuroendocrine lineage. DACH1‐IgG is rare and represents a novel proposed biomarker of neurological autoimmunity and cancer. ANN NEUROL 2022;91:670–675

Introduction: Venous neointimal hyperplasia (VNH) is a major cause of hemodialysis arteriovenous fistula (AVF) vascular access failure. CX3CR1 mediates macrophage infiltration into the vasculature. Mice were used which had been genetically engineered to knock in the human CX3CR1 gene. Hypothesis: The hypothesis to be tested is that increased CX3CR1 gene expression results in VNH formation associated with AVF. Methods: We used 50 CX3CR1 knock in mice which were divided into 2 groups: 1. CX3CR1 antibody (30 mg/kg administered intraperitoneally two times per week) or vehicle (equal amount of volume used for CX3CR1 antibody administered intraperitoneally). AVFs were created by connecting the carotid artery to ipsilateral jugular vein 28 days after nephrectomy was performed to induce chronic kidney disease. Histology and Immunohistochemistry analysis at the outflow vein of AVF were performed to assess the pathological changes. Results: There was a significant decrease in neointima area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (43085 ± 12678 vs. 58963.4 ± 9273 μm 2 , P<0.05). There was a significant decrease in the ratio of neointima/media+adventitia area in the outflow veins of AVF in CX3CR1 antibody group compared to the vehicle group at day 28 (0.34 ± 0.1 vs. 0.61 ± 0.1, P<0.05). Cell density in neointima area in CX3CR1 group outflow veins was significantly lower than the vehicle group at day 14 (9600 ± 1000 vs. 13000 ± 3000 /mm 2 , P<0.01). There was a significant decrease in the average CD68-positive cell density in the CX3CR1 group outflow veins compared to the vehicle group (0.96 ± 0.34 vs.15.9 ±10, P<0.001) at day 14. Conclusion: Decreasing CX3CR1 significantly reduces macrophages infiltration and results in a significant reduction in VNH. This study provides a rationale for using CX3CR1 antibody in reducing VNH formation.

Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are important mechanisms in airway smooth muscle (ASM) intracellular Ca 2+ [Ca 2+ ] i regulation. In other tissues, there is increasing evidence for a role of mitochondria in [Ca 2+ ] i buffering, especially following SR Ca 2+ release. Mitochondrial buffering may also influence Ca 2+ influx. The purpose of the present study was to examine the role of mitochondria in [Ca 2+ ] i buffering in human ASM. Cells were loaded with fura‐2 and exposed to 10 µM histamine in "0" Ca 2+ HBSS containing 1mM lanthanum and 1µM nifedipine (to block Ca 2+ influx/efflux) and in the additional presence of 1µM RU360 (mitochondrial Ca 2+ uniporter inhibitor) or 1µM CGP37157 (CGP; mitochondrial Na + /Ca 2+ exchange inhibitor). Compared to controls, peak [Ca 2+ ] i levels were increased by 30% in the presence of CGP, while RU360 had not effect. Both CGP and RU360 significantly increased store‐operated Ca 2+ entry. In cells co‐loaded with fura‐2 and rhod‐2 (mitochondrial Ca 2+ indicator), exposure to 5 µM caffeine showed instantaneous SR Ca 2+ release followed by an increase in [Ca 2+ ] mito . Exposure to 1 µM ACh resulted in ACh‐induced [Ca 2+ ] i oscillations that were reflected by mitochondria (but with a time delay). In ASM cells co‐labeled for mitochondria (MitoTracker) and SR (fluorescent ryanodine) migration of mitochondria towards SR was observed upon exposure to ACh or cyclopiazonic acid. This migration was completely inhibited by phalloidin (actin inhibitor). These data demonstrate an important role of mitochondria in [Ca 2+ ] i buffering in ASM. Supported by NIH grant RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Significance Statement Fractalkine receptor 1 (CX3CR1) mediates macrophage infiltration into the vasculature. In this study, we used humanized mice knocked in with the human CX3CR1 gene and inhibited CX3CR1 signaling using a variable domains of camelid heavy-chain-only molecule (BI 655088) to test the hypothesis that blockade of CX3CR1 results in less of the venous neointimal hyperplasia formation that is associated with arteriovenous fistula (AVF) failure. We also used human samples removed from failed AVFs combined with cell culture experiments. Our results demonstrate a novel role for CX3CR1 in reducing venous stenosis formation in AVFs. 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- κ B as potential targets of CX3CR1 inhibition. In KI-A–treated vessels compared with KI-V, there was decreased gene expression of Tnf- α , Mcp-1 , and Il-1 β ; with reduction of Cx3cl1 , NF- κ B, 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.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

Reactive airway diseases are significant sources of pulmonary morbidity in neonatal and pediatric patients. Supplemental oxygen exposure in premature infants contributes to airway diseases such as asthma and promotes development of airway remodeling, characterized by increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Decreased plasma membrane caveolin-1 (CAV1) expression has been implicated in airway disease and may contribute to airway remodeling and hyperreactivity. Here, we investigated the impact of clinically relevant moderate hyperoxia (50% O2) on airway remodeling and caveolar protein expression in a neonatal mouse model. Within 12 h of birth, litters of B6129SF2J mice were randomized to room air (RA) or 50% hyperoxia exposure for 7 days with or without caveolin-1 scaffolding domain peptide (CSD; caveolin-1 mimic; 10 µl, 0.25 mM daily via intraperitoneal injection) followed by 14 days of recovery in normoxia. Moderate hyperoxia significantly increased airway reactivity and decreased pulmonary compliance at 3 wk. Histologic assessment demonstrated airway wall thickening and increased ASM mass following hyperoxia. RNA from isolated ASM demonstrated significant decreases in CAV1 and cavin-1 in hyperoxia-exposed animals while cavin-3 was increased. Supplementation with intraperitoneal CSD mitigated both the physiologic and histologic changes observed with hyperoxia. Overall, these data show that moderate hyperoxia is detrimental to developing airway and may predispose to airway reactivity and remodeling. Loss of CAV1 is one mechanism through which hyperoxia produces these deleterious effects. Supplementation of CAV1 using CSD or similar analogs may represent a new therapeutic avenue for blunting hyperoxia-induced pulmonary damage in neonates.

Abstract Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a “do-it-yourself” basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide.