Singh, Avishek

Background: Percutaneous transluminal angioplasty (PTA) is the first-line treatment for arteriovenous fistula (AVF) failure and immaturation. High rate of restenosis occurs post PTA procedure caused by venous neointimal hyperplasia (VNH), and decrease the durability of it. Failed AVF showed increased gene expression of Ier3. Poly lactic- co -glycolic acid nanoparticles (PLGA) loaded with 1,25-dihydroxyvitamin D 3 (1,25 NP) can inhibit Ier3 gene expression. Hypothesis: Adventitial delivery of 1,25 NP in hydrogel will reduce venous stenosis in a murine PTA model. Methods: Male C57BL/6J mice underwent partial nephrectomy at day -42 and arteriovenous fistula (AVF) creation at day -14. At day 0, mice were randomized to vehicle group (PTA + PLGA hydrogel) or treatment group (PTA + 1,25 NP hydrogel). Mice were euthanized at day 3 for gene expression analysis and at day 21 for histopathological analysis. Doppler ultrasound was performed weekly to evaluate AVF patency and function. Results: At day 3, PTA + 1,25 NP treated vessels had a significant decrease in the gene expression of Ier3 compared to controls. At day 21, 1,25 NP treated outflow veins had increased lumen vessel area, apoptosis, and higher peak systolic velocity, but decrease in neointimal area/media area ratio, fibroblasts, pro-inflammatory macrophages (M1), fibrosis (picrosirius red staining) and cellular proliferation. Conclusion: Adventitial delivery of 1,25 NP plus PTA has the ability to reduce restenosis and VNH formation. This protective effect is related to inhibition of Ier3 gene expression which modulates vascular inflammation and fibrosis post PTA.

Patients with hereditary hemorrhagic telangiectasia (HHT) have arteriovenous malformations (AVMs) with genetic mutations involving the activin-A receptor like type 1 ( ACVRL1 or ALK1 ) and endoglin ( ENG ). Recent studies have shown that Neuropilin-1 (NRP-1) inhibits ALK1. We investigated the expression of NRP-1 in livers of patients with HHT and found that there was a significant reduction in NRP-1 in perivascular smooth muscle cells (SMCs). We used Nrp1 SM22KO mice ( Nrp1 was ablated in SMCs) and found hemorrhage, increased immune cell infiltration with a decrease in SMCs, and pericyte lining in lungs and liver in adult mice. Histologic examination revealed lung arteriovenous fistulas (AVFs) with enlarged liver vessels. Evaluation of the retina vessels at P5 from Nrp1 SM22KO mice demonstrated dilated capillaries with a reduction of pericytes. In inflow artery of surgical AVFs from the Nrp1 SM22KO versus WT mice , there was a significant decrease in Tgfb1 , Eng , and Alk1 expression and phosphorylated SMAD1/5/8 (pSMAD1/5/8), with an increase in apoptosis. TGF-β1–stimulated aortic SMCs from Nrp1 SM22KO versus WT mice have decreased pSMAD1/5/8 and increased apoptosis. Coimmunoprecipitation experiments revealed that NRP-1 interacts with ALK1 and ENG in SMCs. In summary, NRP-1 deletion in SMCs leads to reduced ALK1, ENG, and pSMAD1/5/8 signaling and reduced cell death associated with AVM formation.

Significance Statement Expression of immediate early response gene X-1 ( Iex-1 ), also known as Ier-3, is increased in venous neointimal hyperplasia (VNH) and stenosis in hemodialysis arteriovenous fistulas (AVF). In a porcine model, 1α,25(OH) 2 D 3 , an inhibitor of Ier3, reduced VNH/stenosis formation. The 1α,25(OH) 2 D 3 released in the perivascular AVF space from poly(lactic-co-glycolic acid) nanoparticles embedded in a Pluronic F127 hydrogel (1,25 NP) reduced Ier3 gene and protein expression, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining. Concomitantly, blood flow, lumen area, pulse wave velocity, and Young’s modulus increased, and neointima area, peak systolic velocity, and wall shear stress decreased in 1,25 NP–treated vessels. RNA sequencing analysis identified 242 genes involved in inflammatory and apoptotic pathways that had decreased expression in 1,25 NP vessels. Background Few therapies prevent venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation in arteriovenous fistulas (AVF). Expression of the immediate early response gene X-1 ( Iex-1 ), also known as Ier3, is associated with VNH and stenosis in murine AVFs. The study aimed to determine if local release of Ier3 long-acting inhibitor 1α,25(OH) 2 D 3 from poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (1,25 NP) could affect VNH/VS formation in a large animal model. Methods Immediately after AVF creation in a porcine model of renal failure, 1,25 NP or vehicle control was injected into the adventitia space of AVF outflow veins. Scanning electron microscopy and dynamic light scattering characterized drug and control nanoparticles. Animals were sacrificed 3 and 28 days later for gene expression, immunohistologic, magnetic resonance imaging and angiography, and ultrasound analyses. Whole transcriptome RNA sequencing with differential gene expression analysis was performed on outflow veins of AVF. Results Encapsulation of 1α,25(OH) 2 D 3 in PLGA nanoparticles formed nanoparticles of uniform size that were similar to nanoparticles without 1α,25(OH) 2 D 3 . The 1,25 NP–treated AVFs exhibited lower VNH/VS, Ier3 gene expression, and IER-3, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining, fibrosis, and proliferation. Blood flow and lumen area increased significantly, whereas peak systolic velocity and wall shear stress decreased. Treatment increased Young’s modulus and correlated with histologic assessment of fibrosis and with no evidence of vascular calcification. RNA sequencing analysis showed changes in the expression of genes associated with inflammatory, TGFβ1, and apoptotic pathways. Conclusions Local release of 1,25 NP improves AVF flow and hemodynamics, and reduces stenosis in association with reduction in inflammation, apoptosis, and fibrosis in a porcine model of arteriovenous fistula.

Background Venous neointimal hyperplasia and venous stenosis (VS) formation can result in a decrease in arteriovenous fistula (AVF) patency in patients with end‐stage renal disease. There are limited therapies that prevent VNH/VS. Systemic delivery of simvastatin has been shown to reduce VNH/VS but local delivery may help decrease the side effects associated with statin use. We determined if microparticles (MP) composed of cyclodextrins loaded with simvastatin (MP‐SV) could reduce VS/VNH using a murine arteriovenous fistula model with chronic kidney disease. Methods and Results Male C57BL/6J mice underwent nephrectomy to induce chronic kidney disease. Four weeks later, an arteriovenous fistula was placed and animals were randomized to 3 groups: 20 μL of PBS or 20 μL of PBS with 16.6 mg/mL of either MP or MP‐SV. Animals were euthanized 3 days later and the outflow veins were harvested for quantitative reverse transcriptase–polymerase chain reaction analysis and 28 days later for immunohistochemistical staining with morphometric analysis. Doppler ultrasound was performed weekly. Gene expression of vascular endothelial growth factor‐A ( Vegf‐A ), matrix metalloproteinase‐9 ( Mmp‐9 ), transforming growth factor beta 1 ( Tgf‐β1 ), and monocyte chemoattractant protein‐1 ( Mcp‐1 ) were significantly decreased in MP‐SV treated vessels compared with controls. There was a significant decrease in the neointimal area, cell proliferation, inflammation, and fibrosis, with an increase in apoptosis and peak velocity in MP‐SV treated outflow veins. MP‐SV treated fibroblasts when exposed to hypoxic injury had decreased gene expression of Vegf‐A and Mmp‐9 . Conclusions In experimental arteriovenous fistulas, periadventitial delivery of MP‐SV decreased gene expression of Vegf‐A , Mmp‐9 , Tgf‐β1 and Mcp‐1, VNH/VS, inflammation, and fibrosis.

Abstract The uptake or expression of hepatitis B virus (HBV) proteins by dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA (miRNA) may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease namely, immune active (IA; n = 20); low replicative (LR; n = 20); HBeAg negative ( n = 20); acute viral hepatitis (AVH, n = 20) and healthy controls ( n = 20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by quantitative polymerase Chain Reaction (qPCR). The miRNA‐messenger RNA (mRNA)regulatory networks identified 19 miRNAs and 12 target gene interactions in major histocompatibility complex and other immune pathways. miR‐2278, miR‐615‐3p, and miR‐3681‐3p were downregulated in the IA group compared to healthy control, miR‐152‐3p and miR‐3613‐3p in the LR group compared to IA group and miR‐152‐3p and miR‐503‐3p in HBe negative compared to LR group. However, miR‐7‐1‐1‐3p, miR‐192‐5p, miR‐195‐5p, and miR‐32‐5p in LR, miR‐342‐3p, and miR‐940 in HBe negative, and miR‐34a‐5p, miR‐130b‐3p, miR‐221‐3p, miR‐320a, miR‐324‐5p, and miR‐484 in AVH were upregulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs‐mRNAs in DCs seems responsible for an impaired immune response during HBV pathogenesis.

Background Women have decreased hemodialysis arteriovenous fistula (AVF) maturation and patency rates. We determined the mechanisms responsible for the sex‐specific differences in AVF maturation and stenosis formation by performing whole transcriptome RNA sequencing with differential gene expression and pathway analysis, histopathological changes, and in vitro cell culture experiments from male and female smooth muscle cells. Methods and Results Mice with chronic kidney disease and AVF were used. Outflow veins were evaluated for gene expression, histomorphometric analysis, Doppler ultrasound, immunohistologic analysis, and fibrosis. Primary vascular smooth muscle cells were collected from female and male aorta vessels. In female AVFs, RNA sequencing with real‐time polymerase chain reaction analysis demonstrated a significant decrease in the average gene expression of BMP7 (bone morphogenetic protein 7) and downstream IL17Rb (interleukin 17 receptor b) , with increased transforming growth factor‐β1 ( Tgf‐β1) and transforming growth factor‐β receptor 1 ( Tgfβ‐r1) . There was decreased peak velocity, negative vascular remodeling with higher venous fibrosis and an increase in synthetic vascular smooth muscle cell phenotype, decrease in proliferation, and increase in apoptosis in female outflow veins at day 28. In vitro primary vascular smooth muscle cell experiments performed under hypoxic conditions demonstrated, in female compared with male cells, that there was increased gene expression of Tgf‐β1 , Tgfβ‐r1 , and Col1 with increased migration. Conclusions In female AVFs, there is decreased gene expression of BMP7 and IL17Rb with increased Tgf‐β1 and Tgfβ‐r1 , and the cellular and vascular differences result in venous fibrosis with negative vascular remodeling.

Failure to mature and venous neointimal hyperplasia formation are the two major causes of hemodialysis arteriovenous fistula (AVF) vascular access failure. Percutaneous transluminal angioplasty (PTA) is the firstline treatment for both of these conditions, but, clinically, women have decreased patency rates compared with men. The hypothesis to be tested in the present study was that female mice after PTA of venous areas of higher intimal thickening have increased gene expression of transforming growth factor-β1 (TGF-β1) and TGF-β receptor 1 (TGFβ-R1) accompanied with histological changes of fibrosis compared with male mice. Seventeen male and eighteen female C57BL/6J mice were used in this study. Chronic kidney disease was induced by partial nephrectomy, and, 28 days later, an AVF was created to connect the left carotid artery to the right jugular vein. Two weeks later, the higher intimal thickening area was treated with PTA, and mice were euthanized 3 days later for gene expression analysis or 14 days later for histopathological analysis. Doppler ultrasound was performed weekly after AVF creation. At day 3, female AVF had significantly higher average gene expression of TGF-β1 and TGFβ-R1 compared with male AVF. At day 14, female outflow veins had a smaller venous diameter, lumen vessel area, decreased wall shear stress, lower average peak systolic velocity, and an increased neointima area-to-media area ratio. Moreover, female outflow veins showed a significant increase in α-smooth muscle actin and fibroblast-specific protein-1. There was a decrease in M1/M2 with an increase in CD68.