Singh, Avishek K

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 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.