Binxia Yang

Simvastatin reduces venous stenosis formation in a murine hemodialysis vascular access model

Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity.

A Polymeric Fastener Can Easily Functionalize Liposome Surfaces with Gadolinium for Enhanced Magnetic Resonance Imaging

Common methods of loading magnetic resonance imaging (MRI) contrast agents into nanoparticles often suffer from challenges related to particle formation, complex chemical modification/purification steps, and reduced contrast efficiency. This study presents a simple, yet advanced process to address these issues by loading gadolinium, an MRI contrast agent, exclusively on a liposome surface using a polymeric fastener. The fastener, so named for its ability to physically link the two functional components together, consisted of chitosan substituted with diethylenetriaminepentaacetic acid (DTPA) to chelate gadolinium, as well as octadecyl chains to stabilize the modified chitosan on the liposome surface. The assembly strategy, mimicking the mechanisms by which viruses and proteins naturally anchor to a cell, provided greater T1 relaxivity than liposomes loaded with gadolinium in both the interior and outer leaflet. Gadolinium-coated liposomes were ultimately evaluated in vivo using murine ischemia models to highlight the diagnostic capability of the system. Taken together, this process decouples particle assembly and functionalization and, therefore, has considerable potential to enhance imaging quality while alleviating many of the difficulties associated with multifunctional particle fabrication.

Increased expression of HIF-1α, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency

PURPOSE A mouse model of renal insufficiency with arteriovenous fistula (AVF) and venous stenosis was created. The authors tested the hypothesis that there is increased gene expression of hypoxia-inducible factor-1 alpha (HIF-1alpha); vascular endothelial growth factor-A (VEGF-A) and its receptors (VEGFR-1, -2); matrix metalloproteinase-2 (MMP-2), -9 (MMP-9); tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, -2); and a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1) at the venous stenosis. MATERIALS AND METHODS Nineteen male C57BL/6 mice underwent a left nephrectomy and a surgical occlusion of the right upper pole to induce renal function characterized in eight animals. Twenty eight days later, an AVF (n = 11) was created from the right carotid artery to ipsilateral jugular vein, and the mice were killed at day 7 (n = 4) and day 14 (n = 4). The outflow and control veins were removed for gene expression. Three mice were killed at day 28 for histologic analysis. RESULTS The mean serum blood urea nitrogen level remained significantly elevated for 8 weeks when compared with baseline (P < .05). By day seven, there was a significant increase in the expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein, with HIF-1alpha and TIMP-1 levels significantly elevated at day 14 (P < .05). By day 28, the venous stenosis was characterized by a thickened vein wall and neointima. CONCLUSIONS A mouse model of renal insufficiency with AVF was developed that had increased expression of HIF-1alpha, VEGF-A, VEGFR-1, VEGFR-2, MMP-2, TIMP-1, and ADAMTS-1 at the outflow vein with venous stenosis by day 28.

Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

The Mouse Arteriovenous Fistula Model

PURPOSE The first aim of the present study was to create a mouse carotid artery-to-jugular vein arteriovenous (AV) fistula model. This model was used to test the hypothesis that there is increased gene expression of matrix metalloproteinase (MMP)-2 and MMP-9 at the venous stenosis. MATERIALS AND METHODS Ten male FVB/NJ mice underwent the creation of an AV fistula between the left carotid artery and ipsilateral jugular vein, with the contralateral vessels serving as controls. Two mice died 1 day after surgery and the other eight were euthanized at day 28. Reverse transcriptase polymerase chain reaction was performed in five mice, with the grafted vein and control vein tissue used to determine the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2. Immunohistochemical analysis of the grafted vein and control vein was performed in three mice. RESULTS Venous stenosis formed at the outflow vein, characterized by a thickened neointima with cells staining positive for alpha-smooth muscle actin. There was increased expression of MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 by day 28 at the venous stenosis compared with control vein. CONCLUSIONS A mouse carotid artery-to-jugular vein AV fistula model was developed and used to demonstrate increased expression of several markers known to be associated with AV fistula stenosis. The model may be useful in investigating mechanisms responsible for AV fistula venous stenoses.

Adventitial delivery of lentivirus-shRNA-ADAMTS-1 reduces venous stenosis formation in arteriovenous fistula.

Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.

The Role of Iex-1 in the Pathogenesis of Venous Neointimal Hyperplasia Associated with Hemodialysis Arteriovenous Fistula

Arteriovenous fistulas (AVFs) used for hemodialysis fail because of venous neointimal hyperplasia (VNH). There are 1,500,000 patients that have end stage renal disease worldwide and the majority requires hemodialysis. In the present study, the role of the intermediate early response gene X-1 (IEX-1), also known as IER-3 in the pathogenesis of VNH was evaluated. In human samples removed from failed AVF, there was a significant increase in IEX-1 expression localized to the adventitia. In Iex-1 −/− mice and wild type (WT) controls, chronic kidney disease was induced and an AVF placed 28 days later by connecting the carotid artery to jugular vein. The outflow vein was removed three days following the creation of the AVF and gene expression analysis demonstrated a significant decrease in vascular endothelial growth factor-A (Vegf-A) and monocyte chemoattractant protein-1 (Mcp-1) gene expression in Iex-1 −/− mice when compared to WT mice (P<0.05). At 28 days after AVF placement, histomorphometric and immune-histochemical analyses of the outflow vein demonstrated a significant decrease in neointimal hyperplasia with an increase in average lumen vessel area associated with a decrease in fibroblast, myofibroblast, and Ly6C staining. There was a decrease in proliferation (Ki-67) and an increase in the TUNEL staining in Iex-1 KO mice compared to WT. In addition, there was a decrease in Vegf-A, Mcp-1, and matrix metalloproteiniase-9 (Mmp-9) staining. Iex-1 expression was reduced in vivo and in vitro using nanoparticles coated with calcitriol, an inhibitor of Iex-1 that demonstrated that Iex-1 reduction results in decrease in Vegf-A. In aggregate, these results indicate that the absence of IEX-1 gene results in reduced VNH accompanied with a decrease in proliferation, reduced fibroblast, myofibroblast, and Ly6C staining accompanied with increased apoptosis mediated through a reduction in Vegf-A/Mcp-1 axis and Mmp-9. Adventitial delivery of nanoparticles coated with calcitriol reduced Iex-1 and VNH.

Tracking and Therapeutic Value of Human Adipose Tissue–derived Mesenchymal Stem Cell Transplantation in Reducing Venous Neointimal Hyperplasia Associated with Arteriovenous Fistula

PURPOSE To determine if adventitial transplantation of human adipose tissue-derived mesenchymal stem cells (MSCs) to the outflow vein of B6.Cg-Foxn1(nu)/J mice with arteriovenous fistula (AVF) at the time of creation would reduce monocyte chemoattractant protein-1 (Mcp-1) gene expression and venous neointimal hyperplasia. The second aim was to track transplanted zirconium 89 ((89)Zr)-labeled MSCs serially with positron emission tomography (PET) for 21 days. MATERIALS AND METHODS All animal experiments were performed according to protocols approved by the institutional animal care and use committee. Fifty B6.Cg-Foxn1(nu)/J mice were used to accomplish the study aims. Green fluorescent protein was used to stably label 2.5 × 10(5) MSCs, which were injected into the adventitia of the outflow vein at the time of AVF creation in the MSC group. Eleven mice died after AVF placement. Animals were sacrificed on day 7 after AVF placement for real-time polymerase chain reaction (n = 6 for MSC and control groups) and histomorphometric (n = 6 for MSC and control groups) analyses and on day 21 for histomorphometric analysis only (n = 6 for MSC and control groups). In a separate group of experiments (n = 3), animals with transplanted (89)Zr-labeled MSCs were serially imaged with PET for 3 weeks. Multiple comparisons were performed with two-way analysis of variance, followed by the Student t test with post hoc Bonferroni correction. RESULTS In vessels with transplanted MSCs compared with control vessels, there was a significant decrease in Mcp-1 gene expression (day 7: mean reduction, 62%; P = .029), with a significant increase in the mean lumen vessel area (day 7: mean increase, 176% [P = .013]; day 21: mean increase, 415% [P = .011]). Moreover, this was accompanied by a significant decrease in Ki-67 index (proliferation on day 7: mean reduction, 81% [P = .0003]; proliferation on day 21: mean reduction, 60%, [P = .016]). Prolonged retention of MSCs at the adventitia was evidenced by serial PET images of (89)Zr-labeled cells. CONCLUSION Adventitial transplantation of MSCs decreases Mcp-1 gene expression, accompanied by a reduction in venous neointimal hyperplasia.

Blood Outgrowth Endothelial Cells Reduce Hypoxia-Mediated Fibroblast to Myofibroblast Conversion by Decreasing Proangiogenic Cytokines

Purpose: Hypoxic conditions cause fibroblasts to differentiate into alpha smooth-muscle cell actin (α -SMA)-positive cells, i.e. myofibroblasts. This process is a hallmark of venous neointimal hyperplasia (VNH) associated with hemodialysis vascular access. The purpose of this study was to determine if blood outgrowth endothelial cells (BOEC) may reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions, and to determine the potential mechanisms involved. Methods: An experimental model was used, in which fibroblasts and BOEC were subjected to hypoxia under contact and transwell conditions to determine if BOEC reduce the conversion of fibroblasts into myofibroblasts under hypoxic conditions. Gene expression under different conditions was performed. In addition, functional assays including cell proliferation and migration were determined. Results: This study demonstrates that contact needs to occur between BOEC and fibroblasts for the reduction of the hypoxia-driven conversion of fibroblasts into α-SMA. This is associated with a decrease in several proangiogenic genes including vascular endothelial growth factor A, platelet-derived growth factor, fibroblast growth factor and matrix metalloproteinase 2 in fibroblasts in contact with BOEC when compared to fibroblasts alone. In addition, migration is significantly reduced while proliferation remains unchanged. Conclusion: This study helps provide rationale for using BOEC delivered to the adventitia of the outflow vein of hemodialysis vascular access to reduce VNH.