Poster 71 - Evaluating the Effects of Sildenafil Citrate Treatment on cGMP Levels and Proliferation Rates in Vein Smooth Muscle Cells for Enhanced Arteriovenous Fistula Maturation

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

AB

Aabir Banerjee (he/him/his)

High School Student Researcher
Rowan University RISER Program
North Brunswick Township, New Jersey, United States

Primary Investigator(s)

PH

Patrick Hwang, PhD (he/him/his)

Assistant Professor
Department of Biomedical Engineering, Rowan-Virtua School of Translational Biomedical Engineering & Sciences, Henry M. Rowan College of Engineering, Glassboro, New Jersey
Cherry Hill, New Jersey, United States

Co-Author(s)

Shahab Edalatian Zakeri, PharmD

PhD Student
Department of Biomedical Engineering, Rowan-Virtua School of Translational Biomedical Engineering & Sciences, Henry M. Rowan College of Engineering, Glassboro, New Jersey, United States

Introduction: About 808,000 people suffer from End Stage Renal Disease (ESRD) in the United States, and 69% of them require dialysis, which requires effective vascular access. The Arteriovenous Fistula (AVF), created by connecting an artery to a vein, is considered the “gold standard” in achieving vascular access. However, 60% of AVFs fail to mature due to improper vessel dilation, poor outward remodeling (OR), and aggressive intimal hyperplasia (IH) of the vasculature, preventing effective dialysis. Vascular OR is an increase in the lumen diameter of the vessel, and IH is an accumulation of smooth muscle cells in the vessel wall. To improve AVF maturation, we are currently developing a natural biopolymer-based hydrogel that will deliver sildenafil to improve vascular OR and reduce aggressive IH. Sildenafil is a Phosphodiesterase-5 (PDE-5) Inhibitor, competing with cyclic GMP (cGMP) to bind to enzymes that would otherwise degrade cGMP into GMP, increasing available cGMP for the pathway that causes vasodilation, improving OR, and allowing cGMP-dependent protein kinases to decrease the overall number of smooth muscle cells, reducing IH and improving the vessel patency. To understand if the scope of our hydrogel study is feasible, this study seeks to perform an analysis of the effect of sildenafil on cGMP levels and cell proliferation rates in Vascular Smooth Muscle Cells (VSMCs). Study Hypotheses (HA): If sildenafil citrate is applied to VSMCs, cGMP will accumulate and therefore increase, and total number of cells will decrease.

Materials and

Methods: This study implemented a series of cGMP enzyme-linked immunosorbent assays (ELISAs) to test cGMP levels in cell wells treated with 10 µM of sildenafil citrate for 2.5 hours, and negative controls left untreated, to determine if the addition of sildenafil citrate impacted cGMP levels at all, and if so, to what extent. A 1 µM treatment of sildenafil citrate was also tested under the same conditions, but results are not pictured below. The study utilized Vascular Smooth Muscle Cells (VSMCs) between P4 and P6 to conduct the assays. The ELISA assay was sourced from Cayman, and colorimetrically analyzed using a spectrophotometer. Cell proliferation was assessed, before conducting the ELISA, using Trypan Blue dye to stain the cells for visibility.

Results, Conclusions, and Discussions: The cGMP level was quantified using the ELISA kit, and there was no significant difference between the untreated and sildenafil treated groups in complete media conditions. Cell proliferation was evaluated and the sildenafil-treated group showed significant reduction of VSMC proliferation compared to the untreated group.

In vitro cGMP levels were unaffected under complete SMC media, with supplemental fetal bovine serum (FBS) and growth factors, culture conditions. This can be attributed to the possibility that some unknown components in FBS included in the cell culture media might interfere with the sildenafil signaling pathway and affect cellular cGMP levels. Thus, we are currently optimizing in vitro conditions to demonstrate the effectiveness of sildenafil treatment on cGMP levels by conducting a sildenafil treatment on VSMCs after overnight serum starvation and performing a series of ELISA analyses. In addition, Nitric Oxide (NO) raises cGMP levels in vivo, so NO donors are being tested in vitro to mimic the natural pathway better. The results of these analyses will be presented in a follow-up report. If sildenafil is found to cause an increase in cGMP levels under serum-starvation conditions, we will progress to the next stage of our trials. Cell proliferation markedly decreased for sildenafil-treated VSMCs, demonstrating potential to reduce IH. In the next stage of trials, we will begin to load a natural biopolymer-based hydrogel with sildenafil and analyze the effects of the released sildenafil from the hydrogel. The findings of this study will later be applied to continue analyzing the efficacy of the sildenafil delivery hydrogel on vessel dilation and other outcomes in vitro and in vivo. This research has great potential to enhance AVF maturation and improve vascular access utilization for hemodialysis clinical outcomes.

Acknowledgements (Optional):

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