Role of the oncogene E26 tranformation-specific sequence-i in arteriovenous fistula maturation failure

About 15% of the adult United States (U.S.) population is affected by chronic kidney disease (CKD) [1]. Almost 70% of end-stage kidney disease patients in the U.S. need hemodialysis to survive [2]. A vascular access point must be created for hemodialysis to occur. The current gold standard for vascular access points is the arteriovenous fistula (AVF) as it lasts the longest [3]. After its creation, an AVF must successfully mature to be useful. AVF maturation failure often occurs due to excess cell proliferation into the AVF's lumen, called neointimal hyperplasia. When AVF maturation failure occurs, hemodialysis can no longer occur because blood cannot flow through the vascular access point at the needed rate. The aim of this project is to investigate whether E26 transformation-specific sequence-1 (ETS-1) causes neointima hyperplasia after arteriovenous fistula creation surgery in a rat model with chronic kidney disease. The leading hypothesis is that ETS-1 inhibition decreases neointima hyperplasia in the AVF following vascular injury. Histological analysis of ETS-1 gene knockout (KO) and wild-type (WT) rats with AVFs in healthy and induced CKD models was performed, and morphometric analysis was used to quantify the open lumen percentage of AVFs in all four groups. No statistically significant difference in open lumen percentage was found at both Week 1 (on average, WT with CKD: 86.3% at n=4; KO with CKD: 78.9% at n=2) and Week 4 (on average, WT with CKD: 31.9% at n=4; KO with CKD: 63.6% at n=2) between the four experimental groups. Further analysis of rats at both Week 1 and Week 4 is needed to draw conclusions regarding the relationship between ETS-1 inhibition and neointimal hyperplasia formation. Further translational research into inhibition of the ETS-1 pathway may be worthwhile and lead to the development of therapies for prevention of neointimal hyperplasia, improved AVF maturation rates, and reduced need for revisit surgeries, enhancing the quality of life for patients with CKD.

ROLE OF THE ONCOGENE E26 TRANFORMATION-SPECIFIC SEQUENCE-1 IN ARTERIOVENOUS FISTULA MATURATION FAILURE by Emma Breen A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biomedical Engineering Approved: Yang-Ting Shiu, PhD Thesis Faculty Supervisor David W. Grainger, PhD Chair, Department of Biomedical Engineering Kelly W. Broadhead, PhD Honors Faculty Advisor Monisha Pasupathi, PhD Dean, Honors College July 2025 Copyright © 2025 All Rights Reserved ABSTRACT About 15% of the adult United States (U.S.) population is affected by chronic kidney disease (CKD) [1]. Almost 70% of end-stage kidney disease patients in the U.S. need hemodialysis to survive [2]. A vascular access point must be created for hemodialysis to occur. The current gold standard for vascular access points is the arteriovenous fistula (AVF) as it lasts the longest [3]. After its creation, an AVF must successfully mature to be useful. AVF maturation failure often occurs due to excess cell proliferation into the AVF’s lumen, called neointimal hyperplasia. When AVF maturation failure occurs, hemodialysis can no longer occur because blood cannot flow through the vascular access point at the needed rate. The aim of this project is to investigate whether E26 transformation-specific sequence-1 (ETS-1) causes neointima hyperplasia after arteriovenous fistula creation surgery in a rat model with chronic kidney disease. The leading hypothesis is that ETS-1 inhibition decreases neointima hyperplasia in the AVF following vascular injury. Histological analysis of ETS-1 gene knockout (KO) and wild-type (WT) rats with AVFs in healthy and induced CKD models was performed, and morphometric analysis was used to quantify the open lumen percentage of AVFs in all four groups. No statistically significant difference in open lumen percentage was found at both Week 1 (on average, WT with CKD: 86.3% at n=4; KO with CKD: 78.9% at n=2) and Week 4 (on average, WT with CKD: 31.9% at n=4; KO with CKD: 63.6% at n=2) between the four experimental groups. Further analysis of rats at both Week 1 and Week 4 is needed to draw conclusions regarding the relationship between ETS-1 inhibition and neointimal hyperplasia formation. ii Further translational research into inhibition of the ETS-1 pathway may be worthwhile and lead to the development of therapies for prevention of neointimal hyperplasia, improved AVF maturation rates, and reduced need for revisit surgeries, enhancing the quality of life for patients with CKD. iii TABLE OF CONTENTS ABSTRACT ii LIST OF ABBREVIATIONS AND KEYWORDS v INTRODUCTION 1 BACKGROUND 4 METHODS 8 RESULTS 12 DISCUSSION 16 ACKNOWLEDGEMENTS 24 REFERENCES 25 iv LIST OF ABBREVIATIONS CKD chronic kidney disease ESKD end-stage kidney disease AVF arteriovenous fistula ETS-1 E26 transformation-specific sequence-1 ETS-DN ETS dominant-negative VVG Verhoeff-van Gieson IEL internal elastic lamina WT wild-type KO knockout KEYWORDS: arteriovenous fistula, maturation failure, chronic kidney disease, gene knockout, neointimal hyperplasia v 1 INTRODUCTION Chronic kidney disease (CKD) affects about 15% of the adult population in the United States (U.S.) [1]. A variety of factors can cause CKD, including genetic factors such as polycystic kidney disease [4], dietary factors such as a high-salt diet [5], or medicines toxic to the kidney such as cancer drugs [6], [7]. However, the most common causes are age and hypertension, with 90% of CKD patients in the U.S. having hypertension [7], [8]. The third leading cause of CKD is diabetes; up to 51% of new dialysis patients have diabetes [9]. CKD has 5 stages, with each successive stage representing a lower glomerular filtration rate. The 5th and last stage is called end-stage kidney disease (ESKD), or kidney failure, and requires transplantation or dialysis therapies because the kidney’s glomerular filtration rate is less than 15 mL/min and is thus no longer functional [10]. Hemodialysis functions via an extracorporeal dialyzer, through which blood flows and excessive fluid is removed. Waste from the blood is extracted by the dialysis machine in a solution and filtered blood returns to the body. Dialysis sessions typically last four hours, and occur three times per week [11], [12]. In the U.S., almost 70% of ESKD patients require hemodialysis for survival [2]. The national costs of treatment for CKD are significant. In the U.S., one patient’s hemodialysis treatment costs $90,000 of Medicare expenditure per year [3]. For hemodialysis to function, a vascular access point must be created where blood is drawn from the body to the dialysis machine, cleaned, and then returned to the body. There are three common vascular access points for dialysis machines: an arteriovenous graft (AVG), a central venous catheter (CVC), and an arteriovenous fistula (AVF) [3]. The most successful and economical vascular access option is the AVF as it lasts the longest 2 and requires fewer surgical interventions [3], [13]. An AVF is surgically created by anastomosing the artery and vein, most often in an upper extremity [3]. After AVF creation, it must mature to become useful. Successful maturation requires sufficient (10- to 80-fold increase in lumen area) outward remodeling with limited inward remodeling to allow for a blood flow rate of 600 mL/min or higher [3], [14]. Outward remodeling refers to cell proliferation away from the lumen, expanding its open area to support increased blood flow [14]. Inward remodeling, or neointimal hyperplasia, refers to the proliferation of smooth muscle cells from the medial layer into the intimal layer and has adverse impacts on AVF maturation success. Neointimal hyperplasia accumulation narrows the lumen and limits blood flow through the AVF. The decreased blood flow rate does not meet the flow requirements for dialysis, indicating maturation failure. Successful AVF maturation is thus characterized by both outward remodeling and limited neointimal hyperplasia to allow for the necessary increase in blood flow rate, from a baseline of 50 mL/min to a minimum of 600 mL/min for hemodialysis to properly occur [14], [15]. AVF maturation has been associated with several transcription factors, including a pro-fibrotic transcription factor called E26 transformation-specific sequence-1, or ETS proto-oncogene 1 (ETS-1). Previously, ETS-1 has been shown to be associated with poor AVF remodeling in a mouse model [16], [17], [18], [19]. The literature has previously established that ETS-1 causes neointima formation after endovascular injury by activating proinflammatory cytokines and adhesion molecules in a mouse model [16], [17], [18], [19]. The rat model can be advantageous for long-term studies such as this, and its larger size can be better at replicating human conditions [20]. Literature on the effects of ETS-1 inhibition in a rat model is not as extensive as that in a mouse model. Studying the rat 3 model could provide further insight into current unknowns such as downstream genetic effects of ETS-1 inhibition in a larger mammal and how the rat’s vascular parallel geometry (which is more similar to that of humans) plays into the effects of AVF remodeling after ETS-1 inhibition. This work investigates whether ETS-1 also causes neointima formation after AVF creation surgery in a rat model. This was done by observing the effects of ETS-1 gene knockout through histological analyses of ETS-1 knockout (KO) rats and wild-type (WT) rats with AVFs and induced CKD. The hypothesis was that rats with CKD and ETS-1 inhibition will have less neointimal hyperplasia than those without ETS-1 inhibition. If this hypothesis is correct, further translational research into inhibition of the ETS-1 pathway may be worthwhile and lead to the development of therapies for prevention of neointimal hyperplasia and promotion of successful AVF maturation. By investigating ETS-1 inhibition as a potential therapy for neointimal hyperplasia formation prevention, this research could lead to improved AVF maturation rates, reducing the need for revisit surgeries and enhancing the quality of life for CKD patients. 4 BACKGROUND Within the current body of research surrounding CKD, there are several factors that have been associated with the disease including age, hypertension, genetics, diet, and toxic medications [7]. However, direct causation remains unsolved. As a result, current treatments target patients’ symptoms, not the cause. Transplantation is the optimal solution. However, the organ transplant system in the United States (U.S.) is extremely inaccessible. Therefore, hemodialysis is the most common treatment for patients with CKD (see Figure 1). While this extracorporeal dialysis can allow patients to live a healthy and extended life, the process is time-consuming and financially debilitating. The individual cost of dialysis treatment was $10,149 per month through private insurance and $3,364 per month through Medicare in 2020 [21]. Dialysis is even more expensive for patients with preexisting comorbidities, such as hypertension, polycystic kidney disease, and diabetes due to the burden of multiple treatment types [22]. Furthermore, costs double for hemodialysis patients whose AVFs fail, due to treatment delay and AVF revisit surgery, compared to those whose AVFs maintain patency over time [21] (see Figure 2). Prevention of AVF maturation failure has benefits for patients’ both physical and financial well-being. Fig. 1. Illustration of the process of hemodialysis [23]. 5 Fig. 2. Left: vasculature of the arm before AVF creation. Right three: various types of AVFs [24]. The figure by Shiu et al. [24] is licensed under CC BY-NC 4.0. Role of ETS-1 ETS-1 belongs to a family of transcription factors (molecules that turn genes “on” or “off” through binding) containing the ETS domain, which is a DNA-binding domain that recognizes the GGAA/T nucleotide sequence [16], [25]. It has been suggested that different members of the DNA-binding ETS domain correlate to unique and individual biological effects, such as the activation of T-cells and common developmental genes [26]. The ETS-1 oncogene is located in the p135 oncoprotein of the E26 retrovirus and exists without its C-terminal inhibitory sequences, unlike its normal gene counterpart which is located on chromosome 11 in the human genome with its C-terminal inhibitory sequences [26], [27]. The oncogenic version of ETS-1 also has higher DNA-binding activity, which could be associated with increased oncogenesis [26]. Another ETS-1 oncogene isoform lacks its N-terminal inhibitory sequence, which also causes it to have a higher DNAbinding ability [26]. These familial factors of ETS-1 are important to consider when performing knockout and analyzing for its expression, as ETS-1 KO could have downstream effects. 6 ETS-1 expression is regulated by multiple other biologic species. Specifically, it is regulated by a positive feedback loop via NADPH-derived reactive oxygen species (ROS). Angiotensin II (Ang II) is a hormone that is released by the kidney to conserve salt and water, but can lead to hypertension [28]. It acts as an important vasoconstrictor and has historically been associated with promoting mesangial cell hypertrophy and proliferation, increased extracellular matrix deposition, and inflammation [29]. Ang II causes production of ROS and subsequently ETS-1 expression [17]. ETS-1 expression itself then causes the production of NADPH oxidase, producing more ROS, establishing a positive feedback loop [17]. In summation, NADPH oxidase-derived ROS from high Ang II levels leads to ETS-1 expression. ETS-1 expression has previously been shown to be higher in rats exposed to Ang II, confirming that this feedback loop exists in rats as it does in humans [29]. In order for the ETS-1 protein to function, its binding domain and active domain must be able to bind to their respective ligands. The binding domain binds to the complementary DNA promoter region sequence and the active domain binds to other proteins. ETS dominant-negative (ETS-DN) peptides inhibit ETS-1 via competitive binding [17]. In a previous study [16], ETS-DN peptides were used to inhibit the ETS-1 transcription factor in a mouse model of AVFs without CKD. Mice with ETS-1 KO exhibited a decrease in neointimal hyperplasia when compared to mice without ETS-1 KO, as expected [16]. This prompted the conclusion that ETS-1, when innately active, causes neointima hyperplasia formation and, therefore, decreased blood flow through the AVF in a mouse model. Rat Model 7 The rat model is useful to study as its larger size more closely represents the AVF maturation process in humans and allows for long-term treatment [20]. Neointimal hyperplasia is characterized by inward smooth muscle cell migration and proliferation, monocyte and macrophage movement, extracellular matrix deposition, and microvessel formation, accompanied by thrombosis and calcification [30]. These symptoms are caused by increased vessel wall pressure from arterial blood flow. Increased arterial blood pressure then activates adhesion molecules, growth factors, cytokines, reactive oxygen species (ROS), and matrix proteins, as mentioned previously [30]. It also activates the mitogen activated protein kinases (MAP) which are associated with cell proliferation. Arterial neointimal hyperplasia is more well studied but venous neointimal hyperplasia can be attributed to low definition of internal venous elastic lamina, low nitric oxide levels, and hemodynamic factors like compliance, turbulence, and shear stress [30]. These processes are important in analyzing neointimal hyperplasia accumulation in rat models, as a positive correlation between shear stress and ETS-1 expression has been established in previous literature [31]. Sprague-Dawley albino rats are historically considered an apt animal model for gene knockout experiments and provide a closer approximation to human CKD due to their larger size as compared to mice [32]. The rat model is also beneficial to study because it exhibits neointimal hyperplasia in the venous limb of the AVF [30] which is an issue that presents in patients with vascular access points created for hemodialysis. By using diseased and healthy rat models in tandem with ETS-1 gene knockout, the role of ETS-1 in the AVF maturation process may be better understood and translated to the clinical environment. Both rats with and without CKD and gene knockout were 8 sacrificed and their AVFs harvested for histological analysis, the details of which are discussed in the following section. 9 METHODS Disease and Knockout Treatment AVFs in rats were surgically created with or without CKD. There were four groups per experimental timepoint: KO without CKD, KO with CKD, wild type (WT) without CKD, and WT with CKD at both Weeks 1 and 4. KO without CKD rats had not been induced with CKD but possessed ETS-1 inhibition. KO with CKD rats had been both induced with CKD through an adenine diet [33] and possessed ETS-1 inhibition. WT control rats without CKD had been treated with nothing. WT CKD rats had been induced with CKD but lacked ETS-1 inhibition. All samples were analyzed for statistical significance. The novel variables of this project are rats with CKD as opposed to previous studies done in mice without CKD [16], [17] [18], [19]. ETS-1 inhibition was achieved through heterozygous gene knockout. Sprague-Dawley rats were used as the wild-type rats for this project; both WT and ETS1 heterozygous KO rats were partitioned into non-CKD and CKD groups. CKD was induced in the latter group with a 0.25% adenine diet (i.e., 0.25% adenine is added to regular rodent chow) for 8 weeks and then switched to a normal diet (i.e., regular rodent chow) [33]. At the end of 8 weeks, the AVF was surgically created by joining the femoral artery and vein [34]. The rats were then sacrificed at 4 weeks after AVF creation (a total of 12 weeks). The AVFs were then harvested, and the tissues were processed for histological evaluation. Histological Preparation The goal of tissue processing is to remove water from the tissues and fix them in a stronger material for analysis. This was achieved by chemically fixing the tissues in 10 formalin (10% formalin solution; Sigma-Aldrich, St. Louis, MO) and then running them through a series of alcohol solutions (85%, 95%, 100% ethanol; Sigma-Aldrich, St. Louis, MO), xylene (Sigma-Alrich, St. Louis, MO), and paraffin wax (Leica Biosystems, Germany). After the tissues were processed to be dehydrated and rigid with paraffin wax, all sections of rat AVF tissue were embedded with an embedding machine (Leica EG1160 Tissue Embedding Center; Leica Biosystems, Germany). After warming the rat AVFs in the embedding machine’s incubator, each AVF was cut into three sections: (1) the proximal artery and proximal vein section, (2) the distal artery section, and (3) the anastomosis section, which is of most interest to this project as this is where stenosis is most susceptible to occurring in patients. Within the anatomy of a rat AVF, the proximal artery and vein are effectively parallel and are therefore cut into a single section and viewed together. It is still important to embed and analyze the distal artery, proximal artery, and vein AVF sections, as hyperplasia can occur in locations away from the anastomosis. Each section was embedded by setting it in a block of paraffin wax held in a mold boat. Each mold boat was labeled with the animal’s identifying number, euthanasia date, and treatment type for future reference during analysis. Each tissue block was then chilled in a freezer overnight for at least 6 hours before proceeding to the next step. Using a microtome (Leica HistoCore Autocut; Leica Biosystems, Germany), each tissue block was cut into 5 µm sections to be stained and analyzed for the percentage of hyperplasia accumulation within the vessel. Each section’s longitudinal position was labeled in µm through the sample for future analysis to mark exactly where hyperplasia is occurring in the vessel. 11 Each section was then stained for histological analysis of the AVF. Verhoeff-Van Geison (VVG) stain (Thermo Fisher Scientific, Waltham, MA) was used to visualize the presence of elastin fibers and Masson Trichrome (MT) stain (Thermo Fisher Scientific, Waltham, MA) to visualize the presence of collagen fibers, to identify the border of the internal elastic lamina (IEL) and proper cell differentiation to support vessel wall maturation, respectively. These staining techniques involve incubating the slides overnight (minimum 8 hours) in an incubation oven at 50-55 °C, performing the stain, and mounting the slides using Toluene Solution (Fisher Chemical Permount Mounting Medium; Thermo Fisher Scientific, Waltham, MA) and coverslip. The VVG staining procedure involves first applying the stain made of iodine, iron chloride, and hematoxylin to visualize the elastin fibers, followed by applying the counterstain which removes excess iodine with sodium thiosulfate for better visualization of the stained sample. All stained slides were imaged using an Axio Scan Z1 slide scanner (Zeiss, Germany) to obtain high-resolution images (325 nm excitation wavelength at 20x, 0.22 µm/pixel) for analysis. Visualization of Open Lumen Area VVG stain was used to visualize the presence of elastin fibers to identify the IEL of the AVF. In a healthy vessel, the areas of the open lumen and IEL should be about equivalent. However, neointimal hyperplasia accumulation causes the open lumen area to be less than that of the cross-sectional IEL [34]. Thus, morphometric analysis was conducted to determine the degree to which neointimal hyperplasia occluded the AVF’s lumen. For VVG-stained samples, the color of the IEL appears black, which was traced and quantified in Image-J (Image-J Version: 1.53c; U.S. NIH, Bethesda, MD). The open lumen area was subsequently traced (see Figure 3) and quantified. 12 Fig. 3. Left: original AVF image with IEL stained black. Middle: IEL traced in blue. Right: lumen filled in blue. VVG staining is for elastin fibers which make up the IEL in black. This area was then normalized by dividing it by the IEL area and deriving a percentage of open lumen area. Unstained versus stained areas were quantified to calculate the open lumen percentage, providing a visual assessment of vessel openness, an indicator of AVF maturation, in each experimental group after AVF creation. Statistical Analysis A one-way ANOVA with Multiple Comparisons test was used to determine statistical significance across the four experimental groups at Week 1 and Week 4 using GraphPad (GraphPad Prism Version: 10.4.0; GraphPad Inc., San Diego, CA). The data were first tested for normal distribution using a Shapiro-Wilk test with GraphPad. A 5% significance level was applied to determine if the open lumen percentage and collagen presence percentage were significantly different across experimental groups. The data were also analyzed using a non-parametric ANOVA Kruskal-Wallis test for all groups at both Weeks 1 and 4 using GraphPad. 13 RESULTS Open Lumen Percentage at Week 1 The AVFs were analyzed for open lumen percentage at Week 1 through morphometric analysis for WT and KO samples with and without CKD (Figure 4). Open lumen area is inversely related to neointimal hyperplasia accumulation. Open lumen areas were normalized across all samples according to their IEL areas to provide open lumen percentages (normalized open lumen areas). All experimental groups were found to have similar open lumen percentages at Week 1 (Figures 5 and 6). Specifically, open lumen percentages were similar between the WT and KO groups without CKD (Figure 5). Open lumen percentages were also similar between the WT and KO groups with CKD (Figure 5). % Open Lumen Area 100 80 60 40 20 0 Fig. 4. Normalized mean open lumen area for each experimental group with standard error of the mean bars at Week 1 (WT Control: n=4; WT CKD: n=4; KO: n=1; KO CKD: n=2). All experimental groups at Week 1 were found to not have significantly different open lumen percentages. 14 200 µm 200 µm Fig. 5. VVG-stained AVFs with IEL traced in blue. Left: WT without CKD (control) at Week 1. Right: KO without CKD at Week 1. Disregarding the natural folding exhibited by the KO without CKD sample, both representative samples have high open lumen percentages. That is, the open lumen area proportional to each sample’s respective IEL area is high in both samples. 200 µm 200 µm Fig. 6. VVG-stained AVFs with IEL traced in blue. Left: WT with CKD at Week 1. Right: KO with CKD at Week 1. Both the WT with CKD and KO with CKD samples shown here have similarly high open lumen percentages. The normalized open lumen area for WT AVFs with CKD was on average 86.3% while that of the KO AVFs with CKD was 78.9% at Week 1. The open lumen percentage of the four experimental groups at Week 1 was not found to be significantly different after statistical analysis. Open Lumen Percentage at Week 4 Morphometric analysis was also conducted at Week 4 to analyze for open lumen percentage of WT AVFs with and without CKD and KO AVFs with CKD. While no viable 15 200 µm 200 µm 200 µm Fig. 7. VVG-stained AVF with IEL traced in blue. Left: WT without CKD (control) at Week 4. Middle: WT with CKD at Week 4. Right: KO with CKD at Week 4. The tissue seen inside the IEL is neointimal hyperplasia accumulation. The representative WT without CKD and KO with CKD samples exhibit less neointimal hyperplasia accumulation than the WT with CKD sample. samples from the KO without CKD group were available for analysis, the WT without CKD group showed little neointimal hyperplasia accumulation (Figure 7). With regards to the other two viable groups at Week 4, visually, there was more neointimal hyperplasia accumulation in the WT with CKD group than the KO with CKD group (Figure 7). At Week 4, there was a lower open lumen percentage across all groups than at Week 1. The normalized open lumen area was found to be higher in the KO samples with CKD than the WT AVFs and even more so than the WT AVFs with CKD, but not significantly so (Figure 8). The normalized open lumen area for the KO AVFs with CKD was on average 63.6% while that of the WT AVFs with CKD was 31.9%. These data were not found to be statistically significant at Week 4 using an ordinary one-way ANOVA test a n d a n o n - p a r a m e t r i c A N O V A K r u s k a l - W a l l i s t e s t (p = 0.05). 16 % Open Lumen Area 80 60 40 20 0 Fig. 8. Normalized mean open lumen area for each experimental group with standard error of the mean bars at Week 4 (WT Control: n=4; WT CKD: n=4; KO CKD: n=2). The open lumen percentages of the WT control group were found to be lower than those of the KO with CKD group. The open lumen percentages of the WT with CKD group were found to be the lowest out of the three groups. These differences were not found to be statistically significant. 17 DISCUSSION More than one in seven adults in the United States (U.S.) have chronic kidney disease (CKD) [1]. Those with CKD who are experiencing kidney failure must receive hemodialysis treatment to properly filter their blood and survive. In order for hemodialysis to function, there must be a vascular access point created through which to filter blood, the most common of which is the arteriovenous fistula (AVF) [3]. However, AVFs often fail to successfully mature, leading to occlusion or stenosis, preventing a patient’s blood from properly filtering. Successful AVF maturation is characterized by lumen expansion and limited inward tissue remodeling, allowing for the blood flow rate necessary for hemodialysis [3]. AVF maturation failure occurs when there is excess inward cell proliferation, termed neointimal hyperplasia, following AVF creation surgery. The gene, E26 transformation-specific sequence-1 (ETS-1), has previously been associated with poor AVF remodeling in a mouse model by activating growth molecules that stimulate neointimal hyperplasia formation [16], [17], [18], [19]. ETS-1 inhibition in the mouse model was associated with decreased neointimal hyperplasia accumulation in the AVF. The approach for this project was to determine if similar results could be achieved in a rat model by inhibiting ETS-1 in groups with and without CKD. Through histological methods and statistical analysis, no significant difference in open lumen percentage was found between rat subjects with and without ETS-1 inhibition and CKD. However, future steps with larger sample sizes will hopefully provide insight into the effects of inhibiting ETS-1 to promote successful AVF maturation for patients who need hemodialysis treatment to survive. 18 The four experimental groups were analyzed at both Week 1 and Week 4 timepoints. All experimental groups were found to have similar open lumen percentages at Week 1, which is to be expected at an early timepoint (Fig. 4). No significant difference was found between the four groups (p=0.05). At Week 4, the wildtype (WT) with CKD group was found to have a lower open lumen percentage than the ETS-1 knockout (KO) with CKD (Fig. 8). While this difference aligns with the project’s hypothesis, the difference between the two groups was not found to be statistically significant (p=0.05) due to a low sample size (WT CKD: n=3; KO CKD: n=2). Furthermore, there were no usable samples to support data for the KO control group without CKD (Fig. 8). While these results are not significant, the qualitatively higher open lumen percentage in the Week 4 KO CKD group (Fig. 7, 8) provides preliminary support for the hypothesis that ETS-1 inhibition decreases neointimal hyperplasia accumulation in rat AVFs. This finding agrees with aforementioned previous findings in a mouse model [16]. This project’s methods and aims are similar to those of a study done by Feng et al. in mice. Specifically, carotid-jugular AVFs were created in C56BL6 mice with normal kidney function. Some mice were then infused with a specific ETS-dominant negative peptide (ETS-DN) to observe the effects of ETS-1 inhibition on AVF remodeling. This ETS-DN has the same binding domain as ETS-1, but has a different, nonfunctional active domain, preventing ETS-1 from binding and therein preventing transcription from initiating [16]. The specific ETS-DN used was synthesized by adding an HIV-1 TAT peptide to the carboxyl terminus of an engineered peptide that contained the ETS-1 gene’s DNA-binding domain [35]. This ETS-DN peptide was similarly used in this project to inhibit ETS-1 gene expression in the ETS-1 KO rat groups. This specific ETS-DN has been 19 Fig. 9. A visualization of the positive feedback loop between ETS-1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species (ROS) [17]. shown to impede the positive feedback loop through which the ETS-1 gene functions [35]. This positive feedback loop functions via ETS-1 expression causing release of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase species, such as p47phox, which leads to the release of reactive oxygen species (ROS), which causes ETS-1 expression [17], (Fig. 9). The specific ETS-DN used by Feng et al. and this project has been shown to inhibit p47phox following angiotensin II (ang II) initialization in human smooth muscle cells [16], [35]. After infusing this ETS-DN into mice, Feng et al. found decreased neointimal hyperplasia accumulation in AVFs at both 1- and 3-week timepoints post AVF creation surgery. They also found decreased expression of NADPH oxidase species [16]. A key difference to note between this project and Feng et al.’s is that this project included rats with CKD and evaluated the difference in neointima formation in groups both with and without ETS-DN infusion and with and without CKD. Feng et al. instead used normal mouse AVFs with and without ETS-DN [16]. Thus, this study builds on previous findings in mouse AVFs without CKD by analyzing the effects of ETS-1 inhibition in rat AVFs with and without CKD. 20 A previous study done by Shiu and Jaimes with a mouse model similarly found decreased inflammation in AVFs with ETS-1 inhibition [17]. Specifically, the same ETSDN peptide was infused in mice without CKD and several vascular inflammatory factors were found to be decreased. Such factors include alpha-smooth muscle actin, TGF-beta, and CTGF. The AVFs also exhibited overall decreased cell proliferation [17]. In the same vein, Shiu and Jaimes also conducted a study using ETS-DN in a rat model. After balloon injury of the rat carotid artery, it was found that ETS-DN successfully decreased neointima formation within the AVF [36]. Similar to this project, Sprague-Dawley rats were used and infused with the same ETS-DN peptide to inhibit ETS-1 expression. ETS-DN was infused at a dosing rate of 10 mg/kg*day. Following sacrifice, the rats’ carotid arteries were fixed, embedded, sectioned, and stained with Verhoeff Van Gieson stain for morphometric analysis, which is the protocol that this project used as well [36]. A key difference in approaches to note is that the mode of injury used was balloon injury in the carotid artery and subsequent analysis of the carotid artery, whereas this project’s mode of injury was AVF creation surgery followed by morphometric analysis of the AVF, particularly the anastomosis portion. Thus, concepts of ETS-1 vascular functionality in the carotid artery can be applied in this project in terms of specifically exploring possible therapeutic options for improving AVF maturation success. An obvious limitation of this study is the lack of KO without CKD samples to compare with the three other experimental groups at the Week 4 timepoint. The importance of including this experimental group is to act as a control group to compare against the WT group. While these two groups were found to have similar open lumen percentages at the Week 1 timepoint, such results are not representative of the long-term effects of ETS-1 21 inhibition, and thus an isolated Week 4 comparison is needed. Of those groups available for analysis at Week 4, the sample sizes were not large enough to achieve statistical significance. Sample sizes were generally lower at Week 4 than at Week 1 due to the nature of disease progression studies such as this. Overall, sample sizes were a limitation of this study. From this concept, a second limitation arises; this project does not account for possible long-term (post-Week 4) effects of ETS-1 inhibition on other aspects of the body, specifically the immune system, which is necessary if this technique for vascular improvement is to reach clinical application. While ETS-1 expression in this field is an established contributor to inward cell proliferation, inflammation, and neointima formation in vasculature, it is also an important mediator of cell-mediated immune responses to infection. For example, Mouly et al. showed that a lack of ETS-1 transcription factors is associated with decreased T-cell function and resultant deprotection against T-cell bowel disease [37]. Another study by Taveirne et al. demonstrated that ETS-1 deficiency is associated with decreased natural killer cell activity [38]. Natural killer cells are an important part of the immune system in protecting against pathogens and cancers. A third study investigated the effects of ETS-1 inhibition in both isolated B cells and other cell types in the body. They found that ETS-1 inhibition in isolated B cells caused mild immunodeficiency symptoms but inhibition in multiple cell types caused more severe immune dysfunction [39]. One study even found that ETS-1 inhibition was associated with contraction of a Staphylococcus aureus infection [40]. T cells, natural killer cells, and B cells are all vital to the immune system’s ability to protect against disease and infection. Considering the risk of infection at the site of vascular access points used for hemodialysis 22 and the comorbidity of CKD with cancer [41], [42], it would be justified to investigate possible long-term effects of ETS-1 inhibition to determine if the benefits to a patient’s vasculature outweigh the risks to their immune system. If these immune response risks were to be studied further, clinical application of a genetically therapeutic technique for improving AVF maturation success rates is promising. For example, [19] found decreased inflammatory factors with ETS-1 inhibition in human aortic smooth muscle cells. Findings such as these give rise to the potential of ETS-1 inhibition as a future therapeutic for decreasing neointima formation in human AVFs and improving their success rates. If future results of this study in rats are able to achieve significant reduction in neointimal hyperplasia, combining these with studies such as a human cell study [19] would logically provide great potential for this method of preventing AVF failure. A technique to prevent AVF failure would greatly benefit all patients who need hemodialysis to survive. Future steps for this project include increasing the sample size, performing Masson’s Trichrome stain analysis, and investigation at longer timepoints. More rats are currently being bred by this lab to determine if the results found are statistically significant or not. Increasing the sample size, especially at the Week 4 timepoint will help better elucidate the effects of ETS-1 inhibition on neointima formation in rat AVFs with and without CKD. Furthermore, Masson’s Trichrome stain analysis will be performed on both the current and future samples in addition to the current VVG stain analysis in this project. MT staining allows for the visualization of collagen cell accumulation which is indicative of successful AVF maturation. This analysis can be compared to VVG analysis to better understand the mechanisms behind AVF remodeling. Future steps beyond the scope of this 23 current project could include sacrificing the rats at later time points to investigate the longterm effects of ETS-1 inhibition. Such a project could also explore possible downstream effects in other areas of the body, specifically with regards to the immune system. This would allow for justification of ETS-1 inhibition as a safe method for clinical use. The results from this project may also be compared to and considered within the context of other projects in the laboratory focusing on the effects of hemodynamic factors, such as wall shear stress, on ETS-1 expression through computational fluid dynamics. ETS-1 is sensitive to hemodynamic changes caused by the increased blood flow rate required of hemodialysis. Specifically, in a study with human varicose veins with characteristic hemodynamic disturbance, ETS-1 expression was found to be upregulated in all layers of the vein [43]. Furthermore, changes in hemodynamic parameters have been established as promoters of neointimal hyperplasia formation. One study using computational fluid dynamics found that increased wall shear stress in AVFs was positively associated with neointimal hyperplasia formation [44]. Overall, contextualizing and integrating the results of this project with others examining the effects of hemodynamic changes after AVF creation would aid understanding of the complex mechanisms by which neointimal hyperplasia and AVF maturation failure occur. While kidney failure is the last stage of a debilitating disease, the costs of the hemodialysis treatment needed to survive are equally as debilitating, costing patients with private insurance upwards of $10,000 per month. A non-functioning kidney is lifethreatening, and for those whose vascular access points fail, the inability to access hemodialysis treatment is equally life-threatening. Current techniques to treat failed AVFs, such as medical devices that act as mechanical anastomoses, angioplasties, and 24 interventional surgeries such as nearby vessel ablation, have extremely variable outcomes [45], [46]. Treating patients after AVF failure is complex due to high patient-specific variability in mode of failure [45]. Therefore, if future investigation into the effects of ETS1 inhibition proves significant in preventing AVF maturation failure in all modes of failure, the use of this gene knockout therapy could help minimize the need for the treatment of failed AVFs and be a much-needed step towards improving CKD patients’ lives. 25 ACKNOWLEDGEMENTS This project was conducted in Dr. Yan-Ting Shiu’s laboratory at the Salt Lake City, United States Department of Veterans Affairs under the University of Utah School of Medicine’s Department of Internal Medicine. Dr. Yuxia He conducted all surgical procedures for this project. Dr. Hannah Northrup and Dr. Bing Li aided in the review process of this paper. 26 [1] REFERENCES “Chronic Kidney Disease Basics | Chronic Kidney Disease Initiative | CDC.” Accessed: May 09, 2024. [Online]. 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