Visual Abstract

Background: There are very few animal models of balloon angioplasty injury in arteriovenous fistula (AVF), hindering insight into the pathophysiologic processes following angioplasty in AVF. The objective of the study was to develop and characterize a rat model of AVF angioplasty injury. Methods: Balloon angioplasty in 12- to 16-week-old Sprague-Dawley rats was performed at the arteriovenous anastomosis 14 days post-AVF creation with a 2F Fogarty balloon catheter. Morphometry and protein expression of endothelial nitric oxide synthase (eNOS), monocyte-chemoattractant protein-1 (MCP-1), alpha-smooth muscle actin (α-SMA), CD68 (macrophage marker), and collagen expression in AVFs with and without angioplasty were assessed. Results: In AVFs with angioplasty versus without angioplasty: (1) angioplasty increased AVF-vein and artery intimal hyperplasia, (2) angioplasty decreased eNOS protein expression in AVF-vein and artery at 21 days post-AVF creation and remained decreased in the AVF-vein angioplasty group at 35 days, (3) angioplasty increased AVF-vein and artery α-SMA expression within the intimal region at 35 days, (4) angioplasty increased the expression of AVF-vein MCP-1 at 21 days and CD68 at 21 and 35 days, and (5) angioplasty increased AVF-vein and artery collagen expression at 35 days. Conclusion: Our findings describe a reproducible rat model to better understand the pathophysiologic mechanisms that ensue following AVF angioplasty.

A functional and durable vascular access is the lifeline for the hemodialysis patient, as it allows for sustained and consistent dialysis treatment thrice weekly. Currently, the preferred vascular access for hemodialysis patients, as recommended by national vascular access guidelines, is the arteriovenous fistula (AVF) [1, 2]. However, AVF maturation failure has remained a significant clinical problem. The National Institutes of Health (NIH)-funded Dialysis Access Consortium study reported that 60% of AVFs created failed to be successfully used for dialysis [3]. More recently, the NIH-funded Hemodialysis Fistula Maturation Consortium reported that only 43.5% of AVFs matured unassisted, while one-third of AVFs required assisted maturation [4]. Not only do AVFs that require assisted maturation frequently prolong central venous catheter use [5], but interventions to promote AVF maturation have also been associated with shortened AVF patency and more frequent interventions to maintain patency [6-8]. Balloon angioplasty remains the primary and standard therapy to treat AVF dysfunction. However, balloon angioplasty procedures can injure the vascular endothelium and lead to recurrent venous intimal hyperplasia development and restenosis [9]. There are very few animal models of balloon angioplasty injury in AVF, hindering insight into the pathogenesis of balloon angioplasty injury in AVF and development of effective therapies to prevent and treat balloon angioplasty injury to prolong AVF patency.

In our present study, we developed a rat AVF model using an end-to-side anastomosis of femoral vein (end) to artery (side), which recapitulates the anastomotic configuration used in human AVF. We performed balloon angioplasty 14 days following AVF creation and compared it to a control group with AVF creation only without angioplasty. Using this rat AVF balloon angioplasty model, the objective of this study was to examine the natural history of balloon angioplasty injury following AVF creation by characterizing the development of intimal hyperplasia, the major histologic lesion present in AVF maturation failure, and protein expression of key markers of vascular remodeling.

Surgical Creation of Rat AVF and Balloon Angioplasty

All studies and experiments were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH. AVFs were created bilaterally in male Sprague-Dawley rats (Taconic Biosciences, Hudson, NY, USA) aged 12–16 weeks. One side served as the control AVF (AVF without angioplasty), while the contralateral side served as the AVF angioplasty group (Fig. 1). After the rats were anesthetized with isoflurane, buprenorphine, xylazine, and ketamine, a midline incision of the surgical area was performed. The femoral artery and vein were then exposed and flushed with heparin. Using 10-0 monofilament microsurgical sutures, an end-to-side anastomosis was created using the femoral vein (end) and femoral artery (side) (Fig. 2a). After unclamping of the femoral vessels, dilation of the arterialized vein and patency was confirmed visually.

Fig. 1.

Schematic of study design. AVFs were created in both femoral artery and vein regions in each rat. One side had balloon angioplasty performed 14 days following AVF creation. The contralateral side did not have angioplasty and served as the control group. Group 1 was followed up until 21 days after initial AVF creation. Group 2 was followed up until 35 days after initial AVF creation.

Fig. 1.

Schematic of study design. AVFs were created in both femoral artery and vein regions in each rat. One side had balloon angioplasty performed 14 days following AVF creation. The contralateral side did not have angioplasty and served as the control group. Group 1 was followed up until 21 days after initial AVF creation. Group 2 was followed up until 35 days after initial AVF creation.

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Fig. 2.

Rat AVF model and angioplasty of AVF. a Representative image of rat AVF immediately following creation. Configuration of AVF is an end-to-side anastomosis of the femoral vein (end) and femoral artery (side). Note vasodilation of the arterial and venous limb of AVF following creation. b Representative figure of rat AVF during balloon angioplasty. Note inflation of balloon at AVF anastomosis. c Verhoeff’s elastic staining of AVF vessels collected immediately following the balloon angioplasty procedure and the contralateral control. Note the removal of venous intimal hyperplasia (black arrows) and damage to the arterial internal elastic lamina (black arrow heads) following the balloon angioplasty procedure.

Fig. 2.

Rat AVF model and angioplasty of AVF. a Representative image of rat AVF immediately following creation. Configuration of AVF is an end-to-side anastomosis of the femoral vein (end) and femoral artery (side). Note vasodilation of the arterial and venous limb of AVF following creation. b Representative figure of rat AVF during balloon angioplasty. Note inflation of balloon at AVF anastomosis. c Verhoeff’s elastic staining of AVF vessels collected immediately following the balloon angioplasty procedure and the contralateral control. Note the removal of venous intimal hyperplasia (black arrows) and damage to the arterial internal elastic lamina (black arrow heads) following the balloon angioplasty procedure.

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Fourteen days after AVF creation, a balloon angioplasty procedure was performed on one AVF side (Fig. 2b), while the contralateral side did not have a balloon angioplasty procedure and served as the control group (Fig. 1). The AVF side where the balloon angioplasty procedure was to be performed was exposed and the AVF identified. A 2F Fogarty balloon catheter (Edwards Lifesciences Corporation, Irvine, CA, USA) was inserted through the distal artery until it reached the AVF anastomosis. Once the balloon angioplasty catheter reached the vein-artery anastomosis, the balloon catheter was inflated 5 times for 1 min during each inflation, and the balloon catheter was subsequently removed (Fig. 2b).

The animals were euthanized at 21 and 35 days following AVF creation for vessel harvesting of the AVF with angioplasty and AVF without angioplasty vessels. At the selected time points for sacrifice, following anesthesia, the AVF was dissected and patency visually assessed (Fig. 2). Anesthetized rats were euthanized by intracardiac perfusion with PBS for protein studies and 10% formalin for histology studies. The AVF was dissected at the anastomosis to separate the artery and vein. Sections were obtained from the AVF at the artery and vein anastomosis up to 4–6 mm thickness from the anastomoses.

Morphometric Analysis

Morphometric analysis, using Verhoeff’s elastic stain, was performed on each artery and vein sample to evaluate histomorphological changes. Briefly, 4–5 slides of 5-μm sections were obtained by selecting the first of every 10 sections beginning at the artery and vein anastomosis of the AVF. Tissue sections were deparaffinized in xylene and rehydrated in graded ethanol, followed by staining in 5% Verhoeff’s hematoxylin for 1 h. Sections were rinsed in deionized water and then differentiated in 2% ferric chloride solution for 30–60 s. Differentiation was stopped by rinsing in tap water and the sections treated with 5% sodium thiosulfate for 1 min. Sections were then counterstained with Van Gieson’s for 2 min, followed by dehydration through graded ethanol and xylene. Digital photographs of each section were taken using an Olympus BX43 microscope and analyzed using cellSens Dimension software (Olympus Life Science) at a final magnification of 4× and 10× to perform morphometric analysis. Average medial and intimal thickness was determined using 8 lines drawn radially in the intimal region (red) and medial region (blue) as shown in Figure 3a. In addition, lumen and intimal hyperplasia areas were measured as shown in Figure 3b, and the maximal lumen area was defined as the combination of the lumen and intimal hyperplasia areas (Fig. 3b). Percentage of open lumen was calculated by taking the ratio of lumen area and maximal lumen area. For each animal, mean values for the intimal and medial thickness, and % of open lumen and maximal lumen area were reported.

Fig. 3.

Morphometric analysis. a Representative figure of the methodology used for morphometric analysis from a rat AVF-vein (left) and artery (right). Area enclosed by the blue line is the combined luminal plus intimal-medial area and the area enclosed by the red line is the intimal area. Radial lines represent the measurements used to quantify the thickness of the intima (red) and media (blue). b Representative figure of the methodology used to quantify the maximal lumen area and the % of open lumen. Percentage of open lumen was calculated by taking the ratio of the lumen area and maximal lumen area. c Representative figure of the methodology used to quantify the length of internal elastic lamina from a rat ballooned AVF-artery. Blue line represents the length of elastin lamina. AVF, arteriovenous fistula.

Fig. 3.

Morphometric analysis. a Representative figure of the methodology used for morphometric analysis from a rat AVF-vein (left) and artery (right). Area enclosed by the blue line is the combined luminal plus intimal-medial area and the area enclosed by the red line is the intimal area. Radial lines represent the measurements used to quantify the thickness of the intima (red) and media (blue). b Representative figure of the methodology used to quantify the maximal lumen area and the % of open lumen. Percentage of open lumen was calculated by taking the ratio of the lumen area and maximal lumen area. c Representative figure of the methodology used to quantify the length of internal elastic lamina from a rat ballooned AVF-artery. Blue line represents the length of elastin lamina. AVF, arteriovenous fistula.

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Internal Elastic Lamina Quantification

Tissue sections stained with Verhoeff’s elastic stain were used to quantify the length of the arterial elastic lamina. In brief, 2–4 digital photographs of each section were taken at a final magnification of 20×. The elastic lamina was quantified by tracing elastins and measuring their length in micrometer (Fig. 3c) by using cellSens Dimension software (Olympus Life Science). For each animal, average length of the internal elastic lamina was reported.

Collagen Quantification

Masson’s trichrome staining was performed on the paraffin-embedded tissues to evaluate collagen expression. In brief, deparaffinized tissue sections were mordant in Bouin’s solution overnight, followed by staining with Weigert’s hematoxylin for 12 min. Sections were then rinsed and stained in Biebrich scarlet-acid fuchsin for 1 min, followed by treatment with phosphotungstic/phosphomolybdic acid solution for 30 min. Aniline blue staining was performed on the rinsed sections, followed by treatment with 1% glacial acetic acid for 4 min and dehydration with ethanol and xylene. Collagen quantification was carried out by measuring the vessel area of blue-stained regions on the digital photographs of each section using cellSens Dimension software (Olympus Life Science) at a final magnification of 20×.

Immunohistochemical Analysis

Paraffin-embedded arterial and venous sections were evaluated to identify the cells contributing to the vascular remodeling following balloon angioplasty. α-SMA (14-9760-82, Invitrogen) and CD68 (NB600-985, Novus Biologicals) primary antibodies were used to verify the presence of smooth muscle cell phenotype and inflammatory cells, respectively. α-SMA expression in the intimal region and CD68 expression in both medial and intimal regions of AVF vessels were quantified using cellSens Dimension software (Olympus Life Science) on digital photographs taken at a final magnification of 20×.

Western Blotting Analysis

Artery and vein segments isolated from AVF with angioplasty and AVF without angioplasty tissues were prepared by crushing in liquid nitrogen and lysed in the cold radioimmunoprecipitation assay lysis buffer (Millipore, Billerica, MA, USA) containing protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (20 μg) were separated on 4–15% polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Proteins were then transferred from the gel to the nitrocellulose membrane (Thermo Scientific, Waltham, MA, USA) using the Bio-Rad Mini-PROTEAN electrophoresis system. The membranes were blocked with 5% BSA for 1 h. Detection of specific proteins was done using antibodies. Primary antibodies were commercially purchased and employed for the following analyses: (1) total endothelial nitric oxide synthase (eNOS) (Cell Signaling, Beverly, MA, USA #9572) and (2) monocyte chemoattractant protein-1 (MCP-1)(Abcam, Cambridge, MA, USA #Ab25124). Equal loading of protein was confirmed by measuring total protein or glyceraldehyde-3-phosphate dehydrogenase expression. Secondary antibodies were goat anti-mouse, goat anti-rabbit, or chicken anti-goat antibodies, respectively, conjugated to horseradish peroxidase. Detection of the protein bands was performed using standard enhanced chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA). Densitometric analysis was performed to quantitatively assess total protein expression from the Western blotting using Image J. The Image J quantitated intensity of bands was normalized to glyceraldehyde-3-phosphate dehydrogenase.

Statistical Analysis

All data are presented as mean ± SEM. An unpaired Student’s t test was used to compare rats in AVF without angioplasty and AVF with angioplasty groups. A p value <0.05 was considered statistically significant. GraphPad Prism 7.0 (La Jolla, CA, USA) was used for statistical analysis.

Histopathology following AVF Creation and Angioplasty

Representative Verhoeff’s elastic staining of AVF vessels immediately after angioplasty suggested a reduction of venous and arterial hyperplasia and substantial damage to the arterial internal elastic lamina due to balloon injury as compared to the AVF without angioplasty vessels (Fig. 2c). Histological analysis demonstrated that intimal hyperplasia, as measured by intimal area thickness, was significantly greater in the AVF-artery with angioplasty group than the AVF-artery without angioplasty group (Fig. 4a, b) at 21 and 35 days post-AVF creation. The AVF-vein with angioplasty group demonstrated a trend toward greater intimal area thickness than the AVF-vein without angioplasty group at 21 and 35 days post-AVF creation (Fig. 4a, c). Medial thickness was significantly reduced in both AVF-artery and vein in the AVF with angioplasty group compared to the AVF without angioplasty group (Fig. 4b, c). In addition, a significant increase in the maximal lumen area of both AVF-artery and vein was observed in the angioplasty group at 21 days post-AVF creation, which remained significantly higher at even 35 days for the AVF-vein (Fig. 4b, c). Furthermore, no significant difference was observed in the AVF-vein percentage of open lumen between the angioplasty and without angioplasty groups at both 21 and 35 days (Fig. 4c). However, at 35 days following AVF creation, the AVF-artery percentage of open lumen in the angioplasty group showed a significant decrease compared to the group without angioplasty (Fig. 4b).

Fig. 4.

Histology of intimal hyperplasia development following AVF creation and angioplasty. a Representative histology of groups 1 and 2. Scale bar = 100 μM. Morphometric analysis from AVF-artery (b) and AVF-vein (c). N = 4 in each group. *p < 0.05. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

Fig. 4.

Histology of intimal hyperplasia development following AVF creation and angioplasty. a Representative histology of groups 1 and 2. Scale bar = 100 μM. Morphometric analysis from AVF-artery (b) and AVF-vein (c). N = 4 in each group. *p < 0.05. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

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Endothelial Nitric Oxide Synthase Expression Reduced following Angioplasty

eNOS is primarily expressed within the endothelial cells of blood vessels and plays an important role in vascular homeostasis and remodeling. In our Western blotting studies, eNOS protein expression at 21 days post-AVF creation was significantly decreased in the AVF-artery with angioplasty group compared to the AVF-artery without angioplasty group (Fig. 5a). In the AVF-vein, eNOS protein expression was significantly decreased in the AVF-vein with angioplasty group compared to the AVF-vein without angioplasty group at both 21 and 35 days post-AVF creation (Fig. 5b).

Fig. 5.

eNOS and MCP-1 Western blotting. Representative Western blots and densitometric analysis of eNOS and MCP-1 protein expression from AVF-artery (a) and AVF-vein (b) without angioplasty and AVF with angioplasty of group 1 and group 2. Equivalence of loading was assessed by immunoblotting for GAPDH. N = 4–6 in each group. *p < 0.05, **p < 0.01. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. eNOS, endothelial nitric oxide synthase; MCP-1, monocyte-chemoattractant protein-1; AVF, arteriovenous fistula; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 5.

eNOS and MCP-1 Western blotting. Representative Western blots and densitometric analysis of eNOS and MCP-1 protein expression from AVF-artery (a) and AVF-vein (b) without angioplasty and AVF with angioplasty of group 1 and group 2. Equivalence of loading was assessed by immunoblotting for GAPDH. N = 4–6 in each group. *p < 0.05, **p < 0.01. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. eNOS, endothelial nitric oxide synthase; MCP-1, monocyte-chemoattractant protein-1; AVF, arteriovenous fistula; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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MCP-1 Expression Enhanced following Angioplasty

MCP-1 is a known mediator of inflammatory processes in vascular disease. In our Western blotting studies, there was no significant difference in MCP-1 expression in the AVF-artery with angioplasty group compared to the AVF-artery without angioplasty group at 21 and 35 days post-AVF creation (Fig. 5a). At 21 days post-AVF creation, MCP-1 expression was significantly increased in the AVF-vein with angioplasty group as compared to the AVF-vein without angioplasty group (Fig. 5b).

α-SMA and CD68 Expression

To identify cells contributing to vascular remodeling, immunohistochemical staining was carried out using α-SMA and CD68 antibodies. There was a significant increase in α-SMA expression within the intimal regions of both AVF-artery and vein at 35 days post-AVF creation in the angioplasty group (Fig. 6a, b). Furthermore, we observed a significant elevation of CD68+ cells in the AVF-vein in the angioplasty group at 21 days post-AVF creation, which remained elevated at the 35-day time point (Fig. 6a, b).

Fig. 6.

α-SMA and CD68 expression. a α-SMA and CD68 immunostaining of representative sections from groups 1 and 2. Scale bar = 100 μM. Higher magnification images are at 40×. b Quantification of α-SMA (intimal region) and CD68 (intimal and medial regions) expression from AVF-artery (top) and AVF-vein (bottom). N = 3–4 in each group. *p < 0.05, **p < 0.001. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula; α-SMA, alpha-smooth muscle actin.

Fig. 6.

α-SMA and CD68 expression. a α-SMA and CD68 immunostaining of representative sections from groups 1 and 2. Scale bar = 100 μM. Higher magnification images are at 40×. b Quantification of α-SMA (intimal region) and CD68 (intimal and medial regions) expression from AVF-artery (top) and AVF-vein (bottom). N = 3–4 in each group. *p < 0.05, **p < 0.001. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula; α-SMA, alpha-smooth muscle actin.

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Collagen Expression

Masson’s trichrome staining was performed to evaluate collagen expression. At 35 days post-AVF creation, a significant increase in collagen expression was observed for both AVF-artery and vein in the angioplasty group compared to the group without angioplasty (Fig. 7a, b).

Fig. 7.

Collagen expression. a Masson’s trichrome staining of representative sections of groups 1 and 2. Scale bar = 100 μM. b Collagen quantification from AVF-artery (left) and AVF-vein (right). N = 3 in each group. *p < 0.05. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

Fig. 7.

Collagen expression. a Masson’s trichrome staining of representative sections of groups 1 and 2. Scale bar = 100 μM. b Collagen quantification from AVF-artery (left) and AVF-vein (right). N = 3 in each group. *p < 0.05. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

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Length of Internal Elastic Lamina

In addition to endothelial cell damage following angioplasty, deep medial injury with fracturing of the internal elastic lamina can also ensue. We examined the length of the internal elastic lamina of the AVF-artery in both the AVF with angioplasty group and the AVF without angioplasty group. Our results demonstrated that the AVF with angioplasty group had significantly reduced length of internal elastic lamina as compared to the AVF without angioplasty group (Fig. 8).

Fig. 8.

Arterial internal elastic lamina length. a Representative figures of internal elastic lamina in AVF and AVF with angioplasty groups from groups 1 and 2. Scale bar = 20 μM. b Analysis of internal elastic lamina length. N = 3–4 in each group. *p < 0.05, **p < 0.001. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

Fig. 8.

Arterial internal elastic lamina length. a Representative figures of internal elastic lamina in AVF and AVF with angioplasty groups from groups 1 and 2. Scale bar = 20 μM. b Analysis of internal elastic lamina length. N = 3–4 in each group. *p < 0.05, **p < 0.001. An unpaired t test was used to test for statistical differences between AVF without angioplasty and AVF with angioplasty groups. AVF, arteriovenous fistula.

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Using a rat femoral artery to femoral vein AVF model, we developed a balloon angioplasty injury model in a rat AVF. Our study presents several key findings comparing the differences in AVFs with angioplasty intervention and those without angioplasty intervention. As compared to AVFs without angioplasty, AVFs with angioplasty experienced (1) greater intimal hyperplasia development, and reduced medial thickness and increased maximal lumen vessel area; (2) increased collagen and α-SMA expression in both AVF-artery and vein at 35 days and CD68 expression in the AVF-vein at both 21 and 35 days; (3) decreased eNOS protein expression in the AVF-vein and artery at 21 days following AVF creation, with eNOS remaining decreased in the AVF-vein angioplasty group at 35 days; (4) increased MCP-1 expression in the AVF-vein; and (5) decreased arterial internal elastic lamina length following angioplasty.

There continues to be aggressive efforts to increase AVF use in hemodialysis patients [1]. Despite the increase in AVF use in the United States, a large proportion of AVFs created still fail to successfully mature for dialysis use, leading to prolonged duration of dialysis catheter use [3]. The most common angiographic lesion seen in AVF maturation failure is a perianastomotic stenosis (narrowing of the arterial anastomosis and juxta-anastomotic areas of AVF) [10, 11]. In the United States, endovascular interventions, specifically balloon angioplasty, is the most common procedure to treat AVF dysfunction, and since 2005, angioplasty procedures have continued to rise annually [12, 13]. A recent study on US Medicare patients has demonstrated that approximately 40% of total annual vascular access costs (over 1 billion US dollars) is spent on just invasive imaging and endovascular interventions [14]. Previous studies have reported that AVFs that require interventions to promote maturation, time from successful AVF use to abandonment, were decreased in patients who required 2 or more angioplasty interventions compared to patients who did not require any interventions for maturation [6]. Thus, there is an urgent need to improve AVF outcomes and restenosis rates in order to reduce costs after AVF creation. In order for this to occur, a better biological understanding of angioplasty injury in the setting of AVFs is required. Thus, our balloon angioplasty model in a rat AVF represents a feasible and reproducible rodent model to study the pathophysiology of AVF angioplasty injury.

It has been well documented from the arterial literature that the purpose of the angioplasty procedure is to induce outward vessel remodeling by rupturing the intima-media layers of the vessel [15-18]. However, as a consequence, the angioplasty intervention causes significant damage to the endothelial and smooth muscle cells, within the intima and media, respectively, resulting in a response that results in aggressive intimal hyperplasia development and inward remodeling, which ultimately leads to lumen reduction [15-18]. Our histological data suggested a reduction in venous intimal hyperplasia immediately after angioplasty, when compared to the AVFs without angioplasty, demonstrating the immediate effects of the procedure (Fig. 2c). At 21 days following AVF creation, a significant increase in the maximal lumen area of AVF-vein and artery was observed in the angioplasty group at 21 days, which remained significantly higher at even 35 days in the AVF-vein. Also, a similar trend was observed for intimal hyperplasia development as indicated by increased intimal thickness and significant decrease in medial thickness in the AVF-vein and artery angioplasty groups. Furthermore, no significant change was observed in the venous percentage of the open lumen area between the AVF without angioplasty and AVF with angioplasty groups at both 21 and 35 days (Fig. 4). Collectively, the correlation between increased maximal lumen area and lack of a significant change in the percentage of open lumen in the AVF-vein between the 2 groups suggests that increase in the maximal lumen area, following angioplasty, accompanies greater intimal hyperplasia development likely due to injury to the AVF-vein.

Balloon angioplasty is associated with endothelial injury, via denudation of the endothelium, rupture of the intima-media layer of the vessel, stretching of the media layer, and overdistension of the adventitia [17, 19-23]. Endothelial cells have a major influence on vessel remodeling through eNOS expression and its role in the synthesis of nitric oxide. In our AVF balloon angioplasty model, we demonstrated that eNOS protein expression was significantly decreased in both the AVF-artery and vein at 21 days following AVF creation in the AVF angioplasty group compared to the AVF without angioplasty group (Fig. 5). eNOS is present primarily within endothelial layers of blood vessels, suggesting our balloon angioplasty intervention damages the endothelial layer of the AVF, which may subsequently impact nitric oxide production. At 35 days, as compared to the AVF without angioplasty group, the AVF-vein angioplasty group continues to demonstrate decreased eNOS protein expression, while eNOS protein expression recovered in the AVF-artery angioplasty group (Fig. 5). This suggests that endothelial repair and regeneration may be delayed in the AVF-vein following angioplasty compared to the AVF-artery and may be one potential mechanism that is associated with high AVF-vein restenosis rates following angioplasty.

It has been previously reported that α-SMA-expressing cells are the predominant cell type present in the restenotic lesion resulting from angioplasty in human AVFs [9]. Similarly, in our present study, we demonstrated a significant increase in α-SMA expression within the intimal regions of both AVF-artery and vein at 35 days post-AVF in the angioplasty group (Fig. 6), suggesting that the majority of intimal cells exhibit a SMC phenotype. Moreover, we observed a significant increase in CD68+ cells in the AVF-veins of the angioplasty group at 21 days post-AVF creation, which remained elevated at the 35-day time point (Fig. 6). Thus, inflammatory cells may also contribute to the greater intimal hyperplasia formation following angioplasty. In contrast to our findings, Cai et al. [24] reported a significant reduction in intimal plus medial area, along with decreased α-SMA and CD68 expressions 14 days following percutaneous transluminal angioplasty in a murine model. Moreover, there is a recent study from Cai et al. [25] demonstrating sex differences in treatment response to angioplasty in a murine AVF model. This study showed that, as compared to male mice with angioplasty of AVF, outflow veins in female mice had increased α-SMA and CD-68 expression, and increased intimal hyperplasia 14 days after percutaneous transluminal angioplasty [25]. Our present study only utilized male rats. The differences seen between our present study and the Cai et al. [25] study may be due to the difference in the species of rodents used, time points assessed, sex differences, and balloon types.

MCP-1 has been proposed to play a key role in inflammatory processes through its role in potentiating chemotaxis of inflammatory cells such as monocytes and macrophages, activation and migration of endothelial cells, and promotion of proliferation and migration of smooth muscle cells [26-29]. A mouse AVF model with genetic deficiency of MCP-1 has demonstrated increased AVF patency 6 weeks after creation, decreased venous wall thickness, and increased luminal area [26]. In this present study, our results in the setting of balloon angioplasty demonstrate that MCP-1 expression is increased at 21 days in the AVF-vein with angioplasty group compared to the AVF-vein without angioplasty group (Fig. 5). Our observations are consistent with an initial burst of inflammation following the angioplasty procedure. At 35 days following AVF creation, the AVF-vein with angioplasty group showed similar MCP-1 expression compared to the AVF-vein without angioplasty group (Fig. 5), demonstrating attenuation of vascular inflammation following this initial burst.

The important role of collagen density in vascular remodeling following angioplasty has been previously reported to be associated with constrictive remodeling and restenosis [30]. Consistent with a previous report from Cai et al. [24], in the present study, we have observed a significant increase of collagen expression in both AVF-artery and vein in the angioplasty group compared to the control at 35 days post-AVF creation (Fig. 7).

In our histological studies, we have observed a substantial damage to the arterial internal elastic lamina due to balloon injury, which persisted at 21 and 35 days as demonstrated by the significant reduction in the length of the internal elastic lamina and the medial thickness compared to the AVF without angioplasty group (Fig. 8). This suggests that our angioplasty procedure induces a vascular wall injury to promote outward remodeling and expansion, with effects that persist several weeks following angioplasty.

Our work has several limitations. (1) Our studies were conducted in healthy rats; thus, our results may not completely mimic human pathology as seen in advanced CKD and dialysis patients, where AVFs and angioplasty procedures are performed in the setting of CKD. (2) We employed a Fogarty balloon catheter to induce angioplasty injury, instead of a percutaneous transluminal angioplasty balloon, which is more routinely used in the clinical setting of AVF dysfunction. These 2 devices apply different pressures to the vascular wall. (3) Our experiments did not measure flow or perform angiograms to detect stenoses, which serve as the main clinical measurement tools to detect AVF dysfunction. (4) Bilateral placement of AVF is a potential limitation in our study. However, we performed bilateral AVF placement in order to eliminate animal variation.

We have developed a balloon angioplasty injury model in the setting of a rodent AVF. Our model induces outward remodeling and subsequent intimal hyperplasia development at both the AVF-artery and vein. Moreover, our model shows that angioplasty results in endothelial damage and activation of vascular inflammation and fibrosis following the angioplasty intervention, and suggests that smooth muscle cells and inflammatory cells, such as macrophages, are involved in the vascular AVF remodeling process following angioplasty injury. Our results demonstrate the need for a better understanding of the pathophysiologic mechanisms that ensue following angioplasty in AVF in order to identify potential therapeutic strategies to mitigate the vascular injury resulting from the angioplasty intervention.

This work was supported by the animal surgical core resource of the UAB-UCSD O’Brien Center (P30 DK079337). We would like to thank Huashi Li and Linh Pham for their technical support with the tissue staining.

All studies and experiments were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH.

Dr. Lee is a consultant for Proteon Therapeutics, Merck, and Boston Scientific. The remaining authors have no disclosures.

Dr. Lee is supported by grant 2R44 DK109789-03 from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); grant 1R01HL139692-03 from the National Heart, Lung, and Blood Institute (NHLBI); and grant 1I01BX003387-04 from a Veterans Affairs Merit Award.

M.S. performed AVF surgery, balloon angioplasty procedure, histology staining and analysis, and data analysis and contributed to conceptual design and manuscript development and revision. T.W. performed Western blotting and contributed to data analysis and manuscript development and review. A.A. provided critical expertise in the resubmission, specifically in data analysis and manuscript development. L.G. assisted with AVF surgery, development of balloon angioplasty procedure, and manuscript development and revision. T.L. was the principal investigator and contributed to the overall conceptual design, analysis of data, and initial draft of the manuscript and subsequent revisions.

1.
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