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Radiology
 
Radiology. May 2016; 279(2): 513–522.
Published online 2015 November 19. doi:  10.1148/radiol.2015150947
PMCID: PMC4836988
NIHMSID: NIHMS752227

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

Abstract

These results indicate that adventitial transplantation of mesenchymal stem cells may be a potential translational therapy for reducing venous stenosis formation.

Abstract

Purpose

To determine if adventitial transplantation of human adipose tissue–derived mesenchymal stem cells (MSCs) to the outflow vein of B6.Cg-Foxn1nu/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 (89Zr)–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-Foxn1nu/J mice were used to accomplish the study aims. Green fluorescent protein was used to stably label 2.5 × 105 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 89Zr-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 89Zr-labeled cells.

Conclusion

Adventitial transplantation of MSCs decreases Mcp-1 gene expression, accompanied by a reduction in venous neointimal hyperplasia.

© RSNA, 2015

Online supplemental material is available for this article.

Introduction

In the United States, more than 570 000 patients have end-stage renal disease, and most patients require hemodialysis for the purification of their blood (1). Optimally functioning hemodialysis vascular access is required so that patients may undergo purification of their blood and removal of electrolytes. The preferred location for hemodialysis vascular access is the arteriovenous fistula (AVF arteriovenous fistula), and less than two-thirds of patients have a well-functioning AVF arteriovenous fistula 1 year after the start of hemodialysis. In most patients, the AVF arteriovenous fistula will fail because of venous neointimal hyperplasia (VNH venous neointimal hyperplasia) and venous stenosis formation (2,3).

There are many factors thought to contribute to the formation of VNH venous neointimal hyperplasia, including hypoxia, shear stress, oxidative stress, and inflammation (4). It is hypothesized that these factors result in elaboration of proinflammatory cytokines, including monocyte chemoattractant protein–1 (MCP-1), and others (57). As a consequence, this leads to accumulation of macrophages, leukocytes, and smooth-muscle cells, as identified with histologic analysis of specimens removed from a venous stenosis (8).

Mesenchymal stem cells (MSC mesenchymal stem cells) have been isolated and expanded from several different sources, including bone marrow, adipose tissue, and cord blood (9). These cells have anti-inflammatory properties that can result in homeostasis, repair, and regeneration in pathologic responses caused by vascular injury (10). In other studies, investigators have demonstrated that MSC mesenchymal stem cell transplantation can reduce fibrosis in the heart, lung, liver, and kidney in experimental animal models (1116). Along with having anti-inflammatory properties, MSC mesenchymal stem cells can inhibit the proliferative effects of monocytes, tumor cells, and cardiac fibroblasts (1720). Finally, MSC mesenchymal stem cells have been shown to reduce hypoxic injury after myocardial infarction because they home to regions of hypoxia (21,22). In animal models of AVF arteriovenous fistula or graft failure and in clinical specimens, increased expression levels of hypoxia-inducible factor–1α (HIF-1α hypoxia-inducible factor–1α) have been observed. Because of these different properties, MSC mesenchymal stem cells have generated interest for the potential application of alleviating vascular injury. We used human adipose tissue–derived MSC mesenchymal stem cells that were manufactured with good manufacturing practice and that are currently being used in several clinical trials at our institution.

Taken collectively, we hypothesized that adventitial transplantation of MSC mesenchymal stem cells to the outflow vein of the AVF arteriovenous fistula at the time of creation would reduce proinflammatory cytokines, including Mcp-1, thereby reducing VNH venous neointimal hyperplasia formation (23,24). The purpose of this study was to determine if adventitial transplantation of human adipose tissue–derived MSC mesenchymal stem cells to the outflow vein of B6.Cg-Foxn1nu/J mice with AVF arteriovenous fistula at the time of creation would reduce Mcp-1 gene expression and VNH venous neointimal hyperplasia. The second aim was to track transplanted zirconium 89 (89Zr)–labeled MSC mesenchymal stem cells serially by means of positron emission tomography (PET) imaging for 21 days.

Materials and Methods

A.B.D. is an inventor of technology and has a leadership position within the company that supplies technology for growing MSC mesenchymal stem cells used in this study. He did not have access to the data. T.R.D., M.K.P., and A. Bansal have a patent pending on 89Zr labeling of cells. S.M. and A.B.D. have a patent pending on using MSC mesenchymal stem cells for preventing stenosis formation in hemodialysis vascular access. The data were under the control of and analyzed by B.Y., A. Brahmbhatt, E.N.T., D.L.M., S.E., A. Bansal, M.K.P., T.R.D., and S.M.

Experimental Animals

All animal experiments were performed according to protocols approved by our institutional animal care and use committee. Housing and handling of the animals was performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, which was revised in 2000. Animals were housed at 22°C temperature, 41% relative humidity, and 12-hour light-dark cycles. Animals were allowed access to water and food ad libitum. Fifty B6.Cg-Foxn1nu/J mice weighing 20–25 g and aged approximately 6–8 weeks were purchased from Charles River Laboratories, Wilmington, Mass. These animals lack a thymus, are unable to produce T cells, and are therefore immunodeficient, which is ideal for xenograft research. Anesthesia was achieved with intraperitoneal injection of a mixture of ketamine hydrochloride (0.1–0.2 mg per gram of body weight) and xylazine (0.02 mg per gram of body weight). AVF arteriovenous fistula between the right carotid artery and the ipsilateral jugular vein was created, as described previously (25). Green fluorescent protein (GFP green fluorescent protein) was used to stably label 2.5 × 105 MSC mesenchymal stem cells in 5 μL of medium, which was injected into the adventitia of the outflow vein at the time of AVF arteriovenous fistula creation in the MSC mesenchymal stem cell group. The 2 × 105 MSC mesenchymal stem cells labeled with GFP green fluorescent protein were injected into the adventitia of the outflow vein at the time of AVF arteriovenous fistula creation (Fig E1 [online]). Eleven mice died after cell transplantation. Animals were sacrificed on day 7 for either histomorphometric or real-time polymerase chain reaction analyses for each of the following groups: AVF arteriovenous fistula only (control group, n = 6) and MSC mesenchymal stem cell (MSC mesenchymal stem cell group, n = 6). Another group of animals was sacrificed at day 21 after fistula placement for histomorphometric and immunohistochemical analyses for the following groups: AVF arteriovenous fistula only (control group, n = 6) and MSC mesenchymal stem cell (n = 6, Fig E2 [online]). In a separate group of experiments, three mice were used for tracking Zr89-labeled MSC mesenchymal stem cells, and three mice had free Zr89 administered to the adventitia of the outflow vein after creation of an AVF arteriovenous fistula.

Human MSC Preparation

Human MSC mesenchymal stem cells from healthy donors were obtained from the Human Cellular Therapy Laboratory, Mayo Clinic, Rochester, Minn. These cells have been characterized with respect to surface markers and described elsewhere (26). Briefly, they are CD73(+), CD90(+), CD105(+), CD44(+), and HLA-ABC(+), and they are being used in several clinical trials.

GFP Transfection

MSC mesenchymal stem cells were transfected with GFP green fluorescent protein lentivirus from Mayo Clinic Rochester Labs, Rochester, Minn. MSC mesenchymal stem cells were grown overnight in media containing the GFP green fluorescent protein lentivirus. The medium was changed to complete growth medium the next day, and cells were checked for fluorescence after 48 hours. Once fluorescence was confirmed, the cells were cultured in complete media that contained 1 μg puromycin per milliliter. Cells containing the plasmid were expanded in complete growth media.

89Zr Labeling and in Vivo Tracking of Stem Cells

Noninvasive PET imaging was used to evaluate the biodistribution of MSC mesenchymal stem cells delivered to the adventitia outside the AVF arteriovenous fistula in CD1-Foxn1nu mice. For this, MSCs were labeled with a biostable radiolabeling synthon, 89Zr-desferrioxamine-N-chlorosuccinimide, as described previously (27). After delivery of 2 × 105 89Zr-labeled MSC mesenchymal stem cells (at a radioactivity concentration of approximately 0.55 MBq per 106 cells) into the adventitia, the 89Zr-labeled MSC mesenchymal stem cells were tracked for 3 weeks by using a small-animal PET/radiography system (Genesys4; Sofie BioSystems, Culver City, Calif). In the control group, 0.28 MBq of 89Zr (HPO4)2 was delivered into the adventitia. PET images were normalized to units of standardized uptake value, which was calculated as follows: tissue radioactivity concentration/(injected dose/body weight in grams).

Immunohistochemistry and Morphometric Analysis

After fixation with formalin and processing, the samples were embedded in paraffin. Histologic sectioning began at the outflow vein segment. Routinely, 80–120 5-μm sections were obtained, and the cuff used to make the anastomosis could be visualized. Every 25 μm, two to four sections were stained with hematoxylin-eosin, Ki-67, α–smooth-muscle actin (α-SMA α–smooth-muscle actin), HIF-1α hypoxia-inducible factor–1α, CD68, fibroblast specific protein–1 (FSP-1 fibroblast specific protein–1), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling) on paraffin-embedded sections from the outflow vein. The EnVision (Dako, Carpinteria, Calif) method was used with a heat-induced antigen retrieval step (28). The following antibodies were used: mouse monoclonal antibody Ki-67 (Dako, 1:400) or rabbit polyclonal antibody for CD68, α-SMA α–smooth-muscle actin, FSP-1 fibroblast specific protein–1, and HIF-1α hypoxia-inducible factor–1α (Abcam, Cambridge, Mass; 1:600). Immunoglobulin G antibody staining was performed to serve as the control. Sections immunostained with hematoxylin-eosin, Ki-67, α-SMA α–smooth-muscle actin, HIF-1α hypoxia-inducible factor–1α, CD68, FSP-1 fibroblast specific protein–1, or TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling were viewed by using an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) equipped with a Neo-Fluor ×20/0.50 objective and digitized to capture a minimum of 1030 × 1300 pixels by using an Axiocam camera (Zeiss). Images were obtained that included the entire cross-section of the venous anastomosis by using KS 400 Image Analysis software (Zeiss). Ki-67, HIF-1α hypoxia-inducible factor–1α, or TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (brown) and total nuclei (brown and blue) were highlighted, in turn, by selecting the appropriate red-green-blue color intensity range and were then counted. The color intensity was adjusted for each section to account for the decreasing intensity of positive staining over time. The Ki-67, HIF-1α hypoxia-inducible factor–1α, or TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling indexes ([positive cells/total cells] × 100) were calculated for each section. This was repeated twice to ensure that intraobserver variability was less than 10%. The area of the neointima and the media and adventitia for the venous anastomosis was determined by manually tracing the different layers of the vessel wall after immunostaining with hematoxylin-eosin. The neointima was defined as the area above the internal elastic lamina, which was easily determined from the media for three to five sections. The medial and adventitial area was defined from the internal elastic lamina to the fat surrounding the vessel wall.

TUNEL Staining

TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling staining was performed on paraffin-embedded sections from the outflow vein of MSC and control groups as specified by the manufacturer (DeadEnd Colorimetric tunnel assay system, G7360; Promega, Madison, Wis). The negative control is shown where the recombinant terminal deoxynucleotidyl transferase enzyme was omitted.

RNA Isolation

The outflow vein was isolated and stored in RNA stabilizing reagent (Qiagen, Gaithersburg, Md) per the manufactures guidelines. To isolate the RNA, the specimens were homogenized, and total RNA from the samples was isolated by using the RNeasy Mini Kit (Qiagen) (25).

Real-time Polymerase Chain Reaction Analysis

Studies have demonstrated that MSC mesenchymal stem cells exert their anti-inflammatory effect through a reduction in gene expression of Mcp-1 (29). We assessed the gene expression of Mcp-1 by performing real-time polymerase chain reaction analysis on day 7 after vessels with transplanted MSC mesenchymal stem cells were compared with control AVF arteriovenous fistulas alone. Expression for the gene of interest was determined by using real-time polymerase chain reaction analysis, as described previously (25). The primers used are shown in the Table.

Table thumbnail
Mouse Primers Used

Statistical Methods

Data are expressed as means ± standard errors of the mean. Multiple comparisons were performed with two-way analysis of variance, followed by the Student t test with post hoc Bonferroni correction. A significant difference from the control value was indicated by a P value less than .05. JMP version 9 software (SAS Institute, Cary, NC) was used for statistical analyses.

Results

Localization of MSCs after Adventitial Delivery to the Outflow Vein of AVF

MSC mesenchymal stem cells were stably transfected with GFP green fluorescent protein so they could be identified after adventitial delivery by using confocal microscopy of the outflow vein, performed at different times. This demonstrated that GFP green fluorescent protein-positive cells from the vessels with transplanted MSC mesenchymal stem cells (blue positive cells, Fig 1a) were present on day 7. However, by day 21, there was no visualization of the GFP green fluorescent protein signal (data not shown).

Figure 1a:
(a) Photomicrographs (original magnification, ×20) show the localization of human adipose tissue–derived MSC mesenchymal stem cells. GFP green fluorescent protein was used to stably transfect 2.5 × 105 MSC mesenchymal stem cells, ...

PET images of mice after adventitial delivery of 89Zr-labeled MSC mesenchymal stem cells showed that more than 90% of administered 89Zr radioactivity was retained at the delivery site on day 4 (Fig 1). Adventitial retention of 89Zr radioactivity cleared slowly from day 4 to day 21, losing approximately 20% over this period (Fig 1b). Most 89Zr radioactivity that was cleared from the adventitia appeared to translocate to bones. This result confirmed the results obtained by using confocal microscopy with GFP green fluorescent protein-labeled cells on day 7. PET imaging of 89Zr-labeled MSC mesenchymal stem cells allowed tracking of cells beyond 7 days, which was not possible with GFP green fluorescent protein-labeled cells. The retention of most of the delivered stem cells at the delivery site on day 21 demonstrates that the effect is longer than what was visualized by using GFP green fluorescent protein labeling. In the case of the control group in which 89Zr (HPO4)2 was administered, a biodistribution similar to that of 89Zr-labeled MSC mesenchymal stem cells was seen, with most of the radioactivity (approximately 80%) retained at the delivery site and the rest redistributing to bones.

Figure 1b:
(a) Photomicrographs (original magnification, ×20) show the localization of human adipose tissue–derived MSC mesenchymal stem cells. GFP green fluorescent protein was used to stably transfect 2.5 × 105 MSC mesenchymal stem cells, ...

Adventitial Transplantation of MSCs Reduces Gene Expression of Mcp-1 at the Outflow Vein

The mean gene expression of Mcp-1 at the outflow vein of vessels with transplanted MSC mesenchymal stem cells was significantly lower than that in the control group (mean reduction, 62%; P = .029; Fig 2).

Figure 2:
Graph shows Mcp-1 gene expression on day 7 in vessels with transplanted MSC mesenchymal stem cells compared with outflow vein vessels removed from control animals. There is a significant decrease in mean Mcp-1 gene expression in the vessels with transplanted ...

Adventitial Transplantation of MSCs to the Outflow Vein Reduces Mean Neointimal Area and Medial and Adventitial Area and Cell Density in the Neointima While Increasing Mean Lumen Vessel Area on Days 7 and 21

We determined the mean lumen vessel area on day 7 and observed that there was a significant increase in the outflow vein removed from vessels with transplanted MSC mesenchymal stem cells versus the control group (mean increase, 176%; P = .013; Fig E3, A [online]). By day 21, it remained significantly increased in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean increase, 415%; P = .011). We next determined the mean of the neointimal area and the medial and adventitial area. By day 21, there was a significant decrease in the neointimal area and the medial and adventitial area in the outflow vein removed from the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 77%; P = .013; Fig 3; Fig E3, B [online]).

Figure 3:
Photomicrographs (original magnification, ×40) show hematoxylin-eosin staining of vessels with transplanted MSC mesenchymal stem cells (right) compared with outflow vein vessels removed from control animals with AVF arteriovenous fistula only ...

By day 7, the mean cell density of the neointima in the MSC mesenchymal stem cell-treated vessels was significantly lower than that the control group (mean reduction, 83%; P = .0007; Fig E3, C [online]). By day 21, it remained lower in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 83%; P < .0001).

Adventitial Transplantation of MSCs to the Outflow Vein Increases TUNEL Staining

We speculated that the decrease in cell density was also due to an increase in apoptosis (30). Apoptosis was evaluated by using TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling staining (Fig 4, upper row). By day 7, the mean density of cells staining positive for TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling (brown-staining nuclei) at the outflow vein of vessels with transplanted MSC mesenchymal stem cells was significantly increased compared with the control group (mean increase, 3061%; P < .0001). By day 21, it remained higher in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean increase, 425%; P < .0001; Fig E4, A [online]). Therefore, these results demonstrate that vessels with transplanted MSC mesenchymal stem cells have increased TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling activity, indicating cellular apoptosis when compared with control vessels.

Figure 4:
Photomicrographs (original magnification, ×100 [top row] and ×40 [bottom row]) show TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling and Ki-67 staining in murine AVF arteriovenous fistula on day 7 after placement of AVF ...

Adventitial Transplantation of MSCs to the Outflow Vein Reduces Cellular Proliferation at the Outflow Vein

Because the cellular density was decreased, we determined if this was associated with a reduction in cellular proliferation that was assessed by using Ki-67 staining (brown-staining nuclei are positive for Ki-67; Fig 4, lower row). Seven days after fistula placement, there was a significant reduction in the mean Ki-67 index in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 81%; P = .0003; Fig E4, B [online]). By day 21, it remained significantly lower in the vessels with transplanted MSC mesenchymal stem cells when compared with the control vessels (mean reduction, 60%; P = .016).

Adventitial Transplantation of MSCs to the Outflow Vein Reduces α-SMA and FSP-1 Staining

We assessed smooth-muscle deposition by using α-SMA α–smooth-muscle actin staining (Fig 5, upper row). By day 21, the mean α-SMA α–smooth-muscle actin staining was significantly lower in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 27%; P = .013; Fig E5, A [online]). FSP-1 fibroblast specific protein–1 has been used as a fibroblast marker (Fig 5, lower panel). In previous studies, investigators have demonstrated fibroblast to myofibroblast (α-SMA α–smooth-muscle actin) differentiation, resulting in VNH venous neointimal hyperplasia (28,31). By day 7, we observed a significant decrease in the mean FSP-1 fibroblast specific protein–1 staining in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 65%; P = .0003; Fig E5, B [online]). By day 21, it remained significantly lower in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group (mean reduction, 42%; P = .0016). Overall, these results indicate that on day 7, there is a reduction in FSP-1 fibroblast specific protein–1 staining, followed by a decrease in α-SMA α–smooth-muscle actin staining by day 21 in vessels with transplanted MSC mesenchymal stem cells when compared with control vessels.

Figure 5:
Photomicrographs (original magnification, ×40) show FSP-1 fibroblast specific protein–1 and α-SMA α–smooth-muscle actin staining in murine AVF arteriovenous fistula control animals on day 21 after placement of AVF ...

Adventitial Transplantation of MSCs to the Outflow Vein Is Associated with a Reduction in HIF-1α Staining

We quantified HIF-1α hypoxia-inducible factor–1α staining to assess whether MSC mesenchymal stem cell transplantation had an effect on the expression of HIF-1α hypoxia-inducible factor–1α at the outflow vein of AVF arteriovenous fistula. Brown-staining nuclei are positive for HIF-1α hypoxia-inducible factor–1α (Fig 6, upper row). By day 7, there was a significant reduction in the mean density of HIF-1α hypoxia-inducible factor–1α–staining vessels with transplanted MSC mesenchymal stem cells when compared with control vessels (mean reduction, 67%; P < .0001; Fig E6, A [online]). By day 21, it remained significantly lower in the MSC mesenchymal stem cell-treated vessels when compared with control vessels (mean reduction, 62%; P = .0005). Overall, these results indicate that there is decreased expression in HIF-1α hypoxia-inducible factor–1α in vessels with transplanted MSC mesenchymal stem cells when compared with control vessels.

Figure 6:
Photomicrographs (original magnification, ×40) show HIF-1α hypoxia-inducible factor–1α and CD68 staining in murine AVF arteriovenous fistula on day 7 after placement in the outflow vein alone (control group) and vessels ...

Adventitial Transplantation of MSCs to the Outflow Vein Is Associated with a Reduction in CD68 Staining

Cells staining brown in the cytoplasm are positive for CD68 (Fig 6, lower row). By day 7, there was significant reduction in the mean density of CD68 staining in the MSC mesenchymal stem cell-treated vessels when compared with control vessels (mean reduction, 51%; P = .033; Fig E6, B [online]). Overall, there is a significant decrease in CD68 staining in the vessels with transplanted MSC mesenchymal stem cells when compared with the control group.

Discussion

In our study, we demonstrated that adventitial transplantation of human adipose tissue–derived MSC mesenchymal stem cells to the outflow vein of AVF arteriovenous fistula in a murine model reduces VNH venous neointimal hyperplasia. This is mediated by a significant decrease in the gene expression of Mcp-1 in the outflow vein with transplanted MSC mesenchymal stem cells compared with the control group on day 7. There is a significant increase in mean TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling staining with a decrease in proliferation. Additionally, there is a significant decrease in FSP-1 fibroblast specific protein–1, CD68, and α-SMA α–smooth-muscle actin staining, accompanied by a decrease in mean HIF-1α hypoxia-inducible factor–1α staining. Finally, Zr89-labeled MSC mesenchymal stem cells can be tracked up to 3 weeks after adventitial delivery.

Previous studies have shown that there is increased gene expression of Mcp-1 at the AVF arteriovenous fistula (57). Genetic deletion of Mcp-1 in experimental animal models of AVF arteriovenous fistula failure was associated with a reduction in venous stenosis formation (5). Mcp-1 was localized to the endothelium, smooth muscle cells, and leukocytes. In the present study, we observed that vessels with transplanted MSC mesenchymal stem cells had reduced expression of Mcp-1, accompanied by a reduction in cells staining positive for FSP-1 fibroblast specific protein–1, α-SMA α–smooth-muscle actin, and CD68. Adventitial transplantation of MSC mesenchymal stem cells to the outflow vein at the time of AVF arteriovenous fistula results in a reduction of several proinflammatory genes, including Mcp-1. Our findings are consistent with previous studies that have demonstrated that human MSC mesenchymal stem cell transplantation can reduce Mcp-1 gene expression in experimental animal models of focal cerebral ischemia (32). Increased expression of the MCP-1/CCL2 axis is associated with the pathogenesis of several different fibrotic models involving the lung, kidney, and liver (3336). Notably, Mcp-1 is implicated in the dysfunction of AVF arteriovenous fistula clinically and in experimental animal models (5,37). Studies conducted in patients with hemodialysis have shown that there are increased serum levels of MCP-1 and that this is considered to be a risk factor for AVF arteriovenous fistula dysfunction (38,39).

VNH venous neointimal hyperplasia is characterized by an increase in cellular proliferation and extracellular matrix deposition and a decrease in apoptosis (8,40,41). Previous studies have demonstrated the inhibitory effects of MSC mesenchymal stem cell on proliferation of target cells, such as monocytes, tumor cells, and cardiac fibroblasts (1720). Conversely, MSC mesenchymal stem cells have been shown to induce proliferation and migration in lung fibroblasts and endothelial cells (42,43). Studies conducted at our laboratories and others have demonstrated that adventitial and medial fibroblasts can convert to myofibroblasts (α-SMA α–smooth-muscle actin[+]) that can proliferate and migrate, leading to VNH venous neointimal hyperplasia formation (31,44).

In our study, we assessed cellular proliferation by performing Ki-67 staining and observed a significant reduction in cellular proliferation in vessels removed from animals with transplanted MSC mesenchymal stem cells when compared with the control group. MSC mesenchymal stem cells can exert an antiproliferative effect mediated in part by decreasing Mcp-1 expression. Our finding is consistent with a study conducted in vitro in which CD14+ cells isolated from peripheral blood–enhanced fibroblast proliferation mediated through MCP-1 (45). In addition to reduced cellular proliferation, we also observed a significant decrease in cellular density, accompanied by a significant increase in TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling staining.

Hypoxic injury to the vessel wall of the outflow vein at the time of AVF arteriovenous fistula placement and arterial bypass grafts can result in VNH venous neointimal hyperplasia (4648). Several studies have demonstrated increased HIF-1α hypoxia-inducible factor–1α expression in animal models of hemodialysis graft failure and in clinical specimens from patients with hemodialysis vascular access failure (23,4951). In our study, we found that adventitial transplantation of MSC mesenchymal stem cells to adventitia of the outflow vein in a murine model results in a significant reduction in the expression of HIF-1α hypoxia-inducible factor–1α. These findings indicate that MSC mesenchymal stem cell transplantation to the adventitia of the outflow vein is associated with a reduction in expression of HIF-1α hypoxia-inducible factor–1α, therefore possibly reducing hypoxia-induced inflammatory response in the vessel wall. It is well known that macrophages can home to areas of vascular injury associated with hypoxia, and we hypothesize that the reduction in HIF-1α hypoxia-inducible factor–1α and thus hypoxia is responsible for the decreased CD68 staining.

PET imaging of 89Zr-labeled MSC mesenchymal stem cells showed most (approximately 80%) of the radiolabel to be retained at the adventitial delivery site, even 3 weeks after delivery. This result confirms that the cells are in contact with the adventitia of the outflow vein for a prolonged period after delivery. The similar imaging findings with 89Zr (HPO4)2, which delivered 89Zr in its “free” cationic form to the adventitia, would appear to indicate that the adventitia is not well perfused. Thus, the slow release of 89Zr-labeled MSC mesenchymal stem cells from the adventitia cannot be interpreted as strong evidence of specific binding of the cells to the adventitia. In contrast, 89Zr-labeled MSC mesenchymal stem cells delivered intravenously home to the lung, followed by the liver and bones, and free 89Zr (HPO4)2 is mainly localized in the liver and bones. These differences in biodistribution provide further evidence that the prolonged retention of both 89Zr-labeled MSC mesenchymal stem cells and 89Zr (HPO4)2 may be explained by the lack of perfusion on the surface of the adventitia due to a less defined vasa vasorum surrounding the vein when compared with the artery (20). Additionally, the use of 89Zr-labeled MSC mesenchymal stem cells was more sensitive than GFP green fluorescent protein labeling for detection of cellular location after delivery.

There are several limitations to our study. We used an immunodeficient mouse, and a potential T cell–mediated mechanism involved in the pathophysiology of VNH venous neointimal hyperplasia could not be evaluated. A murine model of normal kidney function was used; thus, the effects of chronic kidney disease could not be evaluated. Finally, these results need to be corroborated by using larger animal models, as the murine model may not simulate the clinical scenario.

In conclusion, we demonstrate that adventitial transplantation of human adipose tissue–derived MSC mesenchymal stem cells to the outflow vein of a murine model of AVF arteriovenous fistula results in a decrease in the gene expression of Mcp-1. This resulted in a reduction in VNH venous neointimal hyperplasia, accompanied by a decrease in cell proliferation and an increase in apoptosis with positive vascular remodeling. The clinical importance of this study is that it provides a rationale for using MSC mesenchymal stem cell transplantation in patients with hemodialysis AVF arteriovenous fistula access for reducing VNH venous neointimal hyperplasia and highlights the fact that MSC mesenchymal stem cells can be tracked after delivery by using PET imaging of 89Zr-labeled MSC mesenchymal stem cells.

Advances in Knowledge

  • ■ In vessels with transplanted mesenchymal stem cells (MSC mesenchymal stem cells) compared with control vessels, there was a significant decrease in monocyte chemoattractant protein-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]) and a significant decrease in the mean neointimal area and the medial and adventitial area (day 21: mean reduction, 77%; P = .013) and in neointimal cell density (day 7: mean reduction, 83% [P = .0007]; day 21: mean reduction, 83% [P < .0001]).
  • ■ 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]), fibroblasts (day 7: mean reduction, 65% [P = .0003]; day 21: mean reduction, 42% [P = .0016]), smooth muscle cells (day 21: mean reduction, 27%; P = .013), and macrophage staining (day 7: mean reduction, 51%; P = .033), with a significant increase in cellular apoptosis (day 7: mean increase, 3061% [P < .0001]; day 21: mean increase, 425% [P < .0001]) when compared with controls.
  • ■ We were able to track transplanted zirconium 89–labeled MSC mesenchymal stem cells with PET imaging for 21 days.

Implication for Patient Care

  • ■ Adipose tissue–derived MSC mesenchymal stem cell transplantation to the adventitia of the outflow vein of arteriovenous fistula may potentially help reduce venous stenosis formation.

SUPPLEMENTAL FIGURES

Figure E1:

Figure E2:

Figure E3:

Figure E4:

Figure E5:

Figure E6:

Acknowledgments

The authors thank Jay Mandrekar, PhD, for his biostatistical expertise in interpreting the results.

Received April 27, 2015; revision requested June 12; revision received July 20; accepted August 6; final version accepted September 14.

A. Brahmbhatt was supported by the Howard Hughes Medical Institute (HHMI-SIRF 2013).

Funding: This research was supported by the National Institutes of Health (grant HL098967).

Disclosures of Conflicts of Interest: B.Y. disclosed no relevant relationships. A. Brahmbhatt disclosed no relevant relationships. E.N.T. disclosed no relevant relationships. B.T. disclosed no relevant relationships. D.L.M. disclosed no relevant relationships. S.E. disclosed no relevant relationships. A. Bansal disclosed no relevant relationships. M.K.P. disclosed no relevant relationships. A.B.D. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: author is an inventor of technology at Mill Creek Life Sciences, which supplies technology for growing MSCs, and has a leadership position within the company. Other relationships: author has a patent pending for methods and materials for reducing VNH of an AVF or graft. E.B.L. disclosed no relevant relationships. T.R.D. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: author has a patent pending for 89Zr-labeled cells. D.M. disclosed no relevant relationships. S.M. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: author has a patent pending for preventing venous stenosis formation with MSCs.

Abbreviations:

α-SMA
α–smooth-muscle actin
AVF
arteriovenous fistula
FSP-1
fibroblast specific protein–1
GFP
green fluorescent protein
HIF-1α
hypoxia-inducible factor–1α
MSC
mesenchymal stem cell
TUNEL
terminal deoxynucleotidyl transferase dUTP nick end labeling
VNH
venous neointimal hyperplasia

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