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TRANSLATIONAL AND CLINICAL RESEARCH Adipose Tissue-Derived Mesenchymal Stem Cells Improve Revascularization Outcomes to Restore Renal Function in Swine Atherosclerotic Renal Artery Stenosis ALFONSO EIRIN,a XIANG-YANG ZHU,a JAMES D. KRIER,a HUI TANG,a KYRA L. JORDAN,a JOSEPH P. GRANDE,a,b AMIR LERMAN,c STEPHEN C. TEXTOR,a LILACH O. LERMANa,c Division of Nephrology and Hypertension, bDepartment of Laboratory Medicine and Pathology, and cDivision of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA a

Key Words. Renal artery stenosis • Progenitor cells • Renal hypertension • Revascularization

ABSTRACT Reno-protective strategies are needed to improve renal outcomes in patients with atherosclerotic renal artery stenosis (ARAS). Adipose tissue-derived mesenchymal stem cells (MSCs) can promote renal regeneration, but their potential for attenuating cellular injury and restoring kidney repair in ARAS has not been explored. We hypothesized that replenishment of MSC as an adjunct to percutaneous transluminal renal angioplasty (PTRA) would restore renal cellular integrity and improve renal function in ARAS pigs. Four groups of pigs (n 5 7 each) were studied after 16 weeks of ARAS, ARAS 4 weeks after PTRA and stenting with or without adjunct intrarenal delivery of MSC (10 3 106 cells), and controls. Stenotic kidney blood flow (renal blood flow [RBF]) and glomerular filtration rate (GFR) were measured using multidetector computer tomography (CT). Renal microvascular architecture (micro-CT), fibrosis, inflammation, and

oxidative stress were evaluated ex vivo. Four weeks after successful PTRA, mean arterial pressure fell to a similar level in all revascularized groups. Stenotic kidney GFR and RBF remained decreased in ARAS (p 5 .01 and p 5 .02) and ARAS 1 PTRA (p 5 .02 and p 5 .03) compared with normal but rose to normal levels in ARAS 1 PTRA 1 MSC (p 5 .34 and p 5 .46 vs. normal). Interstitial fibrosis, inflammation, microvascular rarefaction, and oxidative stress were attenuated only in PTRA 1 MSC-treated pigs. A single intrarenal delivery of MSC in conjunction with renal revascularization restored renal hemodynamics and function and decreased inflammation, apoptosis, oxidative stress, microvascular loss, and fibrosis. This study suggests a unique and novel therapeutic potential for MSC in restoring renal function when combined with PTRA in chronic experimental renovascular disease. STEM CELLS 2012;30:1030–1041

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Renal artery stenosis (RAS) is one of the reversible mechanisms for hypertension. Atherosclerosis is the most common cause of RAS, accounting for 90% of the cases [1]. Based upon community-based screening, atherosclerotic RAS (ARAS) exceeding 60% lumen occlusion averages 6.8% in the elderly population [2]. ARAS can accelerate hypertension and lead to loss of kidney function, which are known to increase cardiovascular morbidity and mortality [3]. Renal revascularization using endovascular percutaneous transluminal renal angioplasty (PTRA) and stenting has been a common treatment strategy in patients with ARAS both to reduce blood pressure and to improve renal function. To date, however, randomized, prospective trials fail to identify major

benefits from restoring blood flow for preservation of renal function [4, 5] compared with medical therapy alone. This might be due to lingering kidney tissue damage that is not reversed by restoring blood flow with PTRA alone. In line with these clinical observations, we have previously shown in a swine model of non-ARAS that PTRA partially restores the renal microvascular network and improves renal function, but vascular wall remodeling and fibrosis are incompletely reversed [6]. The presence of an atherosclerotic environment compounds these effects. Renal revascularization in a swine model of ARAS normalizes blood pressure levels but fails to improve tubulointerstitial injury, microvascular rarefaction, and renal function in the stenotic kidney [7]. This dissociation between the effects of revascularization on blood pressure and renal function underscores the need to identify more effective

Author contributions: A.E.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; X.Z.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; J.D.K., H.T., and K.L.J.: collection and/or assembly of data and data analysis and interpretation; J.P.G.: final approval of manuscript; A.L. and S.C.T.: manuscript writing and final approval of manuscript; L.O.L.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. Correspondence: Lilach O. Lerman, Ph.D., M.D., Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. Telephone: 507-266-9376; Fax: 507-266-9316; e-mail: [email protected] Received October 4, 2011; Revised December 23, 2011; accepted for publication January 9, 2012; first published online in STEM CELLS EXPRESS January 30, 2012. C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1047 V

STEM CELLS 2012;30:1030–1041 www.StemCells.com

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Table 1. Systemic characteristics (mean 6 SEM) in normal, ARAS, ARAS 1 PTRA, and ARAS 1 PTRA 1 MSC pigs (n 5 7 each) 4 weeks after PTRA or sham Normal

Body weight (kg) Degree of stenosis (%) Mean arterial pressure (mmHg) Serum creatinine (mg/dl) PRA (ng/ml per hour) Total cholesterol (mg/dl) Triglycerides (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) 8-Isoprostane (pg/ml) Interleukin-1b (pg/ml) Urinary albumin (lg/ml)

54.0 6 0 97.3 6 1.30 6 0.14 6 92.5 6 7.8 6 43.7 6 47.3 6 103.2 6 28.6 6 3.65 6

6.2 11.9 0.14 0.09 16.0 1.9 12.8 8.6 9.9 14.5 1.07

ARAS

55.1 87.2 145.3 1.83 0.13 481.0 9.1 159.3 302.6 195.3 373.3 3.80

6 6 6 6 6 6 6 6 6 6 6 6

4.3 21.1*,† 19.6*,† 0.33*,† 0.12 80.9* 2.3 57.9* 81.4* 0.8*,† 145.7*,† 0.67

ARAS þ PTRA

ARAS þ PTRA þ MSC

55.9 6 7.1 0 102.7 6 5.9 1.89 6 0.31*,† 0.19 6 0.08 415.7 6 126.6* 8.2 6 4.8 148.2 6 62.0* 265.9 6 101.6* 187.0 6 47.9*,† 234.6 6 98.3*,† 3.14 6 1.46

55.8 6 0 97.8 6 1.45 6 0.15 6 403.4 6 6.6 6 137.9 6 291.6 6 124.0 6 71.5 6 3.22 6

5.6 4.8 0.14 0.08 99.2* 1.7 48.4* 85.9* 11.95 41.5 0.33

*p < .05 vs. normal. p < .05 vs. ARAS þ PTRA þ MSC. Abbreviations: ARAS, atherosclerotic renal artery stenosis; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MSC, mesenchymal stem cell; PRA, plasma renin activity; PTRA, percutaneous transluminal renal angioplasty. †

strategies to restore the structures within the stenotic kidney in ARAS in addition to PTRA. Our previous studies demonstrated that intrarenal delivery of autologous hematopoietic endothelial progenitor cells (EPCs) can increase neovascularization and mitigate renal injury in non-atherosclerotic RAS [8]. However, the capacity of this cell-based therapy to reverse the more profound damage observed in the ARAS kidney was more limited in that EPC only partially improved microvascular density and failed to fully restore renal blood flow (RBF) and glomerular filtration rate (GFR) [9]. We speculated that both securing renal arterial patency and at the same time improving the regenerative capacity of the poststenotic kidney using cell-based therapy might be a more effective strategy to preserve the stenotic kidney. As a practical matter, autologous EPCs are difficult to isolate and expand. Mesenchymal stem cells (MSCs) are undifferentiated nonembryonic stem cells present in adult tissues, which have the ability to differentiate into a broad spectrum of cell lineages [10]. Moreover, MSC can be isolated from a variety of tissues, including adipose tissue and bone marrow, and possess immunomodulatory properties that decrease inflammation and immune responses [11]. Previous studies showed that MSCs restore renal structure and function in experimental rodent models of acute renal failure [12]. Whether MSC might augment renal function and structure improvement in response to PTRA in a large animal model remains unknown. Thus, we hypothesized that intrarenal infusion of allogeneic MSC at the time of revascularization would restore renal cellular integrity and repair mechanisms in experimental ARAS.

RESULTS Six weeks after induction of RAS and before PTRA, all ARAS pigs demonstrated hemodynamically significant stenosis (79.4% 6 2.7%, p ¼ .26 analysis of variance) [13], and mean arterial pressure (MAP) was elevated compared with normal pigs (p < .01 in all). The systemic characteristics in all pigs 4 weeks after PTRA or sham are summarized in Table 1. Total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were elevated in all ARAS groups compared with normal. As common in chronic ARAS [14, 15], plasma renin activity (PRA) levels were similar among the groups. www.StemCells.com

PTRA Successfully Reduced Blood Pressure There was no residual stenosis at 16 weeks in PTRA-treated pigs (Fig. 1A). Continuously measured MAP decreased immediately after PTRA and persisted at normal levels until the end of the study (p < .05 vs. ARAS, p > .05 vs. normal) (Table 1, Fig. 1B).

MSC Characterization and Culture MSC displayed a fibroblast-like, spindle-shaped morphology (Supporting Information Fig. 2As), expressed CD44, CD90, and CD105 markers (Supporting Information Fig. 2Cs), and secreted vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-a in the culture media (Supporting Information Fig. 2Bs). Furthermore, successful transdifferentiation of MSC into osteocytes, chondrocytes, and adipocytes in vitro supported their mesenchymal origin (Supporting Information Fig. 3As).

MSC Home to the Stenotic Kidney The MSC retention rate in the kidney 4 weeks after intra-arterial administration was 13.1% 6 2.2% (1–2 cells per field (40) or 800–1,000 per an entire slide). Chloromethylbenzamido-DiI-labeled MSCs were mostly detected at the renal cortical interstitium (cortical-medullary engraftment ratio ¼ 5:1) 4 weeks after injection, although cytokeratin staining showed that some MSCs were incorporated into renal tubules (Fig. 1C). In addition, few MSC costained with the endothelial marker CD31 and the proliferating cell nuclear antigen (PCNA) marker (Supporting Information Fig. 4s). In contrast to the stenotic kidney, very few (1–2 per an entire slide) cells were detected in the contralateral kidney (Supporting Information Fig. 3sB) and heart (Supporting Information Fig. 3sC), as previously shown [8, 16]. Histological analysis showed no evidence of cellular rejection (e.g., CD3 clusters), microinfarcts, or tumors in tissue sections from ARAS þ PTRA þ MSC pigs.

MSCs Restore Renal Hemodynamics and Function Basal stenotic kidney GFR and RBF were similarly attenuated in ARAS and ARAS þ PTRA (Fig. 1D, p < .05 vs. normal) but not different than normal levels in ARAS þ PTRA þ MSC, although the large variability of RBF might have contributed to its apparent increase. GFR responses to acetylcholine (Ach) were normalized in MSC-treated pigs, while RBF responses remained blunted (p ¼ .32 vs. baseline). Serum

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Figure 1. PTRA þ MSC restored blood pressure, renal hemodynamics, and function. (A): Renal angiography in a pig with ARAS before (left) and 4 weeks after (right) revascularization with PTRA. (B): Mean arterial pressure measured using telemetry decreased after PTRA. (C): Red fluorescence of chloromethylbenzamido-DiI (arrows, 40) MSC cytokeratin (green)-stained stenotic kidney 4 weeks after administration. Blue: 40 ,6diamidino-2-phenylindole nuclear stain. (D): Single-kidney RBF and GFR were restored in MSC-treated pigs, although RBF response to Ach remained blunted. *, p < .05 versus normal, †, p < .05 versus ARAS þ PTRA þ MSC, and ‡, p < .05 versus baseline. Abbreviations: Ach, acetylcholine; ARAS, atherosclerotic renal artery stenosis; GFR, glomerular filtration rate; MSC, mesenchymal stem cell; PTRA, percutaneous transluminal renal angioplasty; RBF, renal blood flow.

creatinine levels were higher in ARAS compared with normal and remained elevated after PTRA (p ¼ .04 vs. normal; p ¼ .77 vs. ARAS). Treatment with MSC led to a fall in serum creatinine to normal levels (p ¼ .14 vs. normal, Table 1).

Microvascular Architecture Is Improved in MSC-Treated Pigs Spatial density of cortical microvessels was similarly diminished in ARAS and ARAS þ PTRA but improved after MSC treatment to levels not different from normal pigs (Fig. 2A,

2B). In particular, the number of small vessels ( .05 vs. normal, Supporting Information Fig. 5sB). Vessel diameter was similarly increased in ARAS and ARAS þ

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Figure 2. Treatment with MSC improved angiogenesis and microvascular architecture. (A): Microcomputer tomography three-dimensional images of the kidney showing improved microvascular architecture in ARAS þ PTRA þ MSC. Spatial density (B) and its classification by vessel size (C), average vessel diameter (D), and tortuosity (E) of renal cortical microvessels. (F): Renal protein expression of VEGF and VEGFR-2, eNOS, and bFGF in normal, ARAS, ARAS þ PTRA, and ARAS þ PTRA þ MSC pigs. *, p < .05 versus normal and †, p < .05 versus ARAS þ PTRA þ MSC. Abbreviations: ARAS, atherosclerotic renal artery stenosis; bFGF, basic fibroblast factor; eNOS, endothelial nitric oxide synthase; MSC, mesenchymal stem cell; PTRA, percutaneous transluminal renal angioplasty; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

PTRA compared with normal but decreased to normal levels in MSC-treated pigs (Fig. 2D, p ¼ .12 vs. normal). Tortuosity (a measure of angiogenesis and vascular remodeling) was increased in MSC-treated pigs compared with normal but was lower than in ARAS (p ¼ .03) and ARAS þ PTRA (Fig. 2E, p ¼ .05) pigs. www.StemCells.com

Angiogenic Factors Were Upregulated in Animals Treated with MSC Expression of VEGF was reduced in ARAS and ARAS þ PTRA (Fig. 2F, p < .05 vs. normal) but restored to normal levels in MSC-treated pigs (p < .05 vs. ARAS and ARAS þ PTRA, p ¼ .61 vs. normal). In addition, expression of the

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Figure 3. Oxidative stress declined after PTRA þ MSC. (A): Renal production of superoxide anion (A), detected by DHE (40), and its quantification (B). (C): Representative immunoblots and renal protein expression of NT and p47 in normal, ARAS, ARAS þ PTRA, and ARAS þ PTRA þ MSC. (D): Renal protein expression of TNF-a, MCP-1, IF-c, and NFjb in normal, ARAS, ARAS þ PTRA, and ARAS þ PTRA þ MSC pigs. *, p < .05 versus normal and †, p < .05 versus ARAS þ PTRA þ MSC. Abbreviations: ARAS, atherosclerotic renal artery stenosis; DHE, dihydroethidium; IF-c, interferon c; MCP, monocyte chemoattractant protein; MSC, mesenchymal stem cell; NFjB, nuclear factor kappa B; NT, nitrotyrosine; PTRA, percutaneous transluminal renal angioplasty; TNF-a, tumor necrosis factor a.

proangiogenic factors, endothelial nitric oxide synthase (eNOS), and basic fibroblast growth factor (bFGF) was downregulated in ARAS and ARAS þ PTRA animals, but treatment with MSC restored their expression to levels not differ-

ent from normal (p > .05 vs. normal). Contrarily, renal expression of VEGF receptor (VEGFR)-2 that was attenuated in ARAS was similarly normalized in both PTRA-treated groups (Fig. 2F, p > .05 vs. normal both).

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Figure 4. PTRA þ MSC decreased renal inflammation. Representative immunostaining (40) of B-T lymphocytes (CD45þ), T lymphocytes (CD3þ), and macrophages (CD163þ) (A) and their quantification in tubular and glomerular compartments (B). *, p < .05 versus normal and † , p < .05 versus ARAS þ PTRA þ MSC. Abbreviations: ARAS, atherosclerotic renal artery stenosis; MSC, mesenchymal stem cell; PTRA, percutaneous transluminal renal angioplasty.

MSC Reduced Oxidative Stress Circulating levels of 8-isoprostanes were significantly higher in sham-treated and PTRA-treated ARAS compared with normal (p ¼ .04 and p ¼ .03, respectively) but were restored to normal levels after renal administration of MSC (Table 1, p > .05 vs. normal). Moreover, in situ production of superoxide anion was www.StemCells.com

similarly increased in ARAS and ARAS þ PTRA (p ¼ .01 vs. normal and p ¼ .99 vs. ARAS) and decreased to levels not different from normal in ARAS þ PTRA þ MSC (Fig. 3A, p ¼ .10 vs. normal). Also, the increased protein expression of the NAD(P)H-oxidase subunit p47phox observed in ARAS and ARAS þ PTRA was normalized after MSC treatment, suggesting

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Figure 5. Renal fibrosis and apoptosis were reduced in MSC-treated pigs. (A): Representative renal trichrome staining (40) in normal, ARAS, ARAS þ PTRA, and ARAS þ PTRA þ MSC pigs. Periglomerular and tubulointerstitial fibrosis (B) and glomerular score (C, % of sclerotic glomeruli) decreased after PTRA þ MSC. *, p < .05 versus normal and †, p < .05 versus ARAS þ PTRA þ MSC. (D): TUNEL staining showing increased number of apoptotic cells (green) in ARAS and ARAS þ PTRA, which decreased in MSC-treated animals. Abbreviations: ARAS, atherosclerotic renal artery stenosis; MSC, mesenchymal stem cell; PTRA, percutaneous transluminal renal angioplasty; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

a decreased potential for superoxide generation. Furthermore, protein expression of nitrotyrosine (NT), which was similarly and significantly elevated in ARAS and ARAS þ PTRA kidneys compared with normal (p < .05), was substantially reduced in MSC-treated pigs (Fig. 3B, p < .05 vs. ARAS and ARASþPTRA, p ¼ .29 vs. normal), implying decreased production of peroxynitrite.

MSC Decreased Inflammation in the Stenotic Kidney The number of CD45þ, CD3þ, and CD163þ cells infiltrating the kidney was similarly increased in ARAS and ARAS þ PTRA pigs compared with normal (p < .05 for both) but decreased to normal levels after treatment with MSC (Fig. 4A–

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Figure 6. Collagen formation diminished after PTRAþ MSC. (A) Sirius red staining, viewed under polarized light (top) and its quantification (bottom) decreased by MSC, as did TGF-b1 expression (B). *p