Original Research. Profound Actions of an Agonist of Growth Hormone Releasing Hormone on Angiogenic Therapy by Mesenchymal Stem Cells

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1 Original Research Profound Actions of an Agonist of Growth Hormone Releasing Hormone on Angiogenic Therapy by Mesenchymal Stem Cells QunChao Ma,* Xiangyang Xia,* Quanwei Tao, Kai Lu, Jian Shen, Qiyuan Xu, Xinyang Hu, Yaoliang Tang, Norman L. Block, Keith A. Webster, Andrew V. Schally, Jian an Wang, Hong Yu Objective The efficiency of cell therapy is limited by poor cell survival and engraftment. Here, we studied the effect of the growth hormone releasing hormone agonist, JI-34, on mesenchymal stem cell (MSC) survival and angiogenic therapy in a mouse model of critical limb ischemia. Approach and Results Mouse bone marrow derived MSCs were incubated with or without 10 8 mol/l JI-34 for 24 hours. MSCs were then exposed to hypoxia and serum deprivation to detect the effect of preconditioning on cell apoptosis, migration, and tube formation. For in vivo, critical limb ischemia was induced by femoral artery ligation. After surgery, mice were received 50 μl phosphate-buffered saline or with MSCs or with JI-34 reconditioned MSCs. Treatment of MSCs with JI-34 improved MSC viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by an increased phosphorylation and nuclear translocation of signal transducer and activator of transcription 3. In vivo, JI-34 pretreatment enhanced the engraftment of MSCs into ischemic hindlimb muscles and augmented reperfusion and limb salvage compared with untreated MSCs. Significantly more vasculature and proliferating CD31 + and CD34 + cells were detected in ischemic muscles that received MSCs treated with JI-34. Conclusions Our studies demonstrate a novel role for JI-34 to markedly improve therapeutic angiogenesis in hindlimb ischemia by increasing the viability and mobility of MSCs. These findings support additional studies to explore the full potential of growth hormone releasing hormone agonists to augment cell therapy in the management of ischemia. (Arterioscler Thromb Vasc Biol. 2016;36: DOI: /ATVBAHA ) Key Words: angiogenesis effects growth hormone releasing hormone mesenchymal stromal cells Cell-based therapies show promise to promote regeneration and angiogenesis and reverse or rescue tissues injured by ischemia or infarct. In this context, various kinds of progenitor cells have been tested clinically, including cardiac progenitor cells, 1 endothelial progenitor cells, 2 induced pluripotent stem cells, 3 embryonic stem cells, 4 bone marrow mononuclear cells, 5 and mesenchymal stem cells (MSCs), 6 with mixed outcomes. MSCs have the advantages of immune privilege, stemness, and ease of handling relative to other cell types and have become prominent vehicles for a wide range of indications. 7 The mechanism of MSC action is thought to be primarily paracrine. 8 When exogenously administered, MSCs can facilitate the formation of new capillaries and mediumsized arteries. However, therapeutic angiogenesis by MSCs is limited by poor survival especially in the hostile microenvironment of ischemic tissue. Many different approaches have been attempted to improve the efficacy of MSC therapy, including pretreating with various chemicals or polypeptides, 9,10 preconditioning with physiological stimuli such as hypoxia, 11 or combination with other cells including endothelial progenitor cells 12 and endothelial cells. 13 Optimal conditions have not been achieved. Growth hormone releasing hormone (GHRH) is a neuropeptide produced by the hypothalamus. It is carried to the anterior pituitary through the portal vessels and stimulates the release of growth hormone after binding to the GHRH receptor (GHRH-R) on cell membranes. 14 Previous studies have shown that GHRH and its receptor are also expressed on cells Received on: January 3, 2016; final version accepted on: January 21, From the Department of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University, and Cardiovascular Key Laboratory of Zhejiang Province, Hangzhou, P.R. China (Q.-C.M., X.X., Q.T., K.L., J.S., Q.X., X.H., J.W., H.Y.); Vascular Biology Center, Georgia Regents University, Augusta (Y.T.); Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL (N.L.B.); Department of Pathology (N.L.B.), Department of Molecular and Cellular Pharmacology (K.A.W., A.V.S.), and Division of Hematology/Oncology (A.V.S.), Leonard M. Miller School of Medicine, University of Miami, FL. *These authors contributed equally to this article. The online-only Data Supplement is available with this article at Correspondence to Hong Yu, Department of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University, and Cardiovascular Key Laboratory of Zhejiang Province, Hangzhou , 88 JieFang Rd, P.R. China. Hong Yu, yuvascular@163.com 2016 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 2 Arterioscler Thromb Vasc Biol April 2016 Nonstandard Abbreviations and Acronyms GHRH growth hormone releasing hormone GHRH-R growth hormone releasing hormone receptor MSCs mesenchymal stem cells STAT3 signal transducer and activator of transcription 3 SVs splice variants in normal human tissues, for example, liver, kidney, lung, and prostate, as well as many tumors. 15,16 GHRH and its agonists have been used to treat disorders of the endocrine system, as well as to promote cell proliferation and functional recovery in extrapituitary tissues. 17,18 Compared with the native GHRH, synthetic agonists are much more potent and longer acting because of increased stability. 14 One such GHRH agonist, JI-38, has been used to accelerate wound healing primarily by acting on wound-associated fibroblasts. 19 The systemic administration of GHRH agonists has been shown to stimulate the recovery of cardiac function after infarction or ischemia/reperfusion injury by reducing apoptosis of cardiomyocytes, enhancing the recruitment of endogenous cardiac stem cells, stimulating angiogenesis in heart, and ameliorating cardiac remodeling. 17,20,21 Because GHRH-R is expressed in MSCs, 22 we hypothesized that pretreatment of MSCs with such agonists may confer improved therapeutic properties of MSCs. Here, we demonstrate that JI-34, a potent GHRH agonist, indeed improves MSC proliferation and homing and, thus, augments therapy by promoting angiogenesis in ischemic skeletal muscle of mouse hindlimb. Materials and Methods Materials and Methods are available in the online-only Data Supplement. Results GHRH-R Is Expressed on Mouse Bone Marrow Derived MSCs GHRH-R expression on mouse bone marrow derived MSCs was detected by Western blot (Figure 1A), flow cytometry (Figure 1B), and immunofluorescence staining (Figure IA in the online-only Data Supplement). The results from both Western blot and immunofluorescence staining demonstrate that MSCs express levels of GHRH-R that are similar to the brain, whereas skeletal muscle was negative for GHRH-R expression. Flow cytometry showed that the mean fluorescence intensity of GHRH-R on MSCs was 224.3±34.6 (Figure 1B), which was not significantly changed after cells were treated with GHRH agonist, JI-34 (Figure IB and IC in the online-only Data Supplement). These results were further confirmed by Western blot assay (Figure ID in the onlineonly Data Supplement). These data confirm that GHRH-R is expressed on MSCs. GHRH Agonist Preconditioning Enhances Proliferation and Survival of MSCs To investigate the effects of JI-34 on cell proliferation and viability, we treated MSCs with increasing concentrations of JI-34 for 48 hours and quantified resulting cell numbers. Figure 1C shows that the proliferation of MSCs was optimally enhanced (114±8%) by JI-34 at a concentration of mol/l. The viability of MSCs after culture under hypoxia (0.5% O 2 ) with serum deprivation for 48 hours was significantly increased by JI-34 pretreatment when compared with the untreated MSCs (Figure 1D). Augmentation of MSC proliferation and viability was optimal at JI-34 concentration of mol/l; therefore, this concentration was used for further studies. Apoptosis of MSCs after culture under the hypoxia with serum deprivation condition for 48 hours was determined by flow cytometric analysis of Annexin V + cells (Figure 1E). The rate of apoptosis was reduced from 50.3%±9.1 for untreated cells to 32.9%±7.5 when the cells were pretreated with JI-34, whereas cytoprotection by JI-34 treatment was blocked by cotreatment with GHRH antagonist, MIA-602 (Figure 1F). JI-34 Increased Phosphorylation and Translocation of STAT3 in MSCs Signal transducer and activator of transcription 3 (STAT3) plays an important role in the promotion of angiogenesis, cell survival, and proliferation. 23 To evaluate potential downstream GHRH-R signaling, the activation of STAT3 was examined. Treatment of MSCs with JI-34 resulted in an increase of STAT3 phosphorylation (Figure 2A). The ratio of phosphorylated STAT3 compared with total STAT3 (P-STAT3/T- STAT3) was significantly increased by 5 minutes by treatment with JI-34 and reached a plateau at 30 minutes (Figure 2B). Immunofluorescence staining of STAT3 (Figure 2C) revealed that the majority of STAT3 was in the cytoplasm in control cultures but translocated to the nucleus after treatment with JI-34. Translocation was blocked by GHRH antagonist, MIA- 602 (Figure 2D). To further investigate the possible targets of JI-34, the expression of c-myc, a downstream target of STAT3, was analyzed. The expression of c-myc transcripts in MSCs was increased 200-fold by JI-34 treatment, and this was blocked by GHRH-R antagonist, MIA-602 (Figure IIA in the online-only Data Supplement). Similarly, c-myc protein was upregulated by JI-34 (Figure IIB in the online-only Data Supplement). JI-34 Enhances Mobility and Proangiogenic Activity of MSCs In Vitro In vitro transwell migration assays (Figure 3A) showed that the mobility of MSCs was significantly increased after the cells were treated with JI-34, and this was blocked by GHRH antagonist, MIA-602 (Figure 3B). To further characterize the possible paracrine effect of MSCs treated with JI-34, MSC-conditioned medium was used to culture human umbilical vein endothelial cells and endothelial tube formation was measured using a Matrigel assay (Figure 3C) as described in Materials and Methods. Tube formation was significantly enhanced by conditioned media from JI-34 treated MSCs (Figure 3D). When the GHRH antagonist, MIA-602, was present during the treatment, the effect of JI-34 on protube formation of human umbilical vein endothelial cell was abolished. Contributions of altered gene expression in JI-34 treated MSCs were assessed by quantifying a panel of angiogenesis-related genes (Table 1 in the

3 Ma et al GHRH Agonist Enhances MSC Ability for Angiogenesis 3 Figure 1. In vitro effects of growth hormone releasing hormone (GHRH) agonist, JI-34, on mesenchymal stem cell (MSC). Expression of growth hormone releasing hormone receptor (GHRH-R) on MSC was detected by (A) Western blot and (B) flow cytometry. Gray line is iso-control; black line is GHRH-R. C, MSCs were treated with JI-34 at different concentrations for 24 hours, and then the medium was replaced with fresh JI-34 free medium. Proliferation of MSCs was evaluated by CCK-8 assay after they were cultured 24 hours in JI-34 free medium. The maximal effect of GHRH-A was observed at 10 nmol/l (n=3). *P<0.05 vs 0 nmol/l. D, The viability of MSC after pretreatment with JI-34 at different concentrations was examined by CCK-8 assay after culture under serum deprivation and hypoxic conditions for 48 hours. (Gray column indicates hypoxia with serum deprivation condition.) *P<0.05 vs 0 nmol/l. E, Flow cytometric analysis of MSC apoptosis. MSC were pretreated with JI-34 (JI) or GHRH antagonist, MIA-602 (MIA), for 24 hours and then cultured under hypoxia and with serum deprivation medium for 48 hours. Apoptotic cells (Annexin V + /PI and Annexin V + /PI + cells) were detected by flow cytometric analysis. F, Quantifications of apoptotic MSC in E (n=5 6 in each group). *P<0.05 vs 0 nmol/l. online-only Data Supplement). The mrna levels of vascular endothelial growth factor-a and stromal-derived factor-1 were increased by 9-fold and 2.5-fold, respectively, by treatment with JI-34 (Figure 3F). This effect was blocked by GHRH antagonist, MIA-602, or WP1066, a STAT3 inhibitor. Meanwhile, we also observed that the protein expression level of HGF, vascular endothelial growth factor, and stromal-derived factor-1 was increased in MSC pretreated with JI-34, and this effect could be abolished by the addition of MIA-602 (Figure 3E). In addition, we studied the effects of another novel GHRH-R agonist, MR-409. Compared with JI-34, MR-409 is superior in stability and activity. 18 MSC proliferation and cell survival were significantly enhanced by treatment with MR-409 at 10 8 mol/l (Figure III in the online-only Data Supplement). MSC migration was enhanced by MR-409, and the effect was also suppressed by MIA-602 or STAT3 inhibitor, WP1066 (Figure IV in the online-only Data Supplement). These results indicate that treatment with MR-409 parallels that of JI-34. GHRH Agonist Augments Survival of MSCs In Vivo To investigate homing and engraftment of MSCs in ischemic muscle, transplantations of sex-mismatched and GFP-labeled cells were performed, and the number of retained donor cells in a recipient was examined by detecting the male-specific sry and GFP genes on days 3, 7, and 14 after injection of cells into ischemic muscles. Significantly more donor cells were detected in the ischemic muscle of mice injected with MSCs preconditioned with JI-34 compared with untreated MSCs 3 and 7 days after cell transplantation, and a similar, but nonsignificant, trend was observed on day 14 post-transplantation (Figure 4A; Figure V in the online-only Data Supplement). This was confirmed by tracking DiI-labeled MSCs (Figure VI in the online-only Data Supplement). In addition, the proliferation of transplanted MSCs was detected by Ki67/DiI costaining at day 7. No Ki67/DiI double-positive cells were detected in all groups, which indicated that JI-34 preconditioning did not promote the proliferation of MSCs in ischemic muscle (Figure VIIA in the online-only Data

4 4 Arterioscler Thromb Vasc Biol April 2016 Figure 2. Activation of signal transducer and activator of transcription 3 (STAT3) in mesenchymal stem cell (MSC) by JI-34 pretreatment. A, Western blot analysis of STAT3 activation in lysates from MSC. Dynamic changes of STAT3 phosphorylation were observed at different time points. B, Expression of phosphorylated STAT3 was quantified as a ratio of phosphorylated STAT3 compared with total STAT3 by integrated optical density measurement (n=3). *P<0.05 vs control. C, Immunofluorescence staining for subcellular localization of STAT3 (green) in MSC cultured without additional agent (control) or with JI-34, JI-34+MIA-602, or MIA-602. Scale bars: 50 μm. D, Quantification of nuclear-localized STAT3 in C by a ratio of fluorescence in nucleus to cytoplasm (n=3 in each group). *P<0.05 vs control. Supplement). The apoptosis of engrafted MSCs was evaluated by terminal deoxynucleotidyl transferase mediated dutp-nick end labeling and DiI costaining at day 3. Our results showed that compared with MSCs, apoptotic MSCs were less in MSC-JI group; however, the difference was not significant (Figure VIIB and VIIC in the online-only Data Supplement). Figure 3. JI-34 pretreatment enhanced mesenchymal stem cell (MSC) migration and proangiogenic effect. A, Representative images of migration of MSC in a transwell assay. Scale bars: 200 μm. B, Quantification of migration of MSC. Cells that migrated to the lower chamber were counted (n=4 in each group). *P<0.05 vs others. C, Representative images showing tube formation of human umbilical vein endothelial cell on Matrigel cultured with conditioned media from the specified MSC. Scale bars: 100 μm. D, Quantification of tube formation in C by measuring branch lengths of formed tube. Only length >200 μm was counted. n=5, *P<0.05 vs others. E, Western blot and quantification of proangiogenic cytokines expressions in MSCs treated with JI-34 or MIA-602 for 24 hours. F, Real-time polymerase chain reaction was performed to detect the effect of JI-34 on mrna expression for vascular endothelial growth factor (VEGF) and stromalderived factor-1 (SDF-1) within MSC (n=4), *P<0.05 vs control.

5 Ma et al GHRH Agonist Enhances MSC Ability for Angiogenesis 5 Figure 4. Mesenchymal stem cell (MSC) retention, blood reperfusion, and limb salvage. A, Donor MSC derived from male mice were injected intramuscularly into female mice. The expression of sry gene in ischemic muscle 3 and 14 days after injection was determined by real-time polymerase chain reaction (n=3 in each group). *P<0.05 vs MSC group. B, The blood flow of the lower limbs was quantitatively analyzed as the ratio of ischemic (right) side to nonischemic (left) side (n=9 15 in each group). *P<0.05 vs PBS; #P<0.05 vs MSC. C, Representative laser Doppler perfusion images show dynamic changes in blood perfusion ischemic limb at indicated time points. Different colors represent the changes in the perfusion. D, Representative photographs of hindlimbs from PBS, MSC, or preconditioned MSCtreated animals at day 21. E, Physiological status of ischemic hindlimbs 21 days after transplantation. n=10 for Sham, 11 for PBS, 13 for MSCs, and 12 for MSC-JI. Pretreatment With JI-34 Enhances MSC Therapy in Ischemic Hindlimb Reperfusion of ischemic mouse hind limbs was measured using laser Doppler perfusion imaging at intervals after femoral artery ligation (Figure 4B). Mice that received JI-34 conditioned MSCs recovered perfusion significantly faster than those that received untreated MSC or control groups (Figure 4C). Toe necrosis in ischemic limbs was also reduced in the JI-34 treated MSC group compared with that in the untreated MSC or control groups (Figure 4D and 4E). Pretreatment of MSCs With JI-34 Augments Angiogenesis and Muscle Regeneration In Vivo Muscle regeneration was analyzed by measuring central-localized nuclei in muscle cells of recovered tissues (Figure 5A). Administration of MSCs treated with agonist significantly increased the number of regenerating myofibers in ischemic gastrocnemius muscles 21 days after surgery (Figure 5B). To investigate whether the recovery of blood perfusion was associated with angiogenic activity, capillary density of ischemic muscles was analyzed by CD31 immunostaining (Figure 5C). Capillary densities were significantly higher in mice that received preconditioned MSCs compared with those that received PBS or untreated MSCs on day 21 after injection (Figure 5D). Arteriole density in the ischemic muscles was also determined by immunostaining of smooth muscle α-actin (Figure 5C). Transplantation of MSCs preconditioned with JI-34 also resulted in the highest arteriole density of ischemic muscle (Figure 5E). However, no direct differentiation of MSCs into endothelial cells was observed (Figure 5F). Enhanced Angiogenesis Is Associated With Increased Proliferation of Endothelial Cells To further explore the mechanism of augmented angiogenesis by MSCs, proliferation of endothelial cells was analyzed by immunostaining for Ki67 and CD31 in the ischemic muscles on day 7 after cell transplantation (Figure 6A). A robustly increased percentage of Ki67-positive cells (Figure 6B) and greater Ki67/CD31 double-positive cells (Figure 6C) was observed in mice that received JI-34 treated MSCs compared with those that received PBS or untreated MSCs. Furthermore, we performed real-time polymerase chain reaction to analyze the proangiogenic cytokines content in ischemic muscle. Results showed increased proangiogenic cytokines expression in MSC-JI group compared with other groups (Figure VIII in the online-only Data Supplement). These data, together with vascular density assessment, indicate that transplantation of MSCs preconditioned with JI-34 promotes endothelial cell proliferation and neovascularization in ischemic muscle. Transplantation of MSCs Preconditioned With JI-34 Enhances Recruitment of CD34 + Cells To determine whether treatment with JI-34 enhanced MSCmediated recruitment of endogenous progenitor cells, CD34- positive cells in ischemic muscles from each group at day 3 postsurgery were quantified (Figure 6D). As expected,

6 6 Arterioscler Thromb Vasc Biol April 2016 Figure 5. Transplantation of pretreated mesenchymal stem cell (MSC) promotes angiogenesis and muscle regeneration in vivo. A, Representative hematoxylin and eosin stained sections of ischemic muscles from each group at 21 days; scale bar: 50 μm, myocytes with centralized nuclei were considered as regenerating myofibers. B, Quantification of regenerating myofibers by counting the myocytes with centralized nuclei as a percentage of total myocytes in a field (n=5). *P<0.05 vs PBS and MSC. C, Immunofluorescent staining of CD31 and smooth muscle α-actin (α-sma) in cryosections of muscles obtained from mice at day 21 after surgery. Endothelial cells were stained with CD31, and smooth muscle cells were stained with α-sma. Scale bars: 100 μm. D and E, Quantification of CD31-positive endothelial cells and α-sma positive arteriole density (n=4 in sham group; n=5 in PBS group, and other group n=6). *P<0.05 vs PBS and MSC. F, Differentiation of MSC in vivo. MSC or MSC preconditioned with JI-34 were stained with DiI (red) and injected into ischemic muscle. The muscles were harvested 21 days later, and cryosections stained with DAPI for nuclei (blue) and antibody against CD31 (green) for endothelial cells. No colocalized DiI with CD31 staining was observed. Scalar bar: 100 μm. JI-34 treated MSCs resulted in significantly more CD34 + cells compared with all other groups (Figure 6E). Reduction of Cell Apoptosis In Vivo by MSCs Preconditioned With JI-34 To determine the mechanism of cell protection by preconditioned MSCs, antiapoptotic proteins were analyzed by Western blot (Figure 7A). Significantly more B-cell lymphoma-2 protein was detected in the ischemic muscles transplanted with MSCs treated with JI-34 compared with those transplanted with untreated MSCs or PBS (Figure 7B).These data implicate the activation of antiapoptotic and inhibition of proapoptotic pathways as mechanisms of cytoprotection by JI-34 in vivo.

7 Ma et al GHRH Agonist Enhances MSC Ability for Angiogenesis 7 Figure 6. Cell proliferation and CD34 + cell recruitment in ischemic region. Ischemic muscles were harvested 3 or 7 days after surgery. Immunofluorescence staining was performed on the frozen sections of the recovered muscles. A, Representative sections of ischemic gastrocnemius muscle from day 7 were stained for Ki67 proliferation marker (green), CD31 (red), and DAPI nuclear (blue). A Ki67 and CD31 colocalized cells (pointed by arrows) were demonstrated as the proliferating endothelial cells. Scale bars: 100 μm. B and C, Quantification of proliferating cells and proliferating endothelial cells, respectively (n=5 in each group). *P<0.05 vs PBS and mesenchymal stem cell (MSC). D, Representative pictures visualized by autofluorescence (green), CD34 (red), and total nuclei (blue) in ischemic gastrocnemius muscle at day 3. E, Quantification of CD34 + cells (n=3 4 per group). Scale bars: 100 μm. *P<0.05 vs PBS and MSC. Discussion In this study, we demonstrated that GHRH agonist, JI-34, significantly enhanced the viability and mobility of MSCs. After treatment with the agonist, the production of cytokines from MSCs was augmented, and the therapeutic effects in a mouse model of critical limb ischemia were significantly augmented compared with untreated MSCs. JI-34 agonist bound to a GHRH-R on MSCs and activated downstream STAT3/cmyc signaling and antiapoptotic pathways. These effects of agonist JI-34 were blocked by the GHRH antagonist, MIA- 602, confirming an essential role for GHRH-R binding. MSCs pretreated with JI-34 displayed improved survival in ischemic muscles relative to untreated MSCs. Administration of MSCs treated with JI-34 into ischemic limbs significantly enhanced local angiogenesis, resulting in better reperfusion and limb salvage. More capillaries and mature vessels were observed in the ischemic muscles that received JI-34 treated MSCs. The effect of MSCs in vivo was associated with enhanced EC proliferation and recruitment of CD34 + progenitor cells. Transdifferentiation of MSCs into vascular cells was rarely observed, indicating a primary paracrine role of MSCs in promoting angiogenesis. Previous work has shown that systemic administration of GHRH agonists stimulates proliferation of cells in peripheral tissues. Dioufa et al 19 reported that the GHRH agonist, JI-38, improved wound healing by activating wound-associated fibroblasts through GHRH-R binding. It has been shown that rat cardiomyocytes express pituitary-type GHRH-R, and administration of exogenous GHRH was cardioprotective by preventing apoptosis and reducing the cardiac scar size. This was attributed to activation of ERK1/2, PI3K/Akt, and adenylate cyclase/camp/protein kinase A signaling pathways. 17 Subcutaneous injection of GHRH agonist, JI-38, into rats with acute myocardial infarction improved angiogenesis and cardiac remodeling. 24 The effects of JI-38 include the augmentation of cardiac precursor cell proliferation without elevating systemic growth hormone levels. 20 In addition, GHRH agonist, MR403, was shown to increase viability and proliferation of islet cells, thereby improving the survival of cultured insulinoma cells, suggesting promise for improved

8 8 Arterioscler Thromb Vasc Biol April 2016 Figure 7. Analysis of proteins related to apoptosis in ischemic muscles. A, Representative blots of apoptotic-related protein expression. B, Quantification of protein expression levels (n=3 in each group). *P<0.05 vs PBS group; #P<0.05 vs mesenchymal stem cell (MSC) group. islet transplantation. 25,26 In this study, we observed that JI-34 preconditioning increased MSC proliferation and survival. However, MSCs preconditioned with high concentration of JI-34 (10 7 mol/l) did not exhibit obvious protective effect. We speculated that JI-34 at low concentration will promote cell proliferation and survival but causes cytotoxicity at high concentration. All GHRH analogs exhibited higher biological activity and are more stable than crude GHRH. 14 The biological activities of both JI-38 and MR403 are similar or virtually identical. 14,18 To our knowledge, this study is the first to describe a positive therapeutic benefit of MSCs by pretreatment with a GHRH agonist. Splice variants (SVs) of GHRH-R have been detected in many extrapituitary tissues, including prostate, 27 pancreatic islet, 26 and heart. 17 It has been demonstrated that functional SVs can replace the functions of GHRH pituitary-type receptor. 28 In this study, we detected the expression of 39 KD GHRH-R SV1 in mouse MSCs (Figure 1). Previous studies provided evidence that SV1 plays a pivotal role in regulating cell proliferation and survival. 29 GHRH agonists seem to exert their actions in extrapituitary tissues through direct binding to SV1 without activation of the canonical downstream pathway of growth hormone/insulin-like growth factor axis. 20 Consistent with previous studies, our in vitro data exhibited similar effects of agonist, JI-34, on enhancement of MSC proliferation and viability, whereas the expression of SV1 was not changed after the JI-34 treatment, indicating that the effects of JI-34 on MSCs were not meditated by upregulation of the SV1 receptor. GHRH can also activate Janus kinase 2/signal transducer and activator of transcription 3 pathway. 30 GHRH binds with its receptor and induces STAT3 phosphorylation at tyrosine. 17,21 Activation of STAT3 has been shown to play a critical role in regulation of angiogenesis and activation of cell survival pathway. 23,31 Upon activation mediated by JI-34, STAT3 translocates from cytoplasm into the nucleus to regulate gene expression. Our results consist with previous studies that JI-34 preconditioning corresponds to STAT3 activation and possible its downstream proangiogenic and cell survival pathway. Our data suggested that STAT3 is an important part of GHRH pathway and participates in preconditioning-mediated cytoprotection actions. MSCs are multipotent stem cells that have been widely used for cell-mediated therapy for various clinical indications including cardiovascular. 32 Transplantation of MSCs has also been used to reduce foot necrosis and to increase perfusion of lower limbs in patients with limb ischemia. 33 Such therapy is limited by poor retention and engraftment of the donor cells. 34 The hostile microenvironment of ischemia leads to massive apoptosis and impaired function of the transplanted cells. Indeed, it has been reported that only 0.35±0.05% of transplanted cells survive 4 weeks after limb transplantation. 35 To overcome these limitations, cell preconditioning by physical, chemical, pharmacological, or genetic modification before transplantation has emerged to augment cell function and therapy. 36 Our previous study showed that hypoxia preconditioning markedly increased the viability and mobility of MSCs through a leptin-mediated mechanism and enhanced therapeutic efficacy of MSCs in a mouse myocardial infarction model. 11 Moreover, preconditioning with chemotactic factor can enhance survival and improve biological function of transplanted cells. 37,38 Here for the first time, we found that preconditioning MSCs with GHRH agonist, JI-34, significantly protected MSCs from apoptosis induced by serum deprivation and hypoxia stress in vitro consistent with antiapoptosis as a mechanism of cytoprotection and improved therapy by JI In agreement with this, Jaszberenyi et al 40 recently reported that GHRH antagonists increased the expression of proapoptosis protein B-cell lymphoma-2 associated agonist of cell death in U-87 MG glioblastoma cells as reverse evidence. By transplantation of sex-mismatched and GFP-labeled MSCs and subsequent quantification of sry gene and GFP gene expression from the injected cells, we found that JI-34 treatment markedly improved MSC survival and retention in ischemic limbs 3 and 7 days after cell transplantation. Taken together, our in vitro and in vivo data verified that treating MSCs with JI-34 augments cell homing and maximized the therapeutic effect of transplantation and enhanced generation of cytoprotective and angiogenic cytokines, including vascular endothelial growth factor and stromalderived factor-1. In support of this, Gomes et al 22 found that GHRH agonists augmented vascular endothelial growth factor- A production in MSCs. However, it is important to note that in our hands, the improved short-term survival by JI-34 treatment was not sustained over a longer period (14 days). Moreover, we failed to detect any MSC-derived endothelial cell or α-smooth muscle cell 21 days after transplantation. Therefore, MSC transdifferentiation may not contribute significantly to therapy in this model. Rather, the effects of MSCs are primarily paracrine, affecting both cell activity and recruitment of progenitor cells. We found an increased proangiogenic cytokines expression in the ischemic limb muscle receiving preconditioned MSCs. The latter is also supported by a significant augmentation of CD34 + cells found in the ischemic muscle that received JI-34 treated MSCs relative to controls. In conclusion, our data demonstrate that preconditioning of MSCs with GHRH agonist, JI-34, enhances the survival and proliferation of MSCs, increases the secretion of

9 Ma et al GHRH Agonist Enhances MSC Ability for Angiogenesis 9 proangiogenic factors, and augments the therapeutic potential of MSCs to promote angiogenesis in ischemia tissue. These results support clinical testing of GHRH agonists as agents to improve MSC therapy for cardiovascular indications including critical limb ischemia. Acknowledgments We thank Lu Yang for animal care and Yan Wu for technical assistance and valuable suggestions. Sources of Funding The work in Hangzhou, China, was supported by grants from the National Natural Science Foundation of China (no for H.Y., for J.W., for Q.X.), Minister of Science and Technology of China (no. 2012CBA1305 for H.Y., 2011ZX for J.W.), Key program of Zhejiang province (no. N ), Innovation team of Zhejiang province (no. 2010R50047), Zhejiang Provincial Natural Science Foundation (no. Y for H.Y.), and the Joint Research Fund for Overseas Natural Science of China (no for J.W.). The work in Miami, FL, was supported by the Medical Research Service of Veterans Affairs Department (to A.V.S.), NIH grants HL and HL44578 (to K.A.W.), and the L. Austin Weeks Family Endowment for Research (to N.L.B.). None. Disclosures References 1. Anversa P, Kajstura J, Rota M, Leri A. Regenerating new heart with stem cells. J Clin Invest. 2013;123: doi: /JCI Fadini GP, Losordo D, Dimmeler S. Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use. Circ Res. 2012;110: doi: /CIRCRESAHA Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, Palecek SP, Thomson JA, Kamp TJ. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res. 2009;104:e30 e41. doi: / CIRCRESAHA Földes G, Mioulane M, Wright JS, Liu AQ, Novak P, Merkely B, Gorelik J, Schneider MD, Ali NN, Harding SE. Modulation of human embryonic stem cell-derived cardiomyocyte growth: a testbed for studying human cardiac hypertrophy? 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Improved autograft survival of mesenchymal stromal cells by plasminogen activator inhibitor 1 inhibition. Stem Cells. 2009;27: doi: /stemcells Cerrada I, Ruiz-Saurí A, Carrero R, Trigueros C, Dorronsoro A, Sanchez- Puelles JM, Diez-Juan A, Montero JA, Sepúlveda P. Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cellsmediated cardiac repair. Stem Cells Dev. 2013;22: doi: / scd Hu X, Wu R, Jiang Z, et al. Leptin signaling is required for augmented therapeutic properties of mesenchymal stem cells conferred by hypoxia preconditioning. Stem Cells. 2014;32: doi: /stem Seebach C, Henrich D, Wilhelm K, Barker JH, Marzi I. Endothelial progenitor cells improve directly and indirectly early vascularization of mesenchymal stem cell-driven bone regeneration in a critical bone defect in rats. Cell Transplant. 2012;21: doi: / X Steiner D, Lampert F, Stark GB, Finkenzeller G. Effects of endothelial cells on proliferation and survival of human mesenchymal stem cells and primary osteoblasts. J Orthop Res. 2012;30: doi: / jor Izdebski J, Pinski J, Horvath JE, Halmos G, Groot K, Schally AV. Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone. Proc Natl Acad Sci U S A. 1995;92: Matsubara S, Sato M, Mizobuchi M, Niimi M, Takahara J. Differential gene expression of growth hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues. Endocrinology. 1995;136: doi: /endo Kahán Z, Arencibia JM, Csernus VJ, Groot K, Kineman RD, Robinson WR, Schally AV. Expression of growth hormone-releasing hormone (GHRH) messenger ribonucleic acid and the presence of biologically active GHRH in human breast, endometrial, and ovarian cancers. J Clin Endocrinol Metab. 1999;84: doi: /jcem Granata R, Trovato L, Gallo MP, Destefanis S, Settanni F, Scarlatti F, Brero A, Ramella R, Volante M, Isgaard J, Levi R, Papotti M, Alloatti G, Ghigo E. Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart. Cardiovasc Res. 2009;83: doi: /cvr/ cvp Cai R, Schally AV, Cui T, Szalontay L, Halmos G, Sha W, Kovacs M, Jaszberenyi M, He J, Rick FG, Popovics P, Kanashiro-Takeuchi R, Hare JM, Block NL, Zarandi M. Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities. Peptides. 2014;52: doi: /j. peptides Dioufa N, Schally AV, Chatzistamou I, Moustou E, Block NL, Owens GK, Papavassiliou AG, Kiaris H. Acceleration of wound healing by growth hormone-releasing hormone and its agonists. Proc Natl Acad Sci U S A. 2010;107: doi: /pnas Kanashiro-Takeuchi RM, Tziomalos K, Takeuchi LM, Treuer AV, Lamirault G, Dulce R, Hurtado M, Song Y, Block NL, Rick F, Klukovits A, Hu Q, Varga JL, Schally AV, Hare JM. Cardioprotective effects of growth hormone-releasing hormone agonist after myocardial infarction. Proc Natl Acad Sci U S A. 2010;107: doi: /pnas Penna C, Settanni F, Tullio F, Trovato L, Pagliaro P, Alloatti G, Ghigo E, Granata R. GH-releasing hormone induces cardioprotection in isolated male rat heart via activation of RISK and SAFE pathways. Endocrinology. 2013;154: doi: /en Gomes SA, Rangel EB, Premer C, Dulce RA, Cao Y, Florea V, Balkan W, Rodrigues CO, Schally AV, Hare JM. S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells. Proc Natl Acad Sci U S A. 2013;110: doi: /pnas Chen Z, Han ZC. STAT3: a critical transcription activator in angiogenesis. Med Res Rev. 2008;28: doi: /med Kanashiro-Takeuchi RM, Takeuchi LM, Rick FG, Dulce R, Treuer AV, Florea V, Rodrigues CO, Paulino EC, Hatzistergos KE, Selem SM, Gonzalez DR, Block NL, Schally AV, Hare JM. Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI). Proc Natl Acad Sci U S A. 2012;109: doi: /pnas Ludwig B, Ziegler CG, Schally AV, Richter C, Steffen A, Jabs N, Funk RH, Brendel MD, Block NL, Ehrhart-Bornstein M, Bornstein SR. Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets. Proc Natl Acad Sci U S A. 2010;107: doi: /pnas Schubert U, Schmid J, Lehmann S, Zhang XY, Morawietz H, Block NL, Kanczkowski W, Schally AV, Bornstein SR, Ludwig B. Transplantation of pancreatic islets to adrenal gland is promoted by agonists of growthhormone-releasing hormone. Proc Natl Acad Sci U S A. 2013;110: doi: /pnas Plonowski A, Schally AV, Busto R, Krupa M, Varga JL, Halmos G. Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers. Peptides. 2002;23: Christodoulou C, Schally AV, Chatzistamou I, Kondi-Pafiti A, Lamnissou K, Kouloheri S, Kalofoutis A, Kiaris H. Expression of growth hormonereleasing hormone (GHRH) and splice variant of GHRH receptors in

10 10 Arterioscler Thromb Vasc Biol April 2016 normal mouse tissues. Regul Pept. 2006;136: doi: /j. regpep Siriwardana G, Bradford A, Coy D, Zeitler P. Autocrine/paracrine regulation of breast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol Endocrinol. 2006;20: doi: /me Siejka A, Schally AV, Barabutis N. Activation of Janus kinase/signal transducer and activator of transcription 3 pathway by growth hormonereleasing hormone. Cell Mol Life Sci. 2010;67: doi: / s y. 31. Liu Y, Li PK, Li C, Lin J. Inhibition of STAT3 signaling blocks the anti-apoptotic activity of IL-6 in human liver cancer cells. J Biol Chem. 2010;285: doi: /jbc.M Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006;98: doi: /jcb Gremmels H, Teraa M, Quax PH, den Ouden K, Fledderus JO, Verhaar MC. Neovascularization capacity of mesenchymal stromal cells from critical limb ischemia patients is equivalent to healthy controls. Mol Ther. 2014;22: doi: /mt Daley GQ. The promise and perils of stem cell therapeutics. Cell Stem Cell. 2012;10: doi: /j.stem Zeng L, Hu Q, Wang X, et al. Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling. Circulation. 2007;115: doi: /CIRCULATIONAHA Haider HKh, Ashraf M. Preconditioning and stem cell survival. J Cardiovasc Transl Res. 2010;3: doi: /s Tan Y, Shao H, Eton D, Yang Z, Alonso-Diaz L, Zhang H, Schulick A, Livingstone AS, Yu H. Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor. Cardiovasc Res. 2007;73: doi: /j.cardiores Shao H, Tan Y, Eton D, Yang Z, Uberti MG, Li S, Schulick A, Yu H. Statin and stromal cell-derived factor-1 additively promote angiogenesis by enhancement of progenitor cells incorporation into new vessels. Stem Cells. 2008;26: doi: /stemcells Chao DT, Korsmeyer SJ. BCL-2 family: regulators of cell death. Annu Rev Immunol. 1998;16: doi: /annurev.immunol Jaszberenyi M, Schally AV, Block NL, Zarandi M, Cai RZ, Vidaurre I, Szalontay L, Jayakumar AR, Rick FG. Suppression of the proliferation of human U-87 MG glioblastoma cells by new antagonists of growth hormone-releasing hormone in vivo and in vitro. Target Oncol. 2013;8: doi: /s y. Significance Clinical trials of mesenchymal stem cell (MSC) therapy have shown promise for the treatment of cardiovascular disease, including heart failure and critical limb ischemia. However, poor cell survival and engraftment into host tissues have limited efficacy of MSC therapy. Here, we show that pretreatment of MSCs with JI-34, an agonist of growth hormone releasing hormone, markedly augmented the activities of signal transducer and activator of transcription 3 and significantly enhanced cytoprotection and mobility of MSCs in vitro and in vivo. JI- 34 pretreated MSCs sustained better engraftment and superior proangiogenic ability when transplanted into ischemic limbs. Our findings support a role for growth hormone releasing hormone agonists in preconditioning of stem cells, before transplantation, to enhance cell activation and homing, and related therapeutic activity.