Profound actions of an agonist of growth hormone releasing hormone on angiogenic therapy by mesenchymal stem cells

Abstract

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 cells (MSCs) 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 50μl phosphate buffer saline or with 1×10⁶ MSCs or with 1×10⁶ JI-34 preconditioned MSCs. Treatment of MSCs with JI-34 improved MSCs viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by increased phosphorylation and nuclear translocation of STAT3. In vivo, JI-34 pre-treatment 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.

Introduction

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 cells¹, endothelial progenitor cells (EPCs)², induced pluripotent stem cells³, embryonic stem cells⁴, bone marrow mononuclear cells⁵, and mesenchymal stem cells (MSCs)⁶, with mixed outcomes. MSCs have the advantages of immune privilege, stem-ness and ease of handling relative to other cell types and have become prominent vehicles for a wide range of indications⁷. The mechanism of MSC action is thought to be primarily paracrine⁸. When exogenously administered, MSCs can facilitate the formation of new capillaries and medium-sized 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 physiologic stimuli such as hypoxia¹¹, or combination with other cells including EPCs¹² and endothelial cells¹³. 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 membranes¹⁴. Previous studies have shown that GHRH and its receptor are also expressed on cells in normal human tissues e.g. 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 extra-pituitary tissues^{17, 18}. Compared with the native GHRH, synthetic agonists are much more potent and longer-acting due to increased stability¹⁴. One such GHRH agonist, JI-38, has been used to accelerate wound healing primarily by acting upon wound-associated fibroblasts¹⁹. The systemic administration of GHRH agonists has been shown to stimulate 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}.

Since GHRH-R is expressed in MSCs ²², we hypothesized that pre-treatment 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.

Results

GHRH Receptor is Expressed on mouse BM-MSCs

GHRH-R expression on mouse bone marrow-derived MSCs was detected by western blot (Figure 1A), flow cytometry (Figure 1B), and immunofluorescence (IF) staining (Supplementary Figure IA). The results from both Western blot and IF 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 (Supplementary Figure IB & IC). These results were further confirmed by Western blot assay (Supplementary Figure ID). These data confirm that GHRH-R is expressed on MSCs.

Figure 1. In vitro effects of GHRH agonist, JI-34, on MSC.

Expression of GHRH-R on MSC was detected by (A) Western blot and (B) flow cytometry. Grey line is iso-control; black line is GHRH-R. (C) MSCs were treated with JI-34 at different concentrations for 24 hrs, 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 hrs in JI-34 free medium. The maximal effect of GHRH-A was observed at 10 nM (n = 3). *, P < 0.05 vs. 0 nM. (D) The viability of MSC after pretreatment with JI-34 at different concentrations was examined by CCK8 assay after culture under serum deprivation and hypoxic conditions for 48h. (Gray column indicates hypoxia with serum deprivation condition), *, P < 0.05 vs. 0 nM. (E) Flow cytometric analysis of MSC apoptosis. MSC were pretreated with JI-34 (JI) or GHRH antagonist, MIA602 (MIA), for 24 hrs, and then cultured under hypoxia and with serum deprivation medium for 48 hrs. 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 nM.

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 1×10⁻⁸ mol/L. The viability of MSCs after culture under hypoxia (0.5% O₂) with serum deprivation (H/SD) for 48 hours was significantly increased by JI-34 pretreatment as compared with the untreated MSCs (Figure 1D). Augmentation of MSC proliferation and viability were optimal at JI-34 concentration of 1×10⁻⁸ mol/L therefore this concentration was used for further studies.

Apoptosis of MSCs after culture under the H/SD condition for 48 hrs was determined by flow cytometric analysis of Annexin V positive 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, while cytoprotection by JI-34 treatment was blocked by co-treatment with GHRH antagonist, MIA-602 (Figure 1F).

JI-34 Increased Phosphorylation and Translocation of STAT3 in MSCs

STAT-3 plays an important role in promotion of angiogenesis, cell survival and proliferation²³. 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 over total STAT3 (P-STAT3/T-STAT3) was significantly increased by 5 mins treatment with JI-34 and reached a plateau at 30 mins (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 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 approximately 200-fold by JI-34 treatment and this was blocked by GHRHR antagonist, MIA-602 (Supplementary Figure IIA). Similarly, c-myc protein was upregulated by JI-34 (Supplementary Figure IIB).

Figure 2. Activation of STAT3 in 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 over 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, or 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.

JI-34 Enhances Mobility and Pro-Angiogenic Activity of MSCs in Vitro

In vitro trans-well 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, MIA602 (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 (HUVECs) and endothelial tube formation was measured using a Matrigel assay (Figure 3C) as described in Methods. Tube formation was significantly enhanced by conditioned media from JI-34-treated MSCs (Figure 3D). When the GHRH antagonist, MIA602, was present during the treatment, the effect of JI-34 on pro-tube formation of HUVEC was abolished. Contributions of altered gene expression in JI-34-treated MSCs were assessed by quantifying a panel of angiogenesis-related genes (Supplementary Table S1). The mRNA levels of Vascular endothelial growth factor-A (VEGF-A) and Stromal-derived-factor-1 (SDF-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 the protein expression level of HGF, VEGF and SDF-1 were increased in MSC pretreated with JI-34, and this effect could be abolished by addition of MIA-602 (Figure 3E).

Figure 3. JI-34 pretreatment enhanced MSC migration and pro-angiogenic 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 HUVEC 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 pro-angiogenic cytokines expressions in MSCs treated with JI-34 or MIA-602 for 24 hrs. (F) Real time PCR was performed to detect the effect of JI-34 on mRNA expression for VEGF and SDF-1 within MSC. (n = 4) *, P < 0.05 vs. Control.

In addition, we studied the effects of another novel GHRHR 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 (Supplementary Figure III). MSC migration was enhanced by MR-409, and the effect was also suppressed by MIA-602 or STAT3 inhibitor, WP1066 (Supplementary Figure IV). 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 gender 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 Day 3, 7, and 14 following 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 non-significant, trend was observed on day 14 post transplantation (Figure 4A and Supplementary Figure V). This was confirmed by tracking DiI-labeled MSCs (Supplementary Figure VI). Additionally, the proliferation of transplanted MSCs was detected by Ki67/DiI co-staining 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 (Supplementary Figure VIIA). The apoptosis of engrafted MSCs were evaluated by terminal deoxynucleotidyl transferase–mediated dUTP nick end - labeling (TUNEL) and DiI co-staining at Day 3. Our results showed that compared with MSCs, apoptotic MSCs were less in MSC-JI group, however the difference was not significant (Supplementary Figure VIIB & VIIC).

Figure 4. 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 PCR (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 LDPI 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 MSC treated 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 Hind Limb

Reperfusion of ischemic mouse hind limbs was measured using Laser Doppler Perfusion Imaging (LDPI) at intervals after femoral artery ligation (Figure 4B). Mice that received JI-34-conditioned MSCs recovered perfusion significantly faster than untreated MSC or control groups (Figure 4C). Toe necrosis in ischemic limbs was also reduced in the JI-34-treated MSC group compared with the untreated MSC or control groups (Figure 4D & 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).

Figure 5. Transplantation of pretreated MSC promotes angiogenesis and muscle regeneration in vivo.

(A) Representative hematoxylin and eosin stained sections of ischemic muscles from each group at 21 day; 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 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: 100μm. (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 cryo-sections stained with DAPI for nuclei (blue) and antibody against CD31 (Green) for endothelial cells. No co-localized DiI with CD31 staining was observed. Scalar bar: 100 μm.

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 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 (α-SMA) (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 following cell transplantation (Figure 6A). A robustly increased percentage of Ki67 positive cells (Figure 6B) and greater Ki67/CD31 double positive cells (Figure 6C) were observed in mice that received JI-34-treated MSCs compared with those that received PBS or untreated MSCs. Furthermore, we performed real-time PCR to analysis the pro-angiogenic cytokines content in ischemic muscle. Results showed increased pro-angiogenic 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.

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 co-localized 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 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.

Transplantation of MSCs Preconditioned with JI-34 Enhances Recruitment of CD34⁺ Cells

To determine whether treatment with JI-34 enhanced MSC-mediated recruitment of endogenous progenitor cells, CD34 positive cells in ischemic muscles from each group at day 3 post surgery were quantified (Figure 6D). As expected, 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, anti-apoptotic proteins were analyzed by Western blot (Figure 7A). Significantly more B-cell lymphoma-2 (Bcl-2) protein was detected in the ischemic muscles transplanted with MSCs treated with JI-34 as compared with those transplanted with untreated MSCs or PBS (Figure 7B). These data implicate the activation of anti-apoptotic and inhibition of pro-apoptotic pathways as mechanisms of cytoprotection by JI-34 in vivo.

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. MSC group.

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 receptor on MSCs and activated downstream STAT3/c-myc signaling and anti-apoptotic pathways. These effects of agonist JI-34 were blocked by the GHRH antagonist, MIA-602, confirming an essential role for GHRH receptor 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. Trans-differentiation 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. reported that the GHRH agonist, JI-38, improved wound healing by activating wound-associated fibroblasts through GHRH receptor binding¹⁹. It has been shown that rat cardiomyocytes express pituitary-type GHRH receptor and administration of exogenous GHRH was cardio-protective 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 pathways¹⁷. Subcutaneous injection of GHRH agonist, JI-38, into rats with acute myocardial infarction improved angiogenesis and cardiac remodeling²⁴. 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 survival of cultured insulinoma cells, suggesting promise for improved islet transplantation^{25, 26}. In the present study, we observed 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 cause cytotoxicity at high concentration. All GHRH analogs exhibited higher biological activity and more stable than crude GHRH¹⁴. The biological activities of both JI-38 and MR403 are similar or virtually identical^{14, 18}. To our knowledge, the present study is the first to describe a positive therapeutic benefit of MSCs by pretreatment with a GHRH agonist.

Splice variants (SVs) of GHRH receptor have been detected in many extra-pituitary tissues, including prostate²⁷, pancreatic islet ²⁶ and heart ¹⁷. It has been demonstrated that functional SVs can replace the functions of GHRH-pituitary type receptor²⁸. In the present study, we detected the expression of 39 KD GHRH-R SV-1 in mouse MSCs (Figure 1). Previous studies have provided evidence that SV1 plays a pivotal role in regulating cell proliferation and survival²⁹. GHRH agonists appear to exert their actions in extra-pituitary tissues through direct binding to SV1 without activation of the canonical downstream pathway of GH/insulin like growth factor axis²⁰. Consistent with previous studies, our in vitro data exhibited similar effects of agonist, JI-34, on enhancement of MSC proliferation and viability, while the expression of SV-1 was not changed after the JI-34 treatment, indicating that the effects of JI-34 on MSCs were not meditated by up-regulation of the SV-1 receptor. GHRH can also activate Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/STAT3) pathway³⁰. 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 translocate 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 pro-angiogenic and cell survival pathway. Our data suggested that STAT3 is an important part of GHRH pathway and participate in preconditioning mediated cyto-protection actions.

MSCs are multipotent stem cells that have been widely used for cell-mediated therapy for various clinical indications including cardiovascular³². Transplantation of MSCs has also been used to reduce foot necrosis and to increase perfusion of lower limbs in patients with limb ischemia³³. Such therapy is limited by poor retention and engraftment of the donor cells³⁴. 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 transplantation³⁵.

To overcome these limitations, cell preconditioning by physical, chemical, pharmacological or genetic modification before transplantation has emerged to augment cell function and therapy³⁶. 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 model¹¹. Moreover, preconditioning with chemotactic factor can enhance survival and improve biologic 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 anti-apoptosis as a mechanism of cytoprotection and improved therapy by JI-34³⁹. In agreement with this, Jaszberenyi et al. recently reported that GHRH antagonists increased expression of pro-apoptosis protein BAD (BCL-2-associated agonist of cell death) in U-87 MG glioblastoma cells ⁴⁰ 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 VEGF and SDF-1. In support of this, Gomes et al. found that GHRH agonists augmented VEGF-A production in MSCs⁴¹. However, it is important to note that in our hands, the improved short-term survival by JI-34 treatment was not sustained over the longer period (14 days). Moreover, we failed to detect any MSCs derived endothelial cell or α-smooth muscle cell 21 days after transplantation. Therefore, MSC trans-differentiation 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 pro-angiogenic 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 pro-angiogenic 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.

Significance Statement.

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 pre-treatment of MSCs with JI-34, an agonist of growth hormone releasing hormone (GHRH), markedly augmented the activities of STAT3, and significantly enhanced cytoprotection and mobility of MSCs in vitro and in vivo. JI-34-pretreated MSCs sustained better engraftment and superior pro-angiogenic ability when transplanted into ischemic limbs. Our findings support a role for GHRH agonists in preconditioning of stem cells, prior to transplantation, to enhance cell activation and homing, and related therapeutic activity.

Nonstandard Abbreviations and Acronyms

MSCs

Mesenchymal stem cells

GHRH

Growth hormone-releasing hormone

GHRH-R

Growth hormone-releasing hormone receptor

SVs

Splice variants

VEGF-A

Vascular endothelial growth factor-A

SDF-1

Stromal-derived-factor-1

STAT3

Signal Transducers and Activators of Transcription