Substance P enhances the therapeutic effect of MSCs by modulating their angiogenic potential

Abstract Bone marrow mesenchymal stem cell (MSC) therapy acts through multiple differentiations in damaged tissue or via secretion of paracrine factors, as demonstrated in various inflammatory and ischaemic diseases. However, long‐term ex vivo culture to obtain a sufficient number of cells in MSC transplantation leads to cellular senescence, deficiency of the paracrine potential, and loss of survival rate post‐transplantation. In this study, we evaluated whether supplementation of MSCs with substance P (SP) can improve their therapeutic potential. SP treatment elevated the secretion of paracrine/angiogenic factors, including VEGF, SDF‐1a and PDGF‐BB, from late passage MSCs in vitro. MSCs supplemented with SP accelerated epidermal/dermal regeneration and neovascularization and suppressed inflammation in vivo, compared to MSCs transplanted alone. Importantly, supplementation with SP enabled the incorporation of transplanted human MSCs into the host vasculature as pericytes via PDGF signalling, leading to the direct engagement of transplanted cells in compact vasculature formation. Our results showed that SP is capable of restoring the cellular potential of senescent stem cells, possibly by modulating the generation of paracrine factors from MSCs, which might accelerate MSC‐mediated tissue repair. Thus, SP is anticipated to be a potential beneficial agent in MSC therapy for inflammatory or ischaemic diseases and cutaneous wounds.

In spite of the mounting evidence for the therapeutic effects of MSCs, the low differentiation potential to damaged tissue and transient persistence of transplanted MSCs have emerged as crucial problems that need to be addressed. A sufficient number of MSCs are required for transplantation, which in turn requires long-term ex vivo culture and makes transplantation of late-passage MSCs inevitable. During ex vivo culture, MSCs become senescent, with aberrant biological characteristics such as morphological changes, reduced proliferation rate, deficient immunosuppressive function and insufficient production of cytokines/growth factors such as stromal cell-derived factor-1 alpha (SDF-1α) or vascular endothelial growth factor (VEGF). [19][20][21][22][23] Moreover, because senescent MSCs encounter severe inflammatory environments in vivo post-transplantation, they do not survive long term. Therefore, transplanted MSCs have impaired therapeutic function. To address these problems, the current research focuses on ways to enhance viability and inhibit senescence of MSCs using specific growth factors, co-culture with supportive cells, hypoxic conditions and genetic modification. [22][23][24][25] However, effective methods to improve the efficacy of stem cell therapy in vivo have not been optimized.
Substance P (SP), an endogenous neuropeptide known to be involved in neuro-immune modulation, can also promote cell proliferation and inhibit cell apoptosis. [26][27][28] Hong et al demonstrated that SP can induce mobilization of MSCs into the circulation by repopulating MSCs in the bone marrow, leading to tissue repair. 29 SP promotes anti-inflammatory responses that ameliorate disease progression in cases of corneal wounds, rheumatoid arthritis, radiation-induced intestinal damage and spinal cord injury. [29][30][31][32][33] When MSCs are treated with SP in vitro, the production of VEGF and fibronectin in MSCs increases. 29 Recent studies have demonstrated that SP can restore the immunosuppressive function of late-passage MSCs by blocking senescence-induced reduction of cytokine secretion from MSCs. 23 Our approach was based on the hypothesis that the addition of SP to transplanted MSCs adequately enhances the efficacy of MSC therapeutics in vivo, possibly by modulating the secretion of paracrine factors and enhancing the survival of MSCs at the injured site.
To determine the effect of SP on the paracrine potential of se-

| Experimental animals
Five-week-old nude mice were obtained from DBL (Daehan Bio Link, Seoul, Korea). Animals were maintained under a 12-h light/dark illumination cycle in the animal facility and allowed to acclimatize to the new environment for 1 week. All animal studies were approved by the Ethical Committees for Experimental Animals at Kyung Hee University (KHMC-IACUC-14-010). and sub-cultured at 2.5 × 10 5 cells per 100-mm dish.

| Cytokine measurements
Secretion of SDF-1α, PDGF-BB and VEGF from MSCs was quantified using ELISA kits in accordance with the manufacturer's instructions.
In brief, MSCs were seeded in 24-well plates at 2 × 10 4 cells/well and treated with SP (100 nM) or PBS (control) for 48 h. Conditioned media were harvested and centrifuged to eliminate cell debris. To quantify cytokine levels, conditioned media and standards were added to 96-well plates coated with anti-SDF-1α, anti-PDGF-BB and anti-VEGF antibodies, followed by incubation for 2 h. After four washes with PBS, horseradish peroxidase-conjugated secondary antibodies were added to each well for 2 h at room temperature.
After three more washes, the substrate solution was added and the reaction was allowed to proceed for 30 min; thereafter, the reaction was terminated using stop solution. Absorbance was measured at 450 nm with an EMax Endpoint Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

| Haematoxylin & Eosin (H&E) staining
At days 2 and 7 post-transplantation, mice were euthanized and the skin and spleen were harvested for further quantitative and histological analysis. The skin and spleen were fixed in 3.7% formaldehyde for 24 h and embedded in paraffin, and the paraffin-embedded tissue blocks were processed with a TP1020 tissue processor (Leica Biosystems, Wetzlar, Germany). For histological analysis, 4-μm-thick sections were deparaffinized in xylene and rehydrated using an alcohol gradient. For H&E staining, nuclei were stained with haematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 1 min, after which the sections were washed in running water for 5 min, and then, the cytoplasm and extracellular matrix were stained with eosin Y (Sigma-Aldrich) for 10 s.

| Masson trichrome (MTC) staining
To observe collagen deposition in the wound region, MTC Staining (IHC World, Woodstock, MD, USA) was performed in accordance with the manufacturer's instructions. In brief, paraffinized tissue sections (4-μm thick) were deparaffinized, hydrated and fixed in Bouin's solution (Sigma-Aldrich) for 1 h at 56°C. After washing in distilled water, the samples were stained with Weigert's haematoxylin for 10 min, Biebrich scarlet-acid fuchsin for 2 min, phosphomolybdic-phosphotungstic acid solution for 10 min, and Aniline Blue for 10 min. After washing with distilled water, samples were treated with 1% acetic acid (Sigma-Aldrich) for 5 min and then dehydrated and observed using a microscope.

| Immunofluorescence staining
Wounded tissues were embedded in optical cutting compound (OCT compound, Sakura Finetek, Tokyo, Japan) and cut into 4-μm-thick sections. To eliminate OCT compound, sections were washed thrice with PBS and incubated in 20% normal goat serum to block nonspecific binding. Sections were probed with anti-mouse CD31 antibody (Abcam) for 2 h at RT, followed by FITC-conjugated secondary antibody for 1 h. After two washes, anti-human α-SMA antibody (Dako, DK) was added, followed by Texas Red-conjugated secondary antibody (Vector Laboratories). Thereafter, samples were mounted with Vectashield mounting medium (Vector Laboratories with DAPI) and observed using a Nuance Multiplex Biomarker Imaging System (Cambridge Research Instrumentation).

| Quantitative histological analysis of the wounded area
For quantitative histologic analysis, the wounded area was assessed using previously described methods, with certain modifications 16 Cell migration in the epithelial layer was analysed by measuring the path length of epithelial cells from the non-wounded edge to the centre of the wound. Granulation tissue formation was evaluated on the basis of the invasion of endothelial cells, fibroblast influx, collagen deposition and additional macrophage accumulation. The thickness of the granulation tissue was measured with respect to the underlying muscle fascia. Wound repair is expressed as an absolute value. All quantitative analyses were performed using ten adjacent fields on temporal slides.

| Statistical analysis
All data are presented as the mean ± standard deviation (SD).

Student's t test (for comparisons between two groups) and one-
way analysis of variance (ANOVA; for comparisons of three or more groups, followed by Tukey's post-hoc test) were performed.

| SP stimulates the secretion of paracrine/ angiogenic factors in late-passage MSCs
Paracrine factors from MSCs, rather than the MSCs themselves, are chiefly responsible for tissue repair. SDF-1α is the primary chemokine that supports mobilization of pro-angiogenic cells, including endothelial progenitor cells, and is capable of accelerating vascularization. 34 VEGF, a mitogen highly specific for vascular endothelial cells, induces proliferation, promotes migration and inhibits apoptosis of endothelial cells. MSCs are known to secrete SDF-1α and VEGF constitutively, but the secretion of these paracrine factors reduces as the passage number increases; this might be the main cause of the low efficacy of MSC therapeutics until now. 14 Before SP treatment, activity of BM MSC was evaluated according to passage number. BM MSCs at early (p3) and late passage (p8) were characterized by FACS analysis and then treated with SP. FACS analysis indicated no differences in cell surface marker expression between the early-and late-passage MSCs (Table S1). Cellular senescence was determined by beta galactosidase staining. Even though its portion was not as high, beta galactosidase + cells appeared from passage 5 BM MSC and it was undeniable at passage 8 BM MSC. That is, passage 8 BM MSC might undergo senescent states ( Figure S2). Moreover, cellular activity was reduced and doubling time was firmly elevated at passage 8 BM MSC. Moreover, immune suppressive function was clearly impaired at passage 8 BM MSC, comparing to passage 3 BM MSC ( Figure S3 and Figure S4).
To determine whether SP can affect the secretion of paracrine/ angiogenic factors from late-passage MSCs, passage 8 BM MSC was treated with SP in this study.
Levels of SDF-1α and VEGF in the culture supernatant were determined by ELISA. As the passage number increased, the release of SDF-1α and VEGF by MSCs decreased ( Figure S5). SP treatment increased the secretion of SDF-1α and VEGF in late-passage MSCs to levels resembling early-passage MSCs ( Figure 1A and B). SP effect was dose-dependent.
Pericytes regulate vasoconstriction and vasodilation within capillary beds to control vascular diameter and capillary blood flow. 18,35,36 Dysfunctional and leaky blood vessels that lack pericytes contribute to the development of pathological conditions in vivo. PDGF-BB is involved in the recruitment of pericytes to a variety of vascular beds such as the brain, kidney, heart, lung and adipose tissue, 35

| SP can improve MSC-induced cutaneous wound healing
SP has been shown to accelerate proliferation and restore the immune modulatory function of MSCs in vitro. 23 As shown in Figure 1,  Note: Wound area was analysed based on the histological analysis. Epithelial migration was measured from wound edge to the epithelium covered wound region. Wound coverage was expressed based on epithelial migration length and defect area. Granulation tissue formation was evaluated based on the invasion of endothelial cells, influx of fibroblasts, collagen deposition and accumulation of additional macrophages. The data are represented as mean ± SD of three independent experiments. N = 8 for each group.
(+) macrophage infiltration into injured sites, which was further lessened by the addition of SP ( Figure S6).
This data suggests that the late MSC transplantation accelerated wound healing by stimulating epidermal migration and granulation tissue regeneration while suppressing inflammation. Importantly, all outcomes from MSC transplantation were enhanced by co-treatment with SP.

| SP accelerates angiogenesis by MSCS at the wound site
Next, we explored whether MSC + SP-induced fast wound healing was accompanied by neovascularization. Angiogenesis at the wound site was determined by immunohistochemical staining for CD31, specific for mouse vascular endothelial cells and vas-  Collectively, these results imply that SP induces the formation of mature vessels by promoting the incorporation of transplanted

| SP contributes to incorporation of transplanted MSCS into the host vasculature
MSCs into newly forming vessels as pericytes.   This study proved that SP could enhance MSC-mediated neovascularization, possibly by providing an SDF-1α-enriched wound bed. Although topical treatment with SP has been shown to stimulate skin wound healing, 30 we did not observe the same effect in our study, possibly because we only administered SP once ( Figure S2) and SP is degraded rapidly at wound site. Collectively, our findings support the development of SP as a supplemental agent for MSC therapy in inflammatory or ischaemic diseases. Further detailed study of the effects of SP supplementation on other stem cell subsets in a variety of diseases is planned.

| CON CLUS IONS
Our findings demonstrated that the supplementation of SP improved the efficacy of MSCs.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset used in this study is available from the corresponding author on reasonable request.