Cxcr6-Based Mesenchymal Stem Cell Gene Therapy Potentiates Skin Regeneration in Murine Diabetic Wounds

Abstract

Mesenchymal stem cell (MSC) therapies for wound healing are often compromised due to low recruitment and engraftment of transplanted cells, as well as delayed differentiation into cell lineages for skin regeneration. An increased expression of chemokine ligand CXCL16 in wound bed and its cognate receptor, CXCR6, on murine bone-marrow-derived MSCs suggested a putative therapeutic relevance of exogenous MSC transplantation therapy. Induction of the CXCL16-CXCR6 axis led to activation of focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinases 1/2 (ERK1/2)-mediated matrix metalloproteinases (MMP)-2 promoter regulation and expression, the migratory signaling pathways in MSC. CXCL16 induction also increased the transdifferentiation of MSCs into endothelial-like cells and keratinocytes. Intravenous transplantation of allogenic stable MSCs with Cxcr6 gene therapy potentiated skin tissue regeneration by increasing recruitment and engraftment as well as neovascularization and re-epithelialization at the wound site in excisional splinting wounds of type I and II diabetic mice. This study suggests that activation of the CXCL16-CXCR6 axis in bioengineered MSCs with Cxcr6 overexpression provides a promising therapeutic approach for the treatment of diabetic wounds.

Keywords: CXCR6; cell recruitment and engraftment; cell transplantation; diabetic wound healing; full thickness excisional splinting wound model; gene therapy; mesenchymal stem cells; molecular signaling; skin regeneration; type I (Streptozotocin-induced) and type II (db/db) diabetic mice.

Figure 1

CXCL16 Induces MMP2-Mediated MSCs Migration (A) CXCL16 treatment in MSCs led to phosphorylation of FAK, Src, and ERK1/2 that was perturbed by CXCL16-NAb. (B) Densitometric analysis of activated signaling mediators in MSCs treated with CXCL16 in the absence or presence of CXCL16 neutralizing antibody. (C) Schematic representation of the signaling pathway. (D) Expression analysis of MMPs as downstream effectors of CXCL16 signaling in MSCs depicted a significant increase in MMP2 expression. (E) CXCL16 treatment led to an increase in MMP2 activity by using gelatin zymography. (n = 3–6 replicates/experiment repeated thrice; p < 0.05 as compared with *control; ^#CXCL16-treated group; ^$CXCL16-treated Cxcr6 overexpression).

Figure 2

CXCR6 Overexpression Potentiated MMP2-Mediated MSCs Migration Cxcr6 overexpression in MSCs led to (A) sequential increase in phosphorylation of FAK, Src, and ERK1/2 by immunoblot analysis, (B) densitometric analysis of activated signaling mediators in MSCs treated with CXCL16 in absence or presence of pathway-specific inhibitors, (C) increased mRNA expression of MMP2, and (D) increased migratory potential that was mitigated in presence of CXCL16-NAb or pharmacological inhibitors of FAK, Src, and ERK1/2. (n = 3–6 replicates/experiment repeated thrice; p < 0.05 as compared with *control; ^#CXCL16-treated group; ^$/ˆˆCXCL16-treated Cxcr6 overexpression).

Figure 3

CXCL16-CXCR6 Signaling Activation Led to Nuclear Translocation of p-ERK1/2 and Transcriptional Regulation of MMP2 Promoter Activity (A) Confocal immunostaining depicting CXCL16-induced nuclear translocation of p-ERK1/2 in MSCs transfected with pCMV or pCMV-CXCR6-Flag-tagged vector that was inhibited in presence of CXCL16-NAb or pharmacological inhibitors of FAK, Src, and ERK1/2, but not by MMP pan inhibitor. (B) In silico analysis revealed the presence of three putative binding sites of Sp1 on MMP2 promoter (+1, transcription start site and ▲, translation start site). (C) Promoter reporter assay revealing that all three Sp1 binding sites are essential for promoter activity of MMP2. (D) CXCL16-induced promoter luciferase activity was further increased in MSCs overexpressing CXCR6 and inhibited in MSCs with CXCR6 gene silencing, as well as ERK1/2 inhibitor, PD98509. (E) ChIP assay suggesting higher binding of p-ERK1/2 at Sp1 binding sites—BS2 and BS3, but not BS1, that was further induced by CXCL16. (n = 3 replicates/experiment repeated thrice; p < 0.05 as compared with *control, and ^#CXCR6 overexpressing MSC group).

Figure 4

Temporal Increase in Engraftment of CXCR6 Overexpressed MSCs at Wound Site (Inset) Graph representing pLJM1-EGFP plasmid standard curve indicating the number of EGFP transgene copies in 500 ng of DNA, which was calculated from their Ct (cycle threshold) values using the linear equation. Genomic PCR for EGFP-positive cells was performed to analyze the temporal infiltration of transplanted cells in (A) blood, (B) heart, (C) lung, (D) liver, and (E) wound tissues. (F) Significant increase in engraftment of EGFP-positive cells from blood to the wound site over the healing period analyzed. (n = 2 replicates per wound with 2 wounds/mice, N = 5 mice/group; p < 0.05 as compared with *pLJM1-EGFP control; ^#pLJM1-Cxcr6-EGFP).

Figure 5

CXCL16-CXCR6 Axis Promotes Re-epithelialization and Collagen Deposition at the Wound Site Representative images of (A) hematoxylin & eosin (H&E) stained regenerated wound skin at PS days 1, 3, and 7, (dotted lines depicting the wound margin; WB indicates the location of wounded/regenerated wound bed). (B) Graph depicting increased number of H&E-stained cells, (C) Sirius red at PS day 7, and (D) graph depicting increased percent area of collagen deposition in regenerated wounds suggesting high re-epithelialization and collagen deposition in groups transplanted with stable MSCs-Cxcr6. CXCL16 (0.5 μg/100 μL, i.d.) at each wound periphery further increased the wound tissue regeneration as compared with other groups. (E, epithelial cells; V, vascular cells; H, hair follicles; and G, sebaceous glands). (n = 3 replicates/wound, N = 5 mice/group; p < 0.05 as compared with ^∗wound control; ^$pLJM1-CXCR6-EGFP Tx).

Figure 6

Engrafted CXCR6 Overexpressed MSCs Are Viable at the Wound Site (A) Confocal co-immunostaining of GFP and Ki67, a proliferation marker depicted an increased recruitment of transplanted MSCs-Cxcr6 were viable at the wound site. MSC-Cxcr6-KD did not get recruited at the wound site suggesting the migratory role of CXCR6. (B) Significant increase in colocalization of GFP with Ki67 in MSC-pLJM1-CXCR6-GFP as compared with MSC-pLJM1-GFP depicted higher engraftment and viability of transplanted MSC. (n = 3 replicates/wound, N = 5 mice/group; p < 0.05 as compared with ^∗pLJM1-EGFP-MSC Tx; ^$pLJM1-CXCR6-EGFP Tx).

Figure 7

CXCL16 Modulates MSCs Transdifferentiation Potential toward Endothelial Cells and Keratinocytes (A) Endothelial transdifferentiation of MSCs in vitro in EGM-2 medium showing higher DiIAcLDL uptake when treated with CXCL16 as compared with control that was perturbed in presence of CXCL16 neutralizing antibody. (B) Quantification of DiIAcLDL uptake in cells. (C) CXCL16-enhanced induction of endothelial cell transdifferentiation was confirmed by confocal immunostaining of endothelial markers expression, CD31, and Tie-2. (D) Similarly, PanCK keratinocyte marker expression also confirmed MSC transdifferentiation into keratinocytes, suggesting enhanced plasticity of MSCs in the presence of CXCL16 as compared with control. (n = 6 replicates/experiment repeated thrice; p < 0.01 as compared with **α-MEM control, ^$EGM control, and ^##CXCL16 treated group).

Figure 8

Transplantation of MSCs with CXCR6 Gene Therapy Efficiently Regenerated Skin in Type I and II Diabetic Mice (A) Flow chart representing type I diabetes model generation. (B) High fasting blood glucose levels (>150 mg/dL) was observed in both type I and II (db/db) diabetic mice. Higher percent wound closure area in diabetic mice transplanted with MSCs-Cxcr6 (C) type I Stz-induced C57BL/6J and (D) type II db/db mice. (E) Higher H&E and Sirius red staining in db/db mice transplanted with MSCs-Cxcr6 depicting an organized layer of dermis with the presence of glands and hair follicles in the regenerated wounds as compared with control MSCs transplanted groups (E, epidermis; D, dermis; H, Hair follicles; G, sebaceous glands; Ad, adipose layer). Increased co-immunostaining of (F) GFP/CXCR6, (G) GFP/CD31, and (H) GFP/PanCK suggesting more recruitment, engraftment, neo-vascularization, and epithelialization in db/db mice transplanted with MSCs-Cxcr6 as compared with MSC-control. (n = 3 replicates/wound, N = 4–5 mice/group; p < 0.05 as compared with *pLJM1-EGFP control; ^#diabetic control).