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. 2018 Jan;141(1):55e-67e.
doi: 10.1097/PRS.0000000000003959.

PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing

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Free PMC article

PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing

Sae Hee Ko et al. Plast Reconstr Surg. .
Free PMC article

Abstract

Background: Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing.

Methods: Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics.

Results: PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05).

Conclusions: Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

Figures

Fig. 1
Fig. 1
Effect of PHD-2 knockdown on bone marrow-derived MSCs. (A) Quantitative real-time PCR of PHD-2 transcript expression in MSCs transduced with different lentiviral constructs (A through C). The results were normalized to the β-actin mRNA level, and shown relative to values from cells transduced with scrambled construct D. Constructs A and B showed statistically significant PHD-2 knockdown compared to shScramble (*p<0.05). (B) Western blot of HIF-1α in MSCs transduced with construct A, shRNA to PHD-2 compared to MSCs transduced with construct D, scramble shRNA under normoxic conditions. (C) VEGF expression as detected by ELISA in MSCs stably transduced with shPHD-2 or shScramble shows increased VEGF protein concentration in conditioned media from cells stably transduced with shPHD-2 (*p<0.05). (D) qRT-PCR analysis of common angiogenic genes in MSCs stably transduced with shPHD-2 construct versus those transduced with shScramble. Results are shown as percent of levels compared to levels of shScramble MSCs (*p<0.05). (E) Angiogenesis antibody array using conditioned medium from MSCs stably transduced with shPHD-2 or shScramble. Membrane images of antibody array treated with conditioned media of stably transduced cells (left). Specific proteins captured on the array are labeled 1 to 13. Semiquantitative profiles of proteins 1 to 13 on the angiogenesis antibody array using reverse image scanning densitometry (right). There was a significant increase in the expression of multiple angiogenic proteins by shPHD-2 MSCs in comparison to shScramble MSCs (*p<0.05).
Fig. 2
Fig. 2
PHD-2 knockdown MSCs enhance in vitro angiogenesis. (A) Representative photographs of human umbilical vein endothelial cells seeded onto Matrigel in conditioned medium from shPHD-2 MSCs or shScramble MSCs (left). Quantitative assessment of branch points (right). Conditioned medium from shPHD-2 significantly increased tube formation compared to controls (*p<0.05). (B) Representative images of shPHD-2 MSCs versus shScramble MSCs in co-culture with bEND.3 endothelial cells on Matrigel (a, b, and c). shPHD-2 MSCs seeded with bEND.3 cells form complex mesh-like tubule structures in marked contrast to control cells that remain largely as poorly organized round cells (quantification shown, d). shPHD-2 MSCs express GFP transgene (green), which can be seen aligning with PKH26 dye-labeled endothelial cells (red) within the tubular structures (e).
Fig. 3
Fig. 3
PHD-2 knockdown MSCs enhanced wound healing. Full-thickness excisional wounds on wild type mice were treated with shPHD-2 MSCs, shScramble MSCs, untransduced MSCs, or PBS/Matrigel (Fig. S4). Wound closure rate over time is depicted in the graph. There is a significant acceleration of wound healing in the PHD-2 knockdown MSC treatment group (n=6) (*p<0.05).
Fig. 4
Fig. 4
shPHD-2-transduced MSCs increase blood vessel formation in the wound bed, contribute to newly formed blood vessels, and remain viable and metabolically active in the wound bed after complete closure. (A) Graph depicting immunofluorescence for endothelial cells. Wound sections were stained with an anti-CD31 antibody and detected with Fluor 594 (red) (Fig. S5). Software-assisted quantification of vessel density in the entire area of residual wounds at day 14, as percent fluorescence. Wounds from shPHD-2 MSC-treated mice had significantly higher vessel density compared with that from PBS/Matrigel-treated mice (*p<0.05). (B) Engraftment of bone marrow-derived MSCs. Immunofluorescence of wounds treated with shPHD-2 MSCs shows that GFP expressing MSCs (green) were found in the dermis of residual wounds. MSCs are scattered in the dermis with little evidence for co-localization of CD31 positive blood vessels (red). Some MSCs appear intimately aligned with blood vessels (arrow). (C) Viability of engrafted MSCs. Immunofluorescence of wounds treated with shPHD-2 MSCs shows that GFP expressing MSCs (green) co-localizes with PCNA markers (red) indicating an active metabolic state of engrafted MSCs. (D) Engrafted GFP expressing MSCs (green) express the pericyte/myofibroblast marker α-SMA (red) as shown by co-localization study of wounds treated with shPHD-2 MSCs.
Fig. 5
Fig. 5
H&E stains of wound tissue after full closure at 14 days post-wounding. PHD-2 knockdown MSC-treated wounds have higher dermal cellularity in the wound bed compared to PBS/Matrigel-treated control wounds. Black box indicates the area of healed wound depicted under high power view. Quantification of dermal cellularity is shown (right) (*p<0.05).

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