**4. Therapeutic targets**

**3.3. Decreased angiogenesis: the role of miR-15b**

14 Wound Healing - New insights into Ancient Challenges

molecule 31 (CD31), a marker of endothelial cells [42].

duced from Xu *et al.* [16] with permission of the authors.)

Successful angiogenesis requires coordinated extracellular matrix production in order to provide an adequate architecture for formation of new blood vessels [31]. However, angio‐ genesis within a wound bed is further regulated by numerous angiogenic factors, with vascular endothelial growth factor (VEGF) considered one of the most prominent [40]. Following injury, hypoxia in the wound bed leads to increased expression of hypoxia inducible factor‐1 (HIF‐1), a transcription factor that increases the expression of numerous proangiogenic proteins, including VEGF [31, 41]. In turn, VEGF attracts endothelial cells to the site of injury, in addition to inducing proliferation and angiogenesis via upregulation of proteins, such as the anti‐ apoptotic B‐cell lymphoma‐2 (BCL‐2) [16, 40]. BCL‐2 is also thought to improve wound healing by stabilizing the alpha subunit of HIF‐1 (HIF‐1α), mediated by heat shock protein 90 (HSP90), thereby increasing HIF‐1 mediated VEGF expression [16]. When compared to nondiabetic murine wounds, diabetic murine wounds have been detailed to have decreased levels of HIF‐1a activity, VEGF gene expression, and BCL‐2 gene expression, as well as significantly decreased number of cells that stain for the presence of platelet endothelial cell adhesion

MiR‐15b negatively regulates angiogenesis by decreasing VEGF expression; this decrease in VEGF expression is associated with decreased cell migration and vascular tubule formation

**Figure 7. Quantification of miRNA-15b, BCL2, and VEGFα gene expression in diabetic and non-diabetic wounds.** Quantification by real‐time PCR of miRNA‐15b (miR‐15b) **(A)**, BCL2 **(B)**, and VEGFα **(C)** gene expression in murine diabetic wounds versus murine non‐diabetic wounds, 3–7 days after wounding. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image repro‐

### **4.1. The impact of cellular therapies on diabetic wound healing**

In the setting of the tremendous clinical and fiscal burden of chronic diabetic wounds, efforts to develop effective wound care strategies are ongoing. The dysregulation of wound healing in patients with diabetes occurs at every stage of healing—whether it be the inflammatory phase, the proliferative phase, or the remodeling phase. Given this widespread dysregulation, therapies directed at individual targets of the wound healing response are unlikely to be completely successful in addressing the diabetic wound healing impairment. As such,

**Figure 8. Treatment of diabetic wounds with MSCs expedites wound closure and upregulates miRNA-146a, 7 days after wounding. (A)** Diabetic wounds treated with PBS (left) or MSCs (right). **(B)** CD45 immunostaining of diabetic (Db) and nondiabetic (Hz) wounds, following treatment with either PBS or MSCs. **(C)** Quantitative assessment of dia‐ betic (Db) and nondiabetic (Hz) wound closure, following treatment with either PBS or MSCs. **(D)** Flourescent image demonstrating GFP positive cells (MSCs), confirming the persistence of MSCs after injection. Blue arrows indicate in‐ jection sites. **(E)** Real‐time PCR confirms the upregulation of miRNA‐146a gene expression in Db treated with MSCs, compared with Db wounds treated with PBS. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image reproduced from Xu *et al.* [17] with permission of the authors.)

attention has been drawn to the use of cell‐based therapies for the treatment of chronic diabetic wounds, with the hopes that multipotent cell therapy will address impaired diabetic wound healing at multiple levels of dysregulation [41]. Specifically, MSCs have been a focus due to their capacity for self‐renewal, multipotency, and their ease of retrieval from autologous bone marrow [44].

### *4.1.1. Impact of mesenchymal stem cell treatment on miR-146a*

Treatment of diabetic and nondiabetic murine wounds with either MSCs or PBS revealed that treatment of diabetic wounds with MSCs corrected the dysregulated inflammation present in diabetic wounds. Seven days after treatment with MSCs, diabetic murine skin demonstrated more rapid wound healing, a decreased concentration of CD45 positive cells in the periwound tissues, increased expression of miR‐146a, and decreased gene expression of IRAK1, TRAF6, NFkB, and the proinflammatory cytokines IL‐6 and IL‐8/MIP‐2 (**Figure 8** and **Figure 9**).

**Figure 9. Impact of MSC treatment on IRAK1, TRAF6, NFkB, IL-6, and MIP2 gene expression.** Real‐time PCR dem‐ onstrating the impact of treatment with either MSCs or PBS on gene expression of IRAK1 **(A)**, TRAF6 **(B)**, NFkB **(C)**, IL‐6 **(D)**, and MIP2 **(E)** 3 and 7 days after wounding in diabetic and non‐diabetic mice Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image repro‐ duced from Xu *et al.* [17] with permission of the authors.)

### *4.1.2. Impact of mesenchymal stem cell treatment on miR-29a*

attention has been drawn to the use of cell‐based therapies for the treatment of chronic diabetic wounds, with the hopes that multipotent cell therapy will address impaired diabetic wound healing at multiple levels of dysregulation [41]. Specifically, MSCs have been a focus due to their capacity for self‐renewal, multipotency, and their ease of retrieval from autologous bone

Treatment of diabetic and nondiabetic murine wounds with either MSCs or PBS revealed that treatment of diabetic wounds with MSCs corrected the dysregulated inflammation present in diabetic wounds. Seven days after treatment with MSCs, diabetic murine skin demonstrated more rapid wound healing, a decreased concentration of CD45 positive cells in the periwound tissues, increased expression of miR‐146a, and decreased gene expression of IRAK1, TRAF6, NFkB, and the proinflammatory cytokines IL‐6 and IL‐8/MIP‐2 (**Figure 8** and **Figure 9**).

**Figure 9. Impact of MSC treatment on IRAK1, TRAF6, NFkB, IL-6, and MIP2 gene expression.** Real‐time PCR dem‐ onstrating the impact of treatment with either MSCs or PBS on gene expression of IRAK1 **(A)**, TRAF6 **(B)**, NFkB **(C)**, IL‐6 **(D)**, and MIP2 **(E)** 3 and 7 days after wounding in diabetic and non‐diabetic mice Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image repro‐

duced from Xu *et al.* [17] with permission of the authors.)

*4.1.1. Impact of mesenchymal stem cell treatment on miR-146a*

16 Wound Healing - New insights into Ancient Challenges

marrow [44].

The expedited diabetic wound healing observed after treatment of diabetic skin with MSCs is not solely associated with decreased inflammation. In addition to upregulating gene expres‐ sion of miR‐146a, treatment with MSCs downregulates miR‐29a expression in diabetic murine wounds when compared with nondiabetic murine wounds. The downregulation in miR‐29a 4 weeks after treatment with MSCs is accompanied by an upregulation in collagen I and collagen III protein content (**Figure 10**).

**Figure 10. Impact of MSC treatment on collagen protein and gene expression. (A)** Real‐time PCR quantification of miRNA‐29a gene expression in diabetic and non‐diabetic murine skin 28 days after treatment with MSCs or PBS. Quantification of **(B)** Collagen I and **(C)** Collagen III protein levels in diabetic skin and non‐diabetic skin 28 days after treatment with either MSCs or PBS. **(D)** Western blot depicting the Collagen I and Collagen III protein content in dia‐ betic and non‐diabetic wounds 28 days after treatment with either MSCs or PBS. Results are presented as a mean + SEM for each cohort at each time point. Asterisk (\*) indicates *p* < 0.05 and \*\* indicates *p* < 0.001. (Image repro‐ duced from Zgheib *et al.* [21] with permission of the authors.)

### *4.1.3. Impact of mesenchymal stem cell treatment on miR-15b*

Treatment with MSCs was also successful at correcting the dysregulated miR‐15b expression, further contributing to the improved healing of diabetic wounds observed following treatment with MSCs. Both 3 and 7 days after wounding and treatment with MSCs, diabetic wounds treated with MSCs demonstrated a significant downregulation in miR‐15b gene expression when compares to untreated diabetic wounds (**Figure 11**). Additionally, three days after wounding, diabetic wounds treated with MSCs demonstrated significant upregulation in CD31 positive cell sin the wound bed, as well as significant upregulation in HIF‐1α, BCL‐2, and VEGF gene expression [16].

**Figure 11. Impact of MSC treatment on angiogenesis and miRNA-15b expression. (A)** CD31 immunostaining in dia‐ betic wounds 3 days after wounding and treatment with either PBS or MSCs. **(B)** Quantification of CD31 positive cells in diabetic (Db) and non‐diabetic (Hz) wounds 3 days after wounding. **(C)** Real‐time PCR quantification of miRNA‐15b gene expression 3 and 7 days after wounding in diabetic (Db) and non‐diabetic (Hz) skin treated with either PBS or MSCs. Results are presented as a mean + SEM for each cohort at each time point. P‐values are included. (Image repro‐ duced from Xu *et al.* [16] with permission of the authors.)

### **4.2. Adverse effects of MSC treatment**

with MSCs. Both 3 and 7 days after wounding and treatment with MSCs, diabetic wounds treated with MSCs demonstrated a significant downregulation in miR‐15b gene expression when compares to untreated diabetic wounds (**Figure 11**). Additionally, three days after wounding, diabetic wounds treated with MSCs demonstrated significant upregulation in CD31 positive cell sin the wound bed, as well as significant upregulation in HIF‐1α, BCL‐2,

**Figure 11. Impact of MSC treatment on angiogenesis and miRNA-15b expression. (A)** CD31 immunostaining in dia‐ betic wounds 3 days after wounding and treatment with either PBS or MSCs. **(B)** Quantification of CD31 positive cells in diabetic (Db) and non‐diabetic (Hz) wounds 3 days after wounding. **(C)** Real‐time PCR quantification of miRNA‐15b gene expression 3 and 7 days after wounding in diabetic (Db) and non‐diabetic (Hz) skin treated with either PBS or MSCs. Results are presented as a mean + SEM for each cohort at each time point. P‐values are included. (Image repro‐

duced from Xu *et al.* [16] with permission of the authors.)

and VEGF gene expression [16].

18 Wound Healing - New insights into Ancient Challenges

Despite the continued emergence of evidence cataloging the benefits of MSCs in the treatment of diabetic wounds, results are also emerging that detail adverse effects regarding the thera‐ peutic use of MSCs [45]. Specifically, Jeong *et al* (2001) describes the development of soft tissue sarcomas at the site of injection during evaluation of the impact of MSC treatment on both diabetic neuropathy and myocardial regeneration after MI [46]. Similarly, after bone marrow transplant that included systemic administration of 3 × 106 MSCs, Tolar *et al.* reported 12 out of 17 (70.5%) mice developed soft tissue sarcomas, including ectopic ossicles and extremity sarcomas [47]. In addition to the risk of malignant transformation following administration of MSCs, the immunosuppressive impact of MSCs therapy may place patients at risk of infection; although this has not been observed *in vivo* [48].

### **4.3. The impact of SDF-1α on diabetic wound healing**

The time and resources required to harvest and prepare an adequate number of MSCs for autologous transplant has led to investigation in to additional means of simulating the robust improvement in wound healing seen after treatment with MSCs. In attempting to define the mechanism by which MSCs and stromal progenitor cells improve wound healing in diabetic mice, it was noticed that the improved wound healing was associated with upregulation of stromal cell‐derived factor‐1α (SDF‐1α). SDF‐1α has long been known as a potent chemokine crucial in the migration and localization of stem cells to wounded tissues [7]. Following injury, SDF‐1α expression is upregulated by HIF‐1α via VEGF in response to hypoxia in the injured tissues [49]. However, SDF‐1α is downregulated in diabetic wounds [7, 22]. We have previously shown that overexpression of SDF‐1α in the wound bed is capable of improving the diabetic wound healing impairment [22]. Furthermore, inhibition of SDF‐1α via transfection with a mutant SDF‐1α that binds the CXCR4 receptor without activation further impairs wound closure, increases inflammatory cytokine production and infiltration of inflammatory cells, and further retards angiogenesis [25]. While these studies support SDF‐1α as a key element in mediating the numerous impairments associated with the diabetic wound healing response, there has been no published evaluation of the impact of SDF‐1α treatment on miRNA dysre‐ gulation in diabetic skin or wounds.
