Junyi Hu

The role of microRNA‐15b in the impaired angiogenesis in diabetic wounds

The impairment in diabetic wound healing represents a significant clinical problem. Decreased angiogenesis is thought to play a central role in the pathogenesis of this impairment. We have previously shown that treatment of diabetic murine wounds with mesenchymal stem cells can improve healing, but the mechanisms are not completely defined. MicroRNA‐15b (miR‐15b) has been implicated in the regulation of the angiogenic response. We hypothesized that abnormal miR‐15b expression may contribute to the impaired angiogenesis observed in impaired diabetic wound healing. To test this hypothesis, we examined the expression of miR‐15b and its target genes in diabetic and nondiabetic mice before and after injury. MiR‐15b expression was significantly up‐regulated in diabetic mouse wounds during the wound healing response. Increased miR‐15b levels also closely correlated with decreased gene expression of its proangiogenic target genes. Furthermore, the correction of the diabetic wound healing impairment with mesenchymal stem cell treatment was associated with a significant decrease in miR‐15b expression level and increased gene expression of its proangiogenic target genes. These results provide the first evidence that increased expression of miR‐15b in diabetic wounds in response to injury may, in part, be responsible for the abnormal angiogenic response seen in diabetic wounds and may contribute to the observed wound healing impairment.

Mammalian Fetal Cardiac Regeneration After Myocardial Infarction Is Associated With Differential Gene Expression Compared With the Adult

BACKGROUND In adults, myocardial infarction (MI) results in a brisk inflammatory response, myocardium loss, and scar formation. We have recently reported the first mammalian large-animal model of cardiac regeneration after MI in fetal sheep. We hypothesize that the ability of the fetus to regenerate functional myocardium after MI is owing to differential gene expression regulating the response to MI in the fetus compared with the adult. METHODS Myocardial infarction was created in adult (n=4) or early gestation fetal (n=4) sheep. Tissue was harvested after 3 or 30 days, and RNA was extracted for microarray, followed by principal component analysis and global gene expression analysis for the following gene ontology terms: response to wounding, inflammatory response, extracellular matrix, cell cycle, cell migration, cell proliferation, and apoptosis. RESULTS Principal component analysis demonstrated that the global gene expression pattern in adult infarcts was distinctly different from the uninfarcted region at 3 days and remained different at 30 days after MI. In contrast, gene expression in the fetal infarct was different from the uninfarcted region at 3 days, but by 30 days it returned to a baseline expression pattern similar to the uninfarcted region. Three days after MI there was an increase in the expression of genes related to all gene ontology terms in fetal and adult infarcts, but this increase was much more pronounced in adults. By 30 days, the fetal gene expression returned to baseline, whereas in the adult it remained significantly elevated. CONCLUSIONS These data demonstrate that the global gene expression pattern is dramatically different in the fetal regenerative response to MI compared with the adult response and may partly be responsible for the regeneration.

Mechanisms of mesenchymal stem cell correction of the impaired biomechanical properties of diabetic skin: The role of miR-29a

Diabetic skin has impaired wound healing properties following injury. We have further shown that diabetic skin has weakened biomechanical properties at baseline. We hypothesize that the biomechanical properties of diabetic skin decline during the progression of the diabetic phenotype, and that this decline is due to the dysregulation of miR‐29a, resulting in decreased collagen content. We further hypothesize that treatment with mesenchymal stem cells (MSCs) may improve diabetic wound healing by correction of the dysregulated miR‐29a expression. We analyzed the biomechanical properties, collagen gene expression, collagen protein production, and miR‐29a levels in skin harvested from 6 to 18 week old mice during the development of the diabetic phenotype. We also examined the correction of these impairments by both MSC treatment and the inhibition of miR‐29a. Diabetic skin demonstrated a progressive impairment of biomechanical properties, decreased collagen content, and increased miR‐29a levels during the development of the diabetic phenotype. MSC treatment decreased miR‐29a levels, increased collagen content, and corrected the impaired biomechanical properties of diabetic skin. Additionally, direct inhibition of miR‐29a also increased collagen content in diabetic skin. This decline in the biomechanical properties of diabetic skin during the progression of diabetes may increase the susceptibility of diabetic skin to injury and miR‐29a appears to play a key role in this process.

SCF increases in utero–labeled stem cells migration and improves wound healing

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF‐1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)‐labeled skin stem cells via in utero intra‐amniotic injection of lenti‐RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin.

Mesenchymal stem cells correct impaired diabetic wound healing by decreasing ECM proteolysis

Impaired diabetic wound healing is associated with a dermal extracellular matrix protein profile favoring proteolysis; within the healing diabetic wound, this is represented by an increase in activated matrix metalloproteinase (MMPs). Treatment of diabetic wounds with mesenchymal stem cells (MSCs) has been shown to improve wound healing; however, there has not yet been an assessment of their ability to correct dysregulation of MMPs in diabetic wounds. Furthermore, there has been no prior assessment of the role of microRNA29b (miR-29b), an inhibitory regulatory molecule that targets MMP-9 mRNA. Using in vitro models of fibroblast coculture with MSCs and in vivo murine wound healing models, we tested the hypothesis that MSCs correct dysregulation of MMPs in a microRNA-29b-dependent mechanism. In this study, we first demonstrated that collagen I and III protein content is significantly reduced in diabetic wounds, and treatment with MSCs significantly improves collagen I content in both nondiabetic and diabetic wounds. We then found that MMP-9 gene expression and protein content were significantly upregulated in diabetic wounds, indicating elevated proteolysis. Treatment with MSCs resulted in a decrease in MMP-9 gene expression and protein content level in diabetic wounds 3 and 7 days after wounding. Zymographic analysis indicated that MSC treatment also decreased the amount of activated MMP-9 present in diabetic wounds. Furthermore, miR-29b expression was inversely associated with MMP-9 gene expression; miR-29b expression was decreased in diabetic wounds and diabetic fibroblast. Following treatment of diabetic wounds with MSCs, as well as in diabetic fibroblasts cocultured with MSCs, miR-29b was significantly increased. These findings suggest a potential mechanism through which MSCs enhance diabetic wound healing by improving collagen I content in diabetic wounds through decreasing MMP-9 expression and increasing miR-29b expression.

Increased fetal wound size results in increased oxidative stress and promotes scar formation in fetal wounds

INTRODUCTION: The adult response to dermal injury results in rapid wound closure but with scar formation. In contrast fetal response is regenerative with restoration of normal tissue architecture and no scar formation. We have shown that increasing fetal wound size can increase inflammation and results in scaring. We hypothesize that increased fetal wound size results in scar formation due to increased oxidative stress.