Despite modern medical advances, the mortality from heart failure following myocardial infarction (MI) has not changed since the 1950’s. We have shown that the fetal response to MI is very different than the adult. The fetal response results in regeneration of myocardium and restoration of cardiac functionand is associated with rapid resolution of the inflammatory response. In contrast, the adult response to MI results in infarct expansion, a progressive loss of function, and is associated with a persistent inflammatory response. Inflammation isregulated by the production of cytokines, such as tumor necrosis factor alpha (TNFa), interleukin-1 (IL-1a), and interleukin 6 (IL-6), interleukin-8 (IL-8), and their upstream effector molecules TNF receptor associated factor6 (TRAF6) andinterleukin-1 receptor associated kinase 1 (IRAK1). We hypothesize that the rapid resolution of inflammation observed in the fetal regenerative response to MI is due to differential regulation of cytokine production and upstream effector molecules compared to the adult response.

Introduction: We have shown that the biomechanical properties of murine and human diabetic skin are significantly inferior to non-diabetic skin at baseline. the major structural component of the skin responsible for skin integrity is type I collagen. MicroRNAs (miRNAs) are regulatory molecules that inhibit the translation of mRNAs at the post-transcriptional level, and have been shown to play an important role in the regulation of collagen content. Specifically, MiRNA-29a has been shown to inhibit the translation of Col1a2 mRNA. We hypothesized that the impaired biomechanical properties of murine diabetic skin at baseline is associated with decreased collagen protein and is due, in part, to increased miRNA-29a production. Methods: To test this hypothesis, we obtained samples from 4 and 20 week old diabetic and non-diabetic murine skin. the skin was processed for isolation of total protein and total cellular RNA. Western blot analysis was performed for type I collagen and real-time PCR performed for Col1a2 and miRNA-29a. Results: At 4 weeks of age diabetic and non-diabetic skin had similar type I collagen protein content, however, by 20 weeks the diabetic skin had significantly decreased type I collagen compared to non-diabetic (p<0.05) or 4 week old skin (p<0.05). at 4 weeks diabetic skin had significantly increased col1a2 and miRNA-29a expression compared to non-diabetic skin, and a col1a2/miRNA-29a ratio of 7.6:1 in diabetics. at 20 weeks the diabetic skin also had increased col1a2 and miRNA-29a expression compared to non-diabetic skin, but the col1a2/miRNA-29a ratio was significantly decreased to 2.7:1 in diabetics. Conclusions: These findings provide the first evidence that the impaired biomechanical properties of diabetic skin may be due, in part, to increased miRNA-29a expression relative to collagen gene expression, resulting in decreased collagen protein production. These results represent a novel potential therapeutic target to improve collagen content and subsequent biomechanical properties of diabetic skin to prevent injury, as well as to improve healing following injury.

Introduction: After injury the healing of diabetic skin is impaired. We have previously shown that murine and human diabetic skin has impaired biomechanical properties at baseline compared to non-diabetic skin. This impairment may play a role in the susceptibility of diabetics to developing chronic wounds. We have previously shown that stromal-derived growth factor 1α (SDF-1α) can increase diabetic wound closure rates when delivered at the time of wounding. We hypothesized that pretreatment with SDF-1α could improve the biomechanical properties of diabetic skin at baseline, and thus decrease the susceptibility to injury. Methods: To test this hypothesis, the flanks of 8-12 week old diabetic (Db/Db) and matched heterozytote (Db/+) mice were shaved and the skin treated with a radial intradermal injection of either lenti-SDF-1α or lenti-GFP. The area of injection was then tattooed for later identification. All mice were sacrificed at day 28 post injection. The skin was then harvested and marked to preserve orientation and subjected to biomechanical testing. Results: Pre-treatment with Lenti-SDF-1α resulted in a significant increase in both the maximum stress (p<0.01) and maximum load (p<0.01) when compared to GFP controls for both diabetic and non-diabetic skin. In addition, there was also a significant improvement in the modulus (p<0.05) in both diabetic and non-diabetic SDF-1α treated skin when compared to GFP controls. Conclusion: In addition to its beneficial wound healing properties, SDF-1α also has the ability to impart increased strength and compliance of the skin at baseline. This preventative approach could have a profound impact on the management of patients prone to chronic wounds such as those with diabetes and peripheral vascular disease. Further studies are needed to determine the molecular mechanisms involved in the improved biomechanical properties of skin treated with SDF-1α.

Introduction: MicroRNAs (miRNAs) are a class of endogenous, small non-coding RNAs that negatively regulate the translation of mRNAs at the post-transcriptional level. Recently, miRNAs have been shown to play pivotal roles in diverse developmental and cellular processes and they have been linked to a variety of inflammatory skin diseases and cancers. Diabetic wounds have been shown to have decreased SDF-1aproduction, decreased angiogenesis, and an abnormal inflammatory response. However, the molecular mechanisms that underly the diabetic wound healing impairment remain unclear. We hypothesized that abnormal miRNA expression may, in part, contribute to the diabetic wound healing impairment. Methods: To test this hypothesis, we examined miRNA expression using a microarray for the majority of known mammalian miRNAs. We examined miRNA expression in diabetic (Db-/Db-) and non-diabetic (Db+/Db-) mice at baseline, and 3 days after creation of an 8mm excisional wound. Microarray results were then validated for selected miRNAs using Real-time PCR analysis. Results: At baseline, we found no significant difference in miRNA expression in diabetic versus non-diabetic skin. However, 3 days after wounding there was a significant difference in miRNA expression in diabetic versus non-diabetic wounds. Among 700 individual miRNAs represented on the microarrays, there were 7 miRNAs which were up-regulated and 10 miRNAs which were down-regulated in the diabetic versus non-diabetic mice. We verified decreased expression of miR-23a and miR-146aby quantitative real-time PCR. Surprisingly, miRNA-23a, which negatively regulates SDF-1a, was down-regulated, suggesting that compensatory miRNA mechanisms regulating SDF-1aon a miRNA level are intact but miRNA down-regulation was insufficient to correct SDF-1aproduction in diabetic wounds. In addition, we found a significant decrease in miRNA 146a, which inhibits the inflammatory response, and may explain, in part, the increased inflammatory response seen in diabetic wounds. Conclusion: These findings demonstrate that miRNA play a role in the diabetic wound healing impairment and provides a new layer of regulatory mechanisms that could be targeted for potential therapeutic intervention.

Introduction: The development of heart failure following myocardial infarction (MI) represents a significant clinical problem. We have recently reported the first mammalian model of cardiac regeneration following MI, in which the fetal response to MI results in regeneration of myocardium and restoration of function, whereas the adult response results in a progressive decline in function and heart failure. MicroRNAs (miRNAs) are evolutionarily conserved small regulatory RNAs that regulate the expression of large number of genes involved in cardiac disease, including the control of myocyte growth, cardiac fibrosis, neoangiogenesis, inflammatory response, and maintenance of cardiac rhythm. We hypothesize that the regenerative fetal response to MI may be due, in part, to differential miRNA expression compared to the adult. Methods: To test our hypothesis we used our novel regenerative fetal and our adult ovine models of MI. In brief, MIs were created in fetal (65-76 days gestation) and adult sheep by ligating the left anterior descending coronary artery (LAD) and appropriate diagonal branches to produce an 25% apical infarct. The animals were sacrificed at either 3 days or 4 weeks after MI. Echocardiography was performed pre-and, post-infarction, and just prior to sacrifice to assess LV function and infarct size. Tissue was processed for miRNA isolation and miRNA expression assessed by quantitative real-time PCR. Results: Consistent with our previous reports, the adult EF decreased significantly from 42% at baseline to 21% by 1 month after infarction (p<0.001). In contrast, by 1 month the fetal EF had returned to preinfarction levels. Increased levels of microRNA-21 (miR21) have been associated with the progression of heart failure. We found significant upregulation of miR-21 in the adult infarct at 3 days and 4 weeks. In contrast miR-21 was undetectable in fetal infarcts. MicroRNA-1 (miR-1) inhibits cardiac myogenesis by down-regulating Hand2. We found significant upregulation (40-fold) of miR-1 in the adult heart at baseline compared to the fetal heart. 3 days following MI, both fetal and adult infarcts had decreased miR-1 expression, but by one month the fetal level had returned to baseline, whereas the adult remained significantly decreased. Conclusion: Our studies demonstrate that miRNA play a role in the differential response to MI between the fetus and the adult, in particular the development of heart failure and myogenesis. Understanding this role and that of other miRNA in regulating this differential response will add a new area of regulatory control for the development a therapeutic strategies to modify the adult response to myocardial infarction and prevent the development of heart failure.