Chronic recalcitrant wounds result from delayed or slowed healing processes. Underlying inflammation is a substantial risk factor for impaired dermal wound healing and often leads to chronic wound-related sequelae. Human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The goal of this project was to improve the outcome of chronic wounds by harvesting the exosomes from hASCs for therapeutic intervention. The results demonstrate that long noncoding RNA GAS5 is highly enriched in hASC exosomes and, further, that GAS5 is central to promoting wound repair in vitro. To evaluate the outcome of wound healing in a chronic low-grade inflammatory environment, lipopolysaccharide-treated HDF cells were evaluated for their response to hASC exosome treatment. Ingenuity pathway analysis identified inflammation pathways and genes affected by exosomes in a GAS5-dependent manner. Using siRNA to deplete GAS5 in HDF, the results demonstrated that Toll-like receptor 7 (TLR7) expression levels were regulated by GAS5. Importantly, the results demonstrate that GAS5 regulates inflammatory pathway genes in a chronic inflammation environment. The results presented here demonstrate that hASC exosomes are a viable therapeutic that accelerate the healing of chronic recalcitrant wounds.

The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKC?I is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKC?I activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKC?I isozymes and a caspase-3 binding site on the PKC?I hinge domain with Schr?dinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKC?I. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKC?I and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKC?I, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKC?I activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKC?I catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKC?I catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKC?I is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3?mediated activation of kinase activity.

Type 2 diabetes mellitus is a metabolic disorder defined by systemic insulin resistance. Insulin resistance in adipocytes, an important regulator of glucose metabolism, results in impaired glucose uptake. The trafficking protein, sortilin, regulates major glucose transporter 4 (Glut4) movement, thereby promoting glucose uptake in adipocytes. Here, we demonstrate the presence of an alternatively spliced sortilin variant (Sort^17b), whose levels increase with insulin resistance in mouse 3T3L1 adipocytes. Using a splicing minigene, we show that inclusion of alternative exon 17b results in the expression of Sort^17b splice variant. Bioinformatic analysis indicated a novel intrinsic disorder region (IDR) encoded by exon 17b of Sort^17b. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) measurements using molecular dynamics demonstrated increased flexibility of the protein backbone within the IDR. Using protein–protein docking and co-immunoprecipitation assays, we show robust binding of Glut4 to Sort^17b. Further, results demonstrate that over-expression of Sort^17b correlates with reduced Glut4 translocation and decreased glucose uptake in adipocytes. The study demonstrates that insulin resistance in 3T3L1 adipocytes promotes expression of a novel sortilin splice variant with thus far unknown implications in glucose metabolism. This knowledge may be used to develop therapeutics targeting sortilin variants in the management of type 2 diabetes and metabolic syndrome.

Ovarian cancer is the deadliest malignant disease in women. Protein Kinase C delta (PRKCD; PKCδ) is serine/threonine kinase extensively linked to various cancers. In humans, PKCδ is alternatively spl...