Abstract
Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk in vivo. However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes (hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math>) from human chronic wound fluid. Furthermore, we studied the significance of hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> in diabetic wounds. LC-MS-MS detection of KRT14 in hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math>. dSTORM nanoimaging identified KRT14+ extracellular vesicles (EVκ<math><mrow><msub><mtext>EV</mtext><mtext>κ</mtext></msub></mrow></math>) in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> Ø as 80-145nm. The abundance of hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> Uptake of hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> from non-diabetics, the addition of diabetic hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of hExoκ<math><mrow><msub><mtext>hExo</mtext><mtext>κ</mtext></msub></mrow></math> from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity.