MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea.

PURPOSE: MiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing. METHODS: Our previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs. RESULTS: Overexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1ß, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a. CONCLUSION: Our study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-ß, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.

Exosomes and cardiovascular cell-cell communication.

Exosomes have become an important player in intercellular signaling. These lipid microvesicles can stably transfer miRNA, protein, and other molecules between cells and circulate throughout the body. Exosomes are released by almost all cell types and are present in most if not all biological fluids. The biologically active cargo carried by exosomes can alter the phenotype of recipient cells. Exosomes increasingly are recognized as having an important role in the progression and treatment of cardiac disease states. Injured cardiac cells can release exosomes with important pathological effects on surrounding tissue, in addition to effecting other organs. But of equal interest is the possible benefit(s) conferred by exosomes released from stem cells for use in treatment and possible repair of cardiac damage.

Electrotaxis of cardiac progenitor cells, cardiac fibroblasts, and induced pluripotent stem cell-derived cardiac progenitor cells requires serum and is directed via PI3'K pathways.

BACKGROUND: The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis (electrically guided cell movement) has been clinically used to improve recovery in a number of tissues but has not been investigated for treating myocardial damage. OBJECTIVE: The purpose of this study was to test the electrotactic behaviors of several types of cardiac cells. METHODS: Cardiac progenitor cells (CPCs), cardiac fibroblasts (CFs), and human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) were used. RESULTS: CPCs and CFs electrotax toward the anode of a direct current electric field, whereas hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinase signaling. In addition, very late antigen-4, an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current electric field. The hiPSC-derived CPCs do not express very late antigen-4, migrate toward the cathode in a voltage-dependent manner, and, similar to CPCs and CFs, require media serum and phosphatidylinositide 3-kinase activity for electrotaxis. CONCLUSION: The electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium.

The ZupT transporter plays an important role in zinc homeostasis and contributes to Salmonella enterica virulence.

Zinc is an essential metal for cellular homeostasis and function in both eukaryotes and prokaryotes. To acquire this essential nutrient, bacteria employ transporters characterized by different affinity for the metal. Several studies have investigated the role of the high affinity transporter ZnuABC in the bacterial response to zinc shortage, showing that this transporter has a key role in adapting bacteria to zinc starvation. In contrast, the role of the low affinity zinc importer ZupT has been the subject of limited investigations. Here we show that a Salmonella strain lacking ZupT is impaired in its ability to grow in metal devoid environments and that a znuABC zupT strain exhibits a severe growth defect in zinc devoid media, is hypersensitive to oxidative stress and contains reduced levels of intracellular free zinc. Moreover, we show that ZupT also plays a role in the ability of S. Typhimurium to colonize the host tissues. During systemic infections, the single zupT mutant strain was attenuated only in Nramp1(+/+) mice, but competition experiments between znuABC and znuABC zupT mutants revealed that ZupT contributes to metal uptake in vivo independently of the presence of a functional Nramp1 transporter. Altogether, the here reported results show that ZupT plays an important role in Salmonella zinc homeostasis, being involved in metal import both in vitro and in infected animals.

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut.

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.