Regenerative functions of exosomes rely on their contents which are influenced by pathological stimuli, including hypoxia, in rotator cuff tendon injuries (RCTI). The hypoxic environment triggers tenocytes and adjacent adipose-derived mesenchymal stem cells (ADMSCs) to release regenerative mediators to the ECM via the exosomes which elicit autocrine/paracrine responses to protect the tendon matrix from injury. We investigated the exosomal protein contents from tenocytes and subcutaneous ADMSCs from the shoulder of Yucatan microswine cultured under hypoxic conditions (2% O 2 ). The exosomal proteins were detected using high-resolution massspectrometry nano-LC-MS/MS Tribrid system and were compiled using 'Scaffold' software. Hypoxic exosomes from tenocytes and ADMSCs carried 199 and 65 proteins, respectively. The key proteins identified by mass spectrometry and associated with ECM homeostasis from hypoxic ADMSCs included MMP2, COL6A, CTSD and TN-C and those from hypoxic tenocytes were THSB1, NSEP1, ITIH4 and TN-C. These findings were confirmed at the mRNA and protein level in the hypoxic ADMSCs and tenocytes. These proteins are involved in multiple signaling pathways of ECM repair/regeneration. This warrants further investigations for their translational significance in the management of RCTI.

Vitamin D functions as a potent immunomodulator by interacting with many immune cells however, its role in regulating inflammation in the epicardial adipose tissue (EAT) is unclear. In the EAT of atherosclerotic microswine that were fed with deficient, sufficient or supplemented levels of vitamin D, we evaluated the phenotype of the macrophages. Vitamin D treatment was continued for 12 months and serum 25(OH)D levels were measured regularly. Infiltration of M1/M2 macrophage was investigated by immunostaining for CCR7 and CD206, respectively in conjunction with a pan macrophage marker CD14. Significant difference in the number of CCR7+ cells was observed in the EAT from vitamin D-deficient swine compared to vitamin D-sufficient or -supplemented swine. Expression of CD206 correlated with high levels of serum 25(OH)D indicating a significant increase in M2 macrophages in the EAT of vitamin D-supplemented compared to -deficient swine. These findings suggest that vitamin D-deficiency exacerbates inflammation by increasing pro-inflammatory M1 macrophages, while vitamin D-supplementation attenuates the inflammatory cytokines and promotes M2 macrophages in EAT. This study demonstrates the significance of vitamin D mediated inhibition of macrophage mediated inflammation in the EAT during coronary intervention in addition to its immunomodulatory role. However, additional studies are required to identify the cellular mechanisms that transduce signals between macrophages and smooth muscle cells during restenosis in the presence and absence of vitamin D.

Vitamin D functions as a potent immunomodulator by interacting with many immune cells however, its role in regulating inflammation in the epicardial adipose tissue (EAT) is unclear. In the EAT of atherosclerotic microswine that were fed with deficient, sufficient or supplemented levels of vitamin D, we evaluated the phenotype of the macrophages. Vitamin D treatment was continued for 12 months and serum 25(OH)D levels were measured regularly. Infiltration of M1/M2 macrophage was investigated by immunostaining for CCR7 and CD206, respectively in conjunction with a pan macrophage marker CD14. Significant difference in the number of CCR7+ cells was observed in the EAT from vitamin D-deficient swine compared to vitamin D-sufficient or -supplemented swine. Expression of CD206 correlated with high levels of serum 25(OH)D indicating a significant increase in M2 macrophages in the EAT of vitamin D-supplemented compared to -deficient swine. These findings suggest that vitamin D-deficiency exacerbates inflammation by increasing pro-inflammatory M1 macrophages, while vitamin D-supplementation attenuates the inflammatory cytokines and promotes M2 macrophages in EAT. This study demonstrates the significance of vitamin D mediated inhibition of macrophage mediated inflammation in the EAT during coronary intervention in addition to its immunomodulatory role. However, additional studies are required to identify the cellular mechanisms that transduce signals between macrophages and smooth muscle cells during restenosis in the presence and absence of vitamin D.Author Contribution StatementDKA conceived and designed the experiments; PG, JPF, MMR performed the experiments; PG, JPF, VJS analyzed and interpreted the results; PG prepared the figures and wrote the initial draft of the manuscript; CSB, DKA revised and edited the revised manuscript.

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Esophageal adenocarcinoma (EAC) has seen a steady and steep rise in prevalence since the 1970's. This increase in incidence is correlated with the increase in gastro-esophageal reflux disease and Barrett's esophagus (BE). Unfortunately, where many other cancers have seen progress with advancements in treatments, EAC has not. This cancer is very resistant to almost all first line chemotherapy treatments including the most common platinum based treatments. Although few targeted therapies have been developed, they only show a modest survival advantage for eligible individuals and are not commonly integrated into patient treatment regimens. In our study, we have identified several markers associated with resistance to platinum based chemotherapies that have not previously been documented in EAC. Our study utilized a large-scale discovery mass spectrometric experiment such that the proteome of the 3 different tissue samples could be analyzed, (EAC, BE, and Normal). Part of the novelty of our experiment was that all the tissues collected were from esophagetomies of patients before they had received any chemotherapy. From the data generated we have identified 4 novel markers that are overexpressed in the BE and EAC tissue compared to the normal tissue. In BE tissue DAD1 was over expressed in 9/10 samples, ISG15 (10/10), S100P (10/10) and UBE2N (10/10). In the EAC samples, DAD1 was significantly overexpressed in 20 /20 samples, ISG15 (20/20), S100P (20/20) and UBE2N (19/20). DAD1 inhibits apoptosis and is upregulated by cisplatin and then contributes to cisplatin resistance. S100P has been shown to increase drug resistance in gastric cancer and decrease chemosensitivity 5-FU in vitro in colorectal cancer cells while facilitating proliferation and inhibiting apoptosis. UBE2N plays a role in glycolytic control and in various cellular damage responses. And ISG15 is important in development and metastatic progression of adenocarcinomas through the enhancement of cancer stem cells. We are confident that expression of these four markers can provide clinical utility in assessment of at risk patients with Barrett's esophagus while simultaneously informing treatment regimens. Our goal is to investigate the specific mechanisms by which these markers contribute to cancerous development and chemotherapy resistance in order to elucidate targets for future treatment of EAC. Citation Format: Jonathan P. Fleegel, Joeseph Abdo, Devendra Agrawal. Identification of 4 novel markers in the chemotherapy resistance of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1976.