Department of Biochemistry and Molecular Biologyhttp://hdl.handle.net/10675.2/8472024-03-29T02:29:19Z2024-03-29T02:29:19ZPlos One Transient Plasma Membrane Disruption Induced Calcium Waves in Mouse and Human Corneal Epithelial Cells - Figure Data FilesZhong, ChenLu, XiaowenWatsky, Mitchellhttp://hdl.handle.net/10675.2/6251192024-03-08T01:57:20ZPlos One Transient Plasma Membrane Disruption Induced Calcium Waves in Mouse and Human Corneal Epithelial Cells - Figure Data Files
Zhong, Chen; Lu, Xiaowen; Watsky, Mitchell
Data Files for figures 2-6 in manuscript titled "Transient Plasma Membrane Disruption Induced Calcium Waves in Mouse and Human Corneal Epithelial Cells" published in PLOS ONE.
OPTIMIZED ISOLATION AND QUANTIFICATION OF IN VIVO DISTRIBUTION OF EXOSOMES FOR POTENTIAL TARGETED THERANOSTIC APPLICATIONRashid, Mohammad Harunhttp://hdl.handle.net/10675.2/6237962021-01-14T19:41:15Z2019-07-01T00:00:00ZOPTIMIZED ISOLATION AND QUANTIFICATION OF IN VIVO DISTRIBUTION OF EXOSOMES FOR POTENTIAL TARGETED THERANOSTIC APPLICATION
Rashid, Mohammad Harun
Exosomes are critical mediators of intercellular crosstalk and regulators of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosome research has been thwarted by our limited knowledge of the most efficient isolation method and the in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes compared to other available methods. We demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents.
Further, we also generated engineered exosomes expressing precision peptide for targeting CD206 positive M2-macrophages. M2-macrophages participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early in vivo
detection and intervention of M2-macrophages may boost the clinical outcome. To determine in vivo distribution of M2-macrophages, we adopted 111In-oxine based radiolabeling of the targeted exosomes and SPECT. When injected these radiolabeled targeted exosomes into 4T1 breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, in the spleen, and liver. Ex vivo quantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed the adherence of exosomes to the CD206 positive macrophages on ex vivo fluorescent microscopy imaging, confirming the targeting efficacy of the exosomes. In addition, we utilized these engineered exosomes to carry the Fc portion of mouse IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages both in vitro and in vivo, and reduce tumor burden in a metastatic breast cancer model. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.
2019-07-01T00:00:00ZTHE ROLE OF GPR109A IN REGULATION OF RETINAL ANGIOGENESIS AND BLOOD-RETINAL BARRIER AS A POTENTIAL THERAPEUTIC TARGET IN DIABETIC RETINOPATHYAbdelrahman, Ammarhttp://hdl.handle.net/10675.2/6237212021-01-11T15:56:44Z2020-12-01T00:00:00ZTHE ROLE OF GPR109A IN REGULATION OF RETINAL ANGIOGENESIS AND BLOOD-RETINAL BARRIER AS A POTENTIAL THERAPEUTIC TARGET IN DIABETIC RETINOPATHY
Abdelrahman, Ammar
Currently, treatments of diabetic retinopathy (DR) have limited therapeutic benefits and limited accessibility to the growing diabetic population at risk because of the high expenses and complicated procedures. Inflammation, endothelial dysfunction, and microvascular damage are common features of diabetic complications including DR. GPR109A is the metabolite sensing receptor of beta-hydroxybutyrate (BHB) the principal ketone body in humans. Our previous studies have shown the role of GPR109A expression in promoting anti-inflammatory response in retinal pigmented epithelial (RPE) cells and the relevance of the receptor in DR. Expression of the GPR109A in microvascular endothelial cells (ECs) has been reported recently. However, the relevance of GPR109A expression and activation to retinal EC functions are yet to be studied. Our goal in this study was to identify the role of GPR109A expression and activation in barrier and angiogenic functions of retinal ECs in context of diabetic retinopathy. We used electrical cell impedance sensing (ECIS) technology to evaluate barrier functions in primary human retinal endothelial cells (HRECs) which constitute the inner BRB. Knocking down GPR109A in HRECs with siRNA decreased the transendothelial electrical resistance (TEER) compared to scrambled siRNA. Treating HRECs with BHB increased their TEER and counteracted VEGF-induced barrier disruption through activation of GPR109A and increasing zonula occludens-1 (ZO-1) expression. Treatment of STZ-diabetic mice with exogenous BHB for one month protected against the pathologic albumin leakage induced by diabetes and improved the visual acuity of this animal model of diabetes. Using the mouse model of oxygen induced retinopathy (OIR), we showed that Gpr109a-/- mice had slower vascular recovery from pathologic angiogenesis compared to age matched wild type mice. Moreover, physiologic revascularization of vaso-oblitrated retinas was impaired by loss of GPR109a and associated with dysregulated inflammatory and angiogenic signaling. Collectively, these data point to a role for GPR109A in the regulation of barrier and angiogenic mechanisms in retinal ECs and, promote the receptor as a potential druggable target for impacting these mechanisms in microvascular retinal diseases such as DR.
Record is embargoed until 12/11/2021
2020-12-01T00:00:00ZA Molecular Basis of Chemoresistance in Bladder CancerLahorewala, Sarrahhttp://hdl.handle.net/10675.2/6237032021-01-11T16:48:40Z2020-12-01T00:00:00ZA Molecular Basis of Chemoresistance in Bladder Cancer
Lahorewala, Sarrah
Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC.
Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models.
Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth.
Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.
Record is embargoed until 11/23/2030
2020-12-01T00:00:00Z