Person: Kim, Leo
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Publication Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy(The Association for Research in Vision and Ophthalmology, 2017) Amarnani, Dhanesh; Machuca-Parra, Arturo Israel; Wong, Lindsay L.; Marko, Christina K.; Stefater, James; Stryjewski, Tomasz; Eliott, Dean; Arboleda-Velasquez, Joseph F.; Kim, LeoPurpose The purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing. Methods: PVR membranes were obtained from six patients with grade C PVR. Membrane fragments were analyzed by gross evaluation, fixed for immunohistologic studies to establish cell identity, or digested with collagenase II to obtain single cell suspensions for culture. PVR-derived primary cultures were used to examine the effects of methotrexate (MTX) on proliferation, migration, and cell death. Results: Gross analysis of PVR membranes showed presence of pigmented cells, indicative of retinal pigment epithelial cells. Immunohistochemistry identified cells expressing α-smooth muscle actin, glial fibrillary acidic protein, Bestrophin-1, and F4/80, suggesting the presence of multiple cell types in PVR. Robust PVR primary cultures (C-PVR) were successfully obtained from human membranes, and these cells retained the expression of cell identity markers in culture. C-PVR cultures formed membranes and band-like structures in culture reminiscent of the human condition. MTX significantly reduced the proliferation and band formation of C-PVR, whereas it had no significant effect on cell migration. MTX also induced regulated cell death within C-PVR as assessed by increased expression of caspase-3/7. Conclusions: PVR cells obtained from human membranes can be successfully isolated, cultured, and profiled in vitro. Using these primary cultures, we identified MTX as capable of significantly reducing growth and inducing cell death of PVR cells in vitro.Publication Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL(The Rockefeller University Press, 2017) Machuca-Parra, Arturo I.; Bigger-Allen, Alex; Sanchez, Angie; Boutabla, Anissa; Cardona-Vélez, Jonathan; Amarnani, Dhanesh; Saint-Geniez, Magali; Siebel, Christian W.; Kim, Leo; D’Amore, Patricia A.; Arboleda-Velasquez, JosephCerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a neurological syndrome characterized by small vessel disease (SVD), stroke, and vascular cognitive impairment and dementia caused by mutations in NOTCH3. No therapies are available for this condition. Loss of mural cells, which encompass pericytes and vascular smooth muscle cells, is a hallmark of CADASIL and other SVDs, including diabetic retinopathy, resulting in vascular instability. Here, we showed that Notch3 signaling is both necessary and sufficient to support mural cell coverage in arteries using genetic rescue in Notch3 knockout mice. Furthermore, we show that systemic administration of an agonist Notch3 antibody prevents mural cell loss and modifies plasma proteins associated with Notch3 activity, including endostatin/collagen 18α1 and Notch3 extracellular domain in mice with the C455R mutation, a CADASIL variant associated with Notch3 loss of function. These findings open opportunities for the treatment of CADASIL and other SVDs by modulating Notch3 signaling.Publication Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy(Molecular Vision, 2015) Kim, Leo; Wong, Lindsay L.; Amarnani, Dhanesh; Bigger-Allen, Alexander A.; Hu, Yang; Marko, Christina K.; Eliott, Dean; Shah, Vinay A.; McGuone, Declan; Stemmer-Rachamimov, Anat; Gai, Xiaowu; D’Amore, Patricia A.; Arboleda-Velasquez, JosephPurpose Epiretinal fibrovascular membranes (FVMs) are a hallmark of proliferative diabetic retinopathy (PDR). Surgical removal of FVMs is often indicated to treat tractional retinal detachment. This potentially informative pathological tissue is usually disposed of after surgery without further examination. We developed a method for isolating and characterizing cells derived from FVMs and correlated their expression of specific markers in culture with that in tissue. Methods: FVMs were obtained from 11 patients with PDR during diabetic vitrectomy surgery and were analyzed with electron microscopy (EM), comparative genomic hybridization (CGH), immunohistochemistry, and/or digested with collagenase II for cell isolation and culture. Antibody arrays and enzyme-linked immunosorbent assay (ELISA) were used to profile secreted angiogenesis-related proteins in cell culture supernatants. Results: EM analysis of the FVMs showed abnormal vessels composed of endothelial cells with large nuclei and plasma membrane infoldings, loosely attached perivascular cells, and stromal cells. The cellular constituents of the FVMs lacked major chromosomal aberrations as shown with CGH. Cells derived from FVMs (C-FVMs) could be isolated and maintained in culture. The C-FVMs retained the expression of markers of cell identity in primary culture, which define specific cell populations including CD31-positive, alpha-smooth muscle actin-positive (SMA), and glial fibrillary acidic protein-positive (GFAP) cells. In primary culture, secretion of angiopoietin-1 and thrombospondin-1 was significantly decreased in culture conditions that resemble a diabetic environment in SMA-positive C-FVMs compared to human retinal pericytes derived from a non-diabetic donor. Conclusions: C-FVMs obtained from individuals with PDR can be isolated, cultured, and profiled in vitro and may constitute a unique resource for the discovery of cell signaling mechanisms underlying PDR that extends beyond current animal and cell culture models.Publication Tamoxifen Toxicity in Cultured Retinal Pigment Epithelial Cells Is Mediated by Concurrent Regulated Cell Death Mechanisms(Association for Research in Vision and Ophthalmology (ARVO), 2014) Kim, Leo; Amarnani, Dhanesh; Gnanaguru, Gopalan; Tseng, Wen Allen; Vavvas, Demetrios; D'Amore, PatriciaPurpose.: To evaluate the mechanism of tamoxifen-induced cell death in human cultured RPE cells, and to investigate concurrent cell death mechanisms including pyroptosis, apoptosis, and necroptosis. Methods.: Human RPE cells were cultured until confluence and treated with tamoxifen; cell death was measured by detecting LDH release. Tamoxifen-induced cell death was further confirmed by 7-aminoactinomycin D (7-AAD) and annexin V staining. Lysosomal destabilization was assessed using lysosomal-associated membrane protein-1 (LAMP-1) and acridine orange staining. The roles of lysosomal enzymes cathepsin B and L were examined by blocking their activity. Caspase activity was evaluated by caspase-1, -3, -8, and -9 specific inhibition. Cells were primed with IL-1α and treated with tamoxifen; mature IL-1β production was quantified via ELISA. Caspase activity was verified with the fluorochrome-labeled inhibitor of caspases (FLICA) probe specific for each caspase. Regulated cell necrosis or necroptosis was examined with 7-AAD and inhibition of receptor-interacting protein 1 (RIP1) kinase using necrostatin-1 (Nec-1). Results.: Cell death occurred within 2 hours of tamoxifen treatment of confluent RPE cells and was accompanied by lysosomal membrane permeabilization. Blockade of cathepsin B and L activity led to a significant decrease in cell death, indicating that lysosomal destabilization and cathepsin release occur prior to regulated cell death. Tamoxifen-induced toxicity was shown to occur through both caspase-dependent and caspase-independent cell death pathways. Treatment of RPE cells with caspase inhibitors and Nec-1 resulted in a near complete rescue from cell death. Conclusions.: Tamoxifen-induced cell death occurs through concurrent regulated cell death mechanisms. Simultaneous inhibition of caspase-dependent and caspase-independent cell death pathways is required to protect cells from tamoxifen. Inhibition of upstream activators, such as the cathepsins, may represent a novel approach to block multiple cell death pathways.Publication Conversion to aflibercept for diabetic macular edema unresponsive to ranibizumab or bevacizumab(Dove Medical Press, 2015) Lim, Laurence S; Ng, Wei Yan; Mathur, Ranjana; Wong, Doric; Wong, Edmund YM; Yeo, Ian; Cheung, Chui Ming Gemmy; Lee, Shu Yen; Wong, Tien Yin; Papakostas, Thanos D; Kim, LeoBackground: The purpose of this study was to determine if eyes with diabetic macular edema (DME) unresponsive to ranibizumab or bevacizumab would benefit from conversion to aflibercept. Methods: This study was conducted as a retrospective chart review of subjects with DME unresponsive to ranibizumab and/or bevacizumab and subsequently converted to aflibercept. Results: In total, 21 eyes from 19 subjects of mean age 62±15 years were included. The majority of subjects were male (63%). The median number of ranibizumab or bevacizumab injections before switching to aflibercept was six, and the median number of aflibercept injections after switching was three. Median follow-up was 5 months after the switch. Mean central foveal thickness (CFT) was 453.52±143.39 mm immediately prior to the switch. Morphologically, intraretinal cysts were present in all cases. Mean CFT after the first injection decreased significantly to 362.57±92.82 mm (Wilcoxon signed-rank test; P<0.001). At the end of follow-up, the mean CFT was 324.17±98.76 mm (P<0.001). Mean visual acuity was 0.42±0.23 logMAR just prior to the switch, 0.39±0.31 logMAR after one aflibercept injection, and 0.37±0.22 log-MAR at the end of follow-up. The final visual acuity was significantly better than visual acuity before the switch (P=0.04). Conclusion: Eyes with DME unresponsive to multiple ranibizumab/bevacizumab injections demonstrate anatomical and visual improvement on conversion to aflibercept.Publication Drainage and analysis of suprachoroidal fluid in a patient with acute systemic lupus erythematous(Elsevier, 2016) Stefater, James; Eliott, Dean; Kim, LeoPurpose To describe a case of a patient with acute systemic lupus erythematous (SLE) causing choroidal effusions and to report a novel technique for evaluation of the choroidal fluid which sheds light on effusion pathogenesis. Observations A 37 year-old woman was referred for decreased vision, eye pain and shortness of breath. The patient had bilateral angle closure glaucoma from choroidal effusions and bilateral pleural effusions. Work-up revealed new onset acute SLE. A technique for obtaining suprachoroidal fluid is described, and the fluid was analyzed using Light's criteria and found to be exudative in nature. Conclusions and importance There has been speculation as to pathogenesis of choroidal effusions in a variety of conditions, and many authors believe the most likely process to be transudative. The exudative nature of the fluid in our patient suggests that choroidal effusions in acute SLE are likely caused by inflammation, and not secondary to hypoalbuminemia or another transudative process. Similar analyses of suprachoroidal fluid in other disease processes may help elucidate the underlying pathogenesis and may possibly guide treatment.