Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world’s working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31+ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5–3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.

PurposeThe 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.MethodsPVR 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.ResultsGross 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.ConclusionsPVR 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.

Purpose The human orbit is an environment that is vulnerable to inflammation and edema in the setting of autoimmune thyroid disease. Our study investigated the tenet that orbital adipose tissue lacks lymphatic vessels and analyzed the clinicopathologic differences between patients with acute and chronic thyroid eye disease (TED). The underlying molecular mediators of blood and lymphatic vessel formation within the orbital fat were also evaluated. Design Retrospective cohort study Participants The study included fat specimens from 26 orbits of 15 patients with TED undergoing orbital decompression. Orbital fat specimens from patients without TED as well as cadaveric orbital fat served as controls. Methods Tissue specimens were processed as formalin-fixed paraffin-embedded sections (FFPE) or frozen cryosections for immunohistochemistry. Total RNA was extracted and analyzed via quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR). Clinicopathological correlation was made by determining the Clinical Activity Score (CAS) of each patient with TED. Main Outcome Measures Samples were examined for vascular and lymphatic markers including podoplanin, LYVE-1, and CD31 by immunohistochemistry, as well as for mRNA levels of VEGF, VEGF receptors, SEMA-3F, NRP-1, NRP-2, podoplanin and LYVE-1 by qRT-PCR. Results Clinicopathological correlation revealed increased staining of CD31-positive blood vessels in patients with acute TED with CAS > 4, as well as rare staining of podoplanin-positive lymphatic vessels within acutely inflamed orbital fat tissue. Additionally, qRT-PCR analysis demonstrated increased expression of vascular endothelial growth factor receptor 2 (VEGFR-2) as well as VEGF signaling molecules: VEGF-A, VEGF-C, and VEGF-D. Conclusions In acute TED, compared to chronic TED and control orbital fat, there is increased blood vessel density suggesting neovascularization and rare lymphatic vessels suggestive of limited lymphangiogenesis. This pro-angiogenic and pro-lymphangiogenic microenvironment is likely due to the increased expression of VEGFR-2 and VEGF-A, VEGF-C, and VEGF-D. These findings imply that orbital edema in acute TED may be mediated, in part, by both the formation of new, immature blood vessels and the formation of lymphatic capillaries that are functionally incapable of draining interstitial fluid.