Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation.

PURPOSE: The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity. METHODS: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation. RESULTS: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis. CONCLUSIONS: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity.

Application of human persistent fetal vasculature neural progenitors for transplantation in the inner retina.

Persistent fetal vasculature (PFV) is a potentially serious developmental anomaly in human eyes, which results from a failure of the primary vitreous and the hyaloid vascular systems to regress during development. Recent findings from our laboratory indicate that fibrovascular membranes harvested from subjects with PFV contain neural progenitor cells (herein called NPPFV cells). Our studies on successful isolation, culture, and characterization of NPPFV cells have shown that they highly express neuronal progenitor markers (nestin, Pax6, and Ki67) as well as retinal neuronal markers (ß-III-tubulin and Brn3a). In the presence of retinoic acid and neurotrophins, these cells acquire a neural morphological appearance in vitro, including a round soma and extensive neurites, and express mature neuronal markers (ß-III-tubulin and NF200). Further experiments, including real-time qRT-PCR to quantify characteristic gene expression profiles of these cells and Ca(2+) imaging to evaluate the response to stimulation with different neurotransmitters, indicate that NPPFV cells may resemble a more advanced stage of retinal development and show more differentiation toward inner retinal neurons rather than photoreceptors. To explore the potential of inner retinal transplantation, NPPFV cells were transplanted intravitreally into the eyes of adult C57BL/6 mice. Engrafted NPPFV cells survived well in the intraocular environment in presence of rapamycin and some cells migrated into the inner nuclear layer of the retina 1 week posttransplantation. Three weeks after transplantation, NPPFV cells were observed to migrate and integrate in the inner retina. In response to daily intraperitoneal injections of retinoic acid, a portion of transplanted NPPFV cells exhibited retinal ganglion cell-like morphology and expressed mature neuronal markers (ß-III-tubulin and synaptophysin). These findings indicate that fibrovascular membranes from human PFV harbor a population of neuronal progenitors that may be potential candidates for cell-based therapy for degenerative diseases of the inner retina.

Interferon gamma-inducible protein-10 (IP-10) and eotaxin as biomarkers in age-related macular degeneration.

PURPOSE: To analyze serum cytokine levels in subjects with different stages of AMD and to study the expression of salient cytokines in postmortem eyes with AMD. METHODS: A suspension array system was used to analyze sera (n = 18 to 20/group) from control subjects and those with early AMD (AREDS stage 1), intermediate dry AMD (AREDS stage 3), advanced AMD with geographic atrophy (GA), or neovascular AMD (CNV). Postmortem eyes with AMD or control eyes were examined immunohistochemically for expression of IP-10 and eotaxin (n = 4 to 8/group). RESULTS: Serum eotaxin and IP-10 levels were significantly elevated in all stages of AMD, except for eotaxin levels in neovascular AMD (P < 0.07). The peak of serum IP-10 concentration was at intermediate dry AMD. In donor eyes, IP-10 and eotaxin expressions were increased in the RPE of eyes with early AMD, GA, and CNV. Eotaxin accumulated within the layer of basal linear/laminar deposits in all stages of AMD, while IP-10 was mainly in eyes with GA and CNV. IP-10 was abundant in the connective tissue matrix associated with CNV, and eotaxin was usually present but more focally and with less intense staining. Both IP-10 and eotaxin were expressed by neovascular endothelial cells. Both IP-10 and eotaxin were expressed in the neurosensory retina, but there was no detectable difference in staining between eyes with or without AMD. CONCLUSIONS: IP-10 and eotaxin may be early biomarkers in AMD. The authors hypothesize that the relative balance between levels of IP-10 and eotaxin is critical in regulating the neovascular response.