Molecular regulation of vascular endothelial growth factor expression in the retinal pigment epithelium

Purpose: Vascular endothelial growth factor (VEGF) plays an important role in homeostasis and diseases of the retinal pigment epithelium (RPE), choriocapillaris, and, most notably, age-related macular degeneration (AMD). Although much is known about VEGF regulation in pathologies, little is known about the control of VEGF expression under normal conditions. VEGF expression has been previously shown to be regulated in coordination with cell differentiation in the muscle and kidney. We therefore tested the hypothesis that VEGF in the adult RPE would similarly be regulated in conjunction with differentiation. Methods: A human retinal pigment epithelium cell line (ARPE-19), a line of immortalized human RPE cells, was used for all experiments. RPE cells were polarized in culture for 4 weeks on laminin-coated Transwells. Levels of VEGF mRNA and protein were determined with real-time PCR and enzyme-linked immunosorbent assay, respectively. VEGF-luciferase reporter constructs were used to identify regions of the VEGF promoter that control VEGF expression in the RPE. Microphthalmia-associated transcription factor (MITF)-Tfe transcription factors were blocked using either a pan MITF-Tfe dominant negative or specific small interfering RNA (siRNA). Results: VEGF mRNA and protein secretion increased over time in the RPE cells cultured on Transwells, with protein secretion occurring in a polarized fashion primarily toward the basolateral side. Overexpression of a dominant negative that targets the MITF-Tfe family resulted in a 50% reduction in VEGF expression. The role of the MITF-Tfe family in VEGF regulation in the RPE was corroborated in studies with the VEGF-luciferase reporter constructs, where deletion of the distal VEGF promoter region containing putative binding sites for the MITF-Tfe family resulted in a 50% reduction in VEGF promoter activity. siRNA knockdown of the MITF-Tfe family individually, and in combination, revealed that downregulation of Tfe3 resulted in reduced VEGF expression. Conclusions: Our results indicate that Tfe3, in conjunction with other MITF-Tfe members, regulates VEGF expression in the RPE and are consistent with the hypothesis that VEGF expression in RPE cells is regulated as part of their differentiation

Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy

Purpose 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

Getting Tie(2)d up in angiogenesis

Angiogenesis, the growth of new vessels from pre-existing blood vessels, requires complex signaling pathways and a high degree of spatial and temporal coordination among various cell types, multiple pro- and anti-angiogenic factors, and their corresponding receptors. Although the targeted disruption of many of these factors leads to embryonic lethal defects in vascular development, the precise actions of these factors at the cellular and molecular levels are not completely defined. Such is the case for angiopoietin-1 (Ang1), angiopoietin-2 (Ang2) and the Ang receptor Tie2. Mice deficient in Ang1, Ang2, or Tie2 exhibit aberrant vascular development, characterized by abnormal interactions between endothelial cells (ECs) and their supporting cells. The abluminal cells of the vasculature, which includes pericytes in the microvasculature and smooth muscle cells (SMCs) in large vessels, are collectively referred to as mural cells (reviewed in ref. 1). In this issue of the JCI, Uemura and colleagues provide some insight into the mechanisms that underlie these defects (2). The authors demonstrate that whereas blocking the function of the PDGF β receptor (PDGFR-β) in the developing retinal vasculature led to mural cell-deficient vessels that were poorly remodeled and leaky, administration of recombinant modified angiopoietin-1 (Ang1*) restored the vascular structure of the larger vessels in the absence of the mural cells

IGF2: Epigenetic regulation and role in development and disease

Na razini Europske unije prepoznata je važnost ulaganja u obrazovanje mladih kako bi se olakšao prijelaz iz obrazovanja u svijet rada. Provedbom metode klaster analize nad zaposlenim mladim stanovništvom Europske unije, s različitim razinama obrazovanja, ostvarili su se ciljevi ovog rada. Dobiveni podaci grupirani su u četiri različita klastera, za sve tri provedene analize, te su rezultati grafički prikazani. U klasteru A nalaze se zemlje s najvišom razinom zaposlenosti dok se u D klasteru nalaze one zemlje koje najviše zaostaju sa stopom zaposlenosti mladih na europskoj razini. Mladi s visokim stupnjem obrazovanja ujedno su i u najvećem postotku zaposleni. Također, rezultati analize pokazali su da su muškarci više zaposleni od žena obzirom na razine obrazovanja u sve tri kategorije. Usklađenost obrazovnog sustava i ponude poslova na tržištu rada važan su faktor za povećanje zaposlenosti mladih.At European union level, the importance of investing in youth education to facilitate the transition from education to the labor market has been recognized. By implementing the cluster analysis method on the employed young population of the European Union with different levels of education, the goals of this paper have been achieved. The obtained data were grouped into four different clusters for all three performed analyzes and the results were graphically presented. Cluster A contains the countries with the highest level of employment, while cluster D contains those countries that lag the most behind the youth employment rate at the european level. Young people with a high level of education are at the same time employed in the largest percentage. Also, the results of the analysis showed that men are more employed than women considering all three levels of education. The harmonization of the education system and the supply of jobs on the labour market are an important factor in increasing youth employment