Temsirolimus Inhibits Proliferation and Migration in Retinal Pigment Epithelial and Endothelial Cells via mTOR Inhibition and Decreases VEGF and PDGF Expression Raffael Liegl*., Susanna Koenig., Jakob Siedlecki, Christos Haritoglou, Anselm Kampik, Marcus Kernt Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany Abstract Due to their high prevalence, retinal vascular diseases including age related macular degeneration (AMD), retinal vein occlusions (RVO), diabetic retinopathy (DR) and diabetic macular edema have been major therapeutic targets over the last years. The pathogenesis of these diseases is complex and yet not fully understood. However, increased proliferation, migration and angiogenesis are characteristic cellular features in almost every retinal vascular disease. The introduction of vascular endothelial growth factor (VEGF) binding intravitreal treatment strategies has led to great advances in the therapy of these diseases. While the predominant part of affected patients benefits from the specific binding of VEGF by administering an anti-VEGF antibody into the vitreous cavity, a small number of non-responders exist and alternative or additional therapeutic strategies should therefore be evaluated. The mammalian target of rapamycin (mTOR) is a central signaling pathway that eventually triggers up-regulation of cellular proliferation, migration and survival and has been identified to play a key role in angiogenesis. In the present study we were able to show that both retinal pigment epithelial (RPE) cells as wells as human umbilical vein endothelial cells (HUVEC) are inhibited in proliferating and migrating after treatment with temsirolimus in non-toxic concentrations. Previous studies suggest that the production of VEGF, platelet derived growth factor (PDGF) and other important cytokines is not only triggered by hypoxia but also by mTOR itself. Our results indicate that temsirolimus decreases VEGF and PDGF expression on RNA and protein levels significantly. We therefore believe that the mTOR inhibitor temsirolimus might be a promising drug in the future and it seems worthwhile to evaluate complementary therapeutic effects with anti-VEGF drugs for patients not profiting from mono anti-VEGF therapy alone. Citation: Liegl R, Koenig S, Siedlecki J, Haritoglou C, Kampik A, et al. (2014) Temsirolimus Inhibits Proliferation and Migration in Retinal Pigment Epithelialand Endothelial Cells via mTOR Inhibition and Decreases VEGF and PDGF Expression. PLoS ONE 9(2): e88203. doi:10.1371/journal.pone.0088203 Editor: Olaf Strauß, Eye Hospital, Charite´, Germany Received July 15, 2013; Accepted January 10, 2014; Published February 26, 2014 Copyright: ß 2014 Liegl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction besides a mechanical defect in different structures, such as the endothelium in retinal vessels and the outer blood retinal barrier Age related macular degeneration (AMD), macular edema (ME) formed by the retinal pigment epithelium and Bruchs membrane, following retinal vein occlusions (RVO) and diabetic macular a common underlying mechanism includes the increased produc- edema (DME) as a complication of diabetic retinopathy (DR) are tion of angiogenic and inflammatory components due to increased major reasons for severe vision loss and legal blindness in the hypoxic retinal conditions [8–11]. Although it is sufficiently clear, western world [1–3]. With an expected increase in patients that the three diseases are different in their development and suffering from diabetes mellitus as well as the rising mean age of pathological sequence, it has been shown, that hypoxia and the the population over the next decades even more patients will be deregulation of a large number of various growth factors such as affected in the future resulting in a tremendous socio-economic platelet derived growth factor (PDGF), placenta derived growth burden [4]. factor (PlGF), connective tissue growth factor (CTGF) and The pathogenesis of retinal vascular disease is complex and yet particularly vascular endothelial growth factor (VEGF) play a not fully understood [5,6]. However, cellular proliferation and crucial role in their etiopathologies [12–15]. Based on this vascular leakage are found in AMD, ME following RVO, as well knowledge selective intravitreal inhibition of VEGF has become as in DME, resulting in pathologic fluid accumulation in the a safe and effective primary treatment approach in the therapy of macular area. Moreover is (sub)retinal neovascularization in AMD neovascular AMD, ME following RVO, and DME, as well as in a severe complication and evidence exists, that this event is mainly several other ocular conditions that are characterized by macular triggered by proliferation and migration of endothelial and retinal edema. Today, the intravitreal injection of VEGF antibodies such pigment epithelial cells [7]. In addition, it is well known that as bevacizumab and ranibizumab or fusion proteins such as PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88203 Temsirolimus: Effect on HUVEC and RPE Cells aflibercept [16,17] have therefore become common clinical Human RPE Cell Culture practice. Large studies (e.g. RESOLVE, MARINA or VIEW) Primary RPE cells from four human donors (aged 34, 47, 51, could clearly show that a significant number of patients suffering and 59 years old, obtained 3-10 h postmortem) without any history from macular edema improved in terms of edema resolution as of eye disease were obtained from the Eye Bank of Ludwig well as visual acuity [18–20]. However, a number of patients do Maximilian University and were prepared as previously de- not improve from this treatment and for these cases alternative scribed.[34] Dulbecco modified Eagle medium (DMEM; Bio- treatment options should be investigated. chrom, Berlin, Germany) supplemented with 10% fetal calf serum Hypoxia plays a central role in the development and (FCS; Biochrom) was used as the cell culture medium. Primary progression of retinal vascular diseases. A number of studies could human RPE cells of passage 3 to 7 were used for experiments. clearly link hypoxia with several retinal diseases characterized by retinal ischemia and subsequent pathological angiogenesis involv- HUVEC Culture ing the up-regulation of VEGF and other VEGF-related Cultures of human umbilical vein endothelial cells (HUVEC) polypeptides such as PlGF [21–23]. The binding of these were purchased from Promocell (Heidelberg, Germany) and VEGF-family proteins to their receptor tyrosine kinases cultured according to the manufacturer’s instructions. Endothelial VEGFR-1, VEGFR-2 and VEGFR-3 is followed by numerous cell growth medium (ECGM; Promocell) with 5% FCS (Biochrom) downstream effects leading to proliferation, cell migration, was used as the cell culture medium. The experiments were vascular permeability and endothelial inflammation. The most conducted on HUVECs of passages 3 to 5. angiogenic effect of VEGF in-vivo is linked with its binding to VEGFR-2 [24]. One of the most important signaling pathways among several downstream of VEGFR-2 is PI3K/Akt [25,26] Treatment of Cell Cultures which subsequently activates the mammalian target of Rapamycin For methylthiotetrazole (MTT; 3-[4,5-dimethylthiazol-2-yl]- (mTOR) [27]. mTOR is a serine-threonine protein kinase that 2,5-diphenyl tetrazolium bromide) assays, cells were seeded in 6- plays an important role in signal transduction pathways that well plates. For all other cell culture experiments, HUVECs or control cell growth and angiogenesis and has been a target to RPE cells were seeded in 35-mm tissue culture dishes and cultured many cancer therapy approaches in-vitro and in-vivo [28,29]. on confluence in darkness. None of the used cell culture substrates Temsirolimus is an ester derivative of sirolimus with enhanced were coated with anything aside of the polycarbonate membrane, pharmaceutical properties including improved stability and that was coated with fibronectin and used in the boyden chamber solubility and binds to a cytoplasmatic protein. This complex assay. binds and inhibits mTOR and proved its potency in several For all experiments except proliferation and migration, before clinical and laboratory studies [30–33]. treatment, HUVECs or RPE cells were kept for 24 h in serum-free We therefore evaluated the effect of temsirolimus on cellular conditions. Then, for MTT testing regarding viability and events associated with retinal vascular diseases such as neovascular proliferation, temsirolimus was added to reach final concentrations AMD and DR on retinal pigment epithelial cells and human of 0.005, 0.05, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 mg/ umbilical vein endothelial cells as a common model for endothelial mL. cells in vision research in an in-vitro model. As a result from viability testing and additional molecular pre- testing, temsirolimus at a final concentration of 0.05 mg/mL was Methods chosen for all further experiments regarding growth factor expression. Ethics The methods of securing human tissue were humane, complied Hypoxic Stimulation