Retinal Pigment Epithelium Signal Research Articles

RGX-019-MMAE, a novel MERTK-targeting antibody-drug conjugate (ADC) with robust anti-tumor efficacy in both solid and liquid tumors.

e15103 Background: MERTK, a member of the TAM family of receptor tyrosine kinases, is overexpressed in numerous solid and hematologic cancers. MERTK expression has been implicated in promoting cellular proliferation, survival and drug resistance. MERTK is also expressed on innate immune cells, predominantly on immune-suppressive M2 macrophages, where it mediates phagocytosis of apoptotic cells and regulates expression of immunosuppressive cytokines. Development of MERTK inhibitors has been hampered by observance of retinal degeneration due to inhibition of MERTK signaling in retinal pigment epithelium (RPE). We previously reported that our monoclonal antibody RGX-019 triggers transient activation of MERTK, followed by MERTK receptor internalization and degradation. RGX-019 also reduces colony formation of MERTK-expressing cancer cells. Methods: RGX-019-MMAE is a humanized IgG1-MMAE ADC with high specificity and affinity for human and cynomolgus monkey MERTK that induces efficient internalization of MERTK. These characteristics and the limited expression of MERTK in normal tissue make RGX-019 an ideal candidate for an ADC approach. In addition, MMAE selectively targets actively dividing cells such as tumor cells, while sparing macrophages and RPE, both of which represent terminally-differentiated non-dividing cells. Results: We have observed various degrees of MERTK expression on cancer cells within solid tumors and on primary AML samples. In AML, significantly higher expression was noted in M5 subtype, those with PTPN11 mutation or t(9;11) translocation and higher expression correlated with significantly inferior survival. In contrast, in normal tissue, MERTK expression was almost exclusively limited to macrophages, with virtually no expression on healthy epithelial tissue, except for the retina. Twenty-eight-day treatment with RGX-019-MMAE caused no retinal degeneration in a transgenic mouse model expressing a human version of MERTK. RGX-019-MMAE induced cytotoxicity in MERTK-expressing cancer cells and in xenografts, RGX-019-MMAE demonstrated anti-tumor efficacy including complete tumor regressions in various solid cancers such as TNBC as well as AML and MM. Anti-tumor efficacy was associated with increased overall survival. Notably, even tumors with low and heterogenous MERTK expression responded to RGX-019-MMAE treatment. In vitro studies demonstrated that RGX-019-MMAE can kill MERTK non-expressing cancer cells through bystander effects when co-cultured with MERTK-positive cells, consistent with the activity we observed in MERTK heterogenous tumors. Conclusions: Collectively, RGX-019-MMAE’s robust anti-tumor efficacy in multiple aggressive tumors, and MERTK’s expression profile on cancer cells and normal tissue support further advancement of RGX-019-MMAE as a drug candidate for development in solid and liquid cancers.

Nano-Graphene Oxide-Promoted Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells through Regulation of Phospholipase D Signaling.

Nano-graphene oxide (Nano-GO) is an extensively studied multifunctional carbon nanomaterial with attractive applications in biomedicine and biotechnology. However, few studies have been conducted to assess the epithelial-to-mesenchymal transition (EMT) in the retinal pigment epithelium (RPE). We aimed to determine whether Nano-GO induces EMT by regulating phospholipase D (PLD) signaling in human RPE (ARPE-19) cells. The physicochemical characterization of Nano-GO was performed using a Zetasizer, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. RPE cell viability assays were performed, and the migratory effects of RPE cells were evaluated. RPE cell collagen gel contraction was also determined. Intracellular reactive oxygen species (ROS) levels were determined by fluorescence microscopy and flow cytometry. Immunofluorescence staining and western blot analysis were used to detect EMT-related protein expression. Phospholipase D (PLD) enzymatic activities were also measured. Nano-GO significantly enhanced the scratch-healing ability of RPE cells, indicating that the RPE cell migration ability was increased. Following Nano-GO treatment, the RPE cell penetration of the chamber was significantly promoted, suggesting that the migratory ability was strengthened. We also observed collagen gel contraction and the generation of intracellular ROS in RPE cells. The results showed that Nano-GO induced collagen gel contraction and intracellular ROS production in RPE cells. Moreover, immunofluorescence staining and western blot analysis revealed that Nano-GO significantly regulated key molecules of EMT, including epithelial-cadherin, neural-cadherin, α-smooth muscle actin, vimentin, and matrix metalloproteinases (MMP-2 and MMP-9). Interestingly, Nano-GO-induced RPE cell migration and intracellular ROS production were abrogated in PLD-knockdown RPE cells, indicating that PLD activation played a crucial role in the Nano-GO-induced RPE EMT process. We demonstrate for the first time that Nano-GO promotes RPE cell migration through PLD-mediated ROS production. We provide preliminary evidence to support the hypothesis that Nano-GO has adverse health effects related to RPE damage.

Photoreceptor outer segment layer contributes to optical coherence tomography signal attenuation beneath neurosensory detachments.

To establish the factors responsible for attenuation of the optical coherence tomography (OCT) signal beneath the neurosensory detachment (NSD). We retrospectively reviewed 33 eyes with acute central serous chorioretinopathy. The thickness of the neurosensory retina, the thickness of the photoreceptor outer segment (PROS) layer, the height of the NSD, and the reflectivity of the underlying retinal pigment epithelium (RPE) were measured at selected points of B-scans exported from 6 × 6 mm OCT angiography protocols. The intensity of the flow signal was measured at the corresponding regions of the choriocapillaris slab. The correlation between the parameters of the NSD and both the reflectivity of underlying RPE and the intensity of the flow signal in the choriocapillaris was calculated. Correlation coefficients between RPE reflectivity and neurosensory retinal thickness, PROS layer thickness, and NSD height were -0.32, -0.64, and -0.25, respectively (p < 0.001). Correlation coefficients between the intensity of the flow signal and neurosensory retinal thickness, PROS layer thickness, and NSD height were -0.24 (p = 0.004), -0.52 (p < 0.001), and 0.13 (p = 0.13), respectively. The thickness of the PROS layer is the most significant factor affecting OCT signal at the level of RPE and OCT angiography flow signal in the choriocapillaris beneath the NSD.

Effects of trans-resveratrol on type 1 diabetes-induced up-regulation of apoptosis and mitogen-activated protein kinase signaling in retinal pigment epithelium of Dark Agouti rats

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins.

Atlas of Human Retinal Pigment Epithelium Organelles Significant for Clinical Imaging.

PurposeTo quantify organelles impacting imaging in the cell body and intact apical processes of human retinal pigment epithelium (RPE), including melanosomes, lipofuscin–melanolipofuscin (LM), mitochondria, and nuclei.MethodsA normal perifovea of a 21-year-old white male was preserved after rapid organ recovery. An aligned image stack was generated using serial block-face scanning electron microscopy and was annotated by expert readers (TrakEM, ImageJ). Acquired measures included cell body and nuclear volume (n = 17); organelle count in apical processes (n = 17) and cell bodies (n = 8); distance of cell body organelles along a normalized apical–basal axis (n = 8); and dimensions of organelle-bounding boxes in apical processes in selected subsamples of cell bodies and apical processes.ResultsIn 2661 sections through 17 cells, apical processes contained 65 ± 24 melanosomes in mononucleate (n = 15) and 131 ± 28 in binucleate cells (n = 2). Cell bodies contained 681 ± 153 LM and 734 ± 170 mitochondria. LM was excluded from the basal quartile, and mitochondria from the apical quartile. Lengths of melanosomes, LM, and mitochondria, respectively were 2305 ± 528, 1320 ± 574, and 1195 ± 294 nm. The ratio of cell body to nucleus volume was 4.6 ± 0.4. LM and mitochondria covered 75% and 63%, respectively, of the retinal imaging plane.ConclusionsAmong RPE signal sources for optical coherence tomography, LM and mitochondria are the most numerous reflective cell body organelles. These and our published data show that most melanosomes are in apical processes. Overlapping LM and previously mitochondria cushions may support multiple reflective bands in cell bodies. This atlas of subcellular reflectivity sources can inform development of advanced optical coherence tomography technologies.

Inhibition of thyroid hormone signaling protects retinal pigment epithelium and photoreceptors from cell death in a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Dry AMD is characterized by a progressive macular degeneration of the retinal pigment epithelium (RPE) and photoreceptors, and the RPE oxidative damage/dystrophy is at the core of the disease. Recent population/patients-based studies have shown an association of high free serum thyroid hormone (TH) levels with increased risk of AMD. This work investigated the effects of TH signaling inhibition on RPE and photoreceptor damage/cell death in an oxidative stress-induced mouse model of AMD. TH signaling inhibition was achieved by anti-thyroid drug treatment and oxidative stress was induced by sodium iodate (NaIO3) administration. Mice treated with NaIO3 showed severe RPE and photoreceptor cell death/necroptosis, destruction, oxidative damage, retinal stress, and reduced retinal function. Treatment with anti-thyroid drug protected RPE and photoreceptors from damage/cell death induced by NaIO3, reduced oxidative damage of RPE and photoreceptors, and preserved retinal function. Gene expression analysis showed that the NaIO3-induced RPE/photoreceptor damage/cell death involves multiple mechanisms, including cellular oxidative stress responses, activation of necroptosis/apoptosis signaling, and inflammatory responses. Treatment with anti-thyroid drug abolished these cellular stress/death responses. The findings of this study demonstrate a role of TH signaling in RPE and photoreceptor cell death after oxidative stress challenge, and support a role of TH signaling in the pathogenesis of AMD.

Inhibiting microRNA-144 potentiates Nrf2-dependent antioxidant signaling in RPE and protects against oxidative stress-induced outer retinal degeneration

The retinal pigment epithelium (RPE) is consistently exposed to high levels of pro-oxidant and inflammatory stimuli. As such, under normal conditions the antioxidant machinery in the RPE cell is one of the most efficient in the entire body. However, antioxidant defense mechanisms are often impacted negatively by the process of aging and/or degenerative disease leaving RPE susceptible to damage which contributes to retinal dysfunction. Thus, understanding better the mechanisms governing antioxidant responses in RPE is critically important. Here, we evaluated the role of the redox sensitive microRNA miR-144 in regulation of antioxidant signaling in human and mouse RPE. In cultured human RPE, miR-144-3p and miR-144-5p expression was upregulated in response to pro-oxidant stimuli. Likewise, overexpression of miR-144-3p and -5p using targeted miR mimics was associated with reduced expression of Nrf2 and downstream antioxidant target genes (NQO1 and GCLC), reduced levels of glutathione and increased RPE cell death. Alternately, some protection was conferred against the above when miR-144-3p and miR-144-5p expression was suppressed using antagomirs. Expression analyses revealed a higher conservation of miR-144-3p expression across species and additionally, the presence of two potential Nrf2 binding sites in the 3p sequence compared to only one in the 5p sequence. Thus, we evaluated the impact of miR-144-3p expression in the retinas of mice in which a robust pro-oxidant environment was generated using sodium iodate (SI). Subretinal injection of miR-144-3p antagomir in SI mice preserved retinal integrity and function, decreased oxidative stress, limited apoptosis and enhanced antioxidant gene expression. Collectively, the present work establishes miR-144 as a potential target for preventing and treating degenerative retinal diseases in which oxidative stress is paramount and RPE is prominently affected (e.g., age-related macular degeneration and diabetic retinopathy).

Effects of Resveratrol on diabetes‐induced up‐regulation of apoptosis and MAPK signaling in retinal pigment epithelium of dark Agouti rats

Although diabetic retinopathy (DRT) is a treacherous outcome of uncontrolled hyperglycemia, the mechanisms which initiate the onset of DRT are not yet clear. The current study investigated modulatory effects of Resveratrol on type 1 diabetes‐induced changes in apoptosis and mitogen‐activated protein kinase (MAPK) signaling in retinal pigmented epithelium (RPE) of dark Agouti rats. Adult male rats (12–14 weeks, n=15) were segregated in duplicates into normal control, Resveratrol‐treated, streptozotocin‐induced diabetic, and Resveratrol‐treated (5mg/kg/d, from the day of confirmation of diabetes to the sample collection day) diabetic groups. The RPE was collected on days 30 and 90. For the latter sampling time, another Resveratrol‐treated (from 45d after the confirmation of diabetes to sampling day 90) diabetic group was included to investigate the effects of different treating protocol of Resveratrol. Resveratrol when administered to normal rats, increased gene expressions (RT‐PCR analysis) of caspases‐3, 8and 9, Bax, Bcl2,p38MAPK, JNK, and p53on 30d (P&lt;0.05). On the other hand, diabetes decreased gene expressions of caspase‐3and 8, Erk, p38MAPK, and JNK(P&lt;0.05). Resveratrol reversed the inhibited gene expressions of caspase‐8,Erk, JNKand p38MAPK to normal control levels in diabetic rats (P&lt;0.05). Resveratrol normalized diabetes‐induced upregulation of proteins (Western blotting analysis) caspase‐3 and 9, cytochrome‐c, Bcl2 and ERK (P&lt;0.05) on 30d. On 90d, Resveratrol recovered diabetes‐induced decreases in caspases‐3 and 8 (18 kDa fragment), cytochrome‐c, and Bcl2 proteins to the control level. Similar effects of Resveratrol were observed when given to diabetic rats only for 45d instead of 90d. In conclusion, Resveratrol when given to normal rats upregulates transcription of apoptosis and MAPK genes, however, their protein levels do not increase suggesting the lack of induction of apoptosis and MAPK‐mediated cell signaling. Resveratrol imparts its protective effects by normalizing apoptosis and MAPK signaling. These results are suggestive of beneficial effects of Resveratrol on the RPE in diabetic rats.Support or Funding InformationCollege of Graduate studies and Research sector, Kuwait UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RS9, an Nrf2 activator, accelerates LC3-assosciated phagocytosis via dual activation of AMPK/mTOR and Nrf2/SQSTM1 signaling in retinal pigment epithelium

RS9, an Nrf2 activator, accelerates LC3-assosciated phagocytosis via dual activation of AMPK/mTOR and Nrf2/SQSTM1 signaling in retinal pigment epithelium

Disruption of outer blood-retinal barrier by Toxoplasma gondii-infected monocytes is mediated by paracrinely activated FAK signaling.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

Torpedo maculopathy: Two case reports and a literature review

Case reportsThe cases concern a 4 year-old boy and 25 year-old female with 20/20 visual acuity, who presented with a unilateral non-pigmented macular lesion, temporal to the fovea, a torpedo shaped defect in the retinal pigment epithelium (RPE). Optical coherence tomography showed attenuation of the RPE signal, and in the second patient there proved to be a neurosensory detachment, RPE atrophy, and thinning of the retinal layers. The lesion was hypoautofluorescent and hyperfluorescent on fluorescein angiography. DiscussionTorpedo maculopathy is an asymptomatic characteristic lesion which should be considered in the differential diagnosis of macular lesions in children and young patients.

Maculopatía en torpedo: presentación de 2 casos clínicos y revisión de la literatura

Case reportsThe cases concern a 4 year-old boy and 25 year-old female with 20/20 visual acuity, who presented with a unilateral non-pigmented macular lesion, temporal to the fovea, a torpedo shaped defect in the retinal pigment epithelium (RPE). Optical coherence tomography showed attenuation of the RPE signal, and in the second patient there proved to be a neurosensory detachment, RPE atrophy, and thinning of the retinal layers. The lesion was hypoautofluorescent and hyperfluorescent on fluorescein angiography. DiscussionTorpedo maculopathy is an asymptomatic characteristic lesion which should be considered in the differential diagnosis of macular lesions in children and young patients.

Automated Analysis of Vitreous Inflammation Using Spectral-Domain Optical Coherence Tomography.

To develop an automated method for quantifying vitreous signal intensity on optical coherence tomography (OCT), with particular application for use in the assessment of vitreous inflammation. This retrospective, observational case-control series comprised 30 patients (30 eyes), with vitreous haze secondary to intermediate, posterior, or panuveitis; 12 patients (12 eyes) with uveitis without evidence of vitreous haze; and 18 patients (18 eyes) without intraocular inflammation or vitreoretinal disease. The presence and severity of vitreous haze was classified according to the National Eye Institute system; other inflammatory indices and clinical parameters were also documented. Spectral-domain OCT images were analyzed using custom VITreous ANalysis software (termed 'VITAN'), which is fully automated and avoids the need for manual segmentation. VITAN performed accurate segmentation in all scans. Automated measurements of the vitreous:retinal pigment epithelium (RPE) signal ratio showed a moderate correlation with clinical vitreous haze scores (r = 0.585, P < 0.001), comparable to that reported using manual segmentation in our previous study (r = 0.566, P = 0.0001). The novel parameter of vitreous:RPE textural ratio showed a marginally stronger correlation (r = 0.604, P < 0.001) with clinical vitreous haze scores than the Vitreous:RPE signal ratio. The custom OCT image analysis software (VITAN) allows rapid and automated measurement of vitreous parameters, that is comparable to our previously reported vitreous:RPE index, and correlates with clinically measured disease activity. Such OCT-based indices may provide the much needed objective markers of vitreous activity, which may be used in both clinical assessment, and as outcome measures in clinical trials for intermediate, posterior, and panuveitis. We describe a rapid automated method for quantifying vitreous signal intensity on optical coherence tomography (OCT) and show that this correlates with clinical assessment of vitreous inflammation. Such OCT-based indices may provide the much needed objective markers of vitreous activity, which may be used both in routine clinical assessment, and as outcome measures in clinical trials for intermediate, posterior, and panuveitis.

Delayed near-infrared analysis permits visualization of rodent retinal pigment epithelium layer in vivo.

Patches of atrophy of the retinal pigment epithelium (RPE) have not been described in rodent models of retinal degeneration, as they have the clinical setting using fundus autofluorescence. We hypothesize that prelabeling the RPE would increase contrast and allow for improved visualization of RPE loss in vivo. Here, we demonstrate a new technique termed “delayed near-infrared analysis (DNIRA)” that permits ready detection of rat RPE, using optical imaging in the near-infrared (IR) spectrum with aid of indocyanine green (ICG) dye. Using DNIRA, we demonstrate a fluorescent RPE signal that is detected using confocal scanning laser ophthalmoscopy up to 28 days following ICG injection. This signal is apparent only after ICG injection, is dose dependent, requires the presence of the ICG filters (795/810 nm excitation/emission), does not appear in the IR reflectance channel, and is eliminated in the presence of sodium iodate, a toxin that causes RPE loss. Rat RPE explants confirm internalization of ICG dye. Together with normal retinal electrophysiology, these findings demonstrate that DNIRA is a new and safe noninvasive optical imaging technique for in vivo visualization of the RPE in models of retinal disease.

Epithelial Ca2+ Model Prediction: Suramin Enhances P2Y2 Receptor Desensitization by Accelerating their Phosphorylation Rate

Ca2+ signaling is essential to most biological functions. To understand Ca2+ signaling in retinal pigment epithelium (RPE), we constructed a computational model of Ca2+ wave propagation in ARPE-19 cells based on Ca2+ imaging measurements after mechanical stimulation. We exploited the extensive experimental material from Abu Khamidakh et al. 2013 Exp Eye Res. The model relied on the assumption that cells experience different conditions depending on their location with respect to the stimulation site. In this context, the model was used to simulate the measured Ca2+ waves and predict the drug effects only by changing the appropriate environmental parameters or drug target components.Our model reproduced the measured Ca2+ dynamics in control cells and in cells after α-glycyrrhetinic acid treatment. As an example of model usage, the model was applied to study suramin inhibition mechanisms in P2Y2 receptors. The model suggested that the suramin inhibition of Ca2+ signal propagation may be related to increased P2Y2 receptor phosphorylation rate: the higher phosphorylation rate causes faster desensitization of the receptors after ligand binding shortening the duration of the Ca2+ elevation. The modeling results showed that a relatively larger inhibition occurs with higher Ca2+ waves near the site of mechanical stimulation.In summary, the model predicted suramin drug effects on P2Y2 receptors suggesting that suramin enhances their desensitization by accelerating the phosphorylation rate. This intriguing modeling result requires further investigation and needs to be confirmed experimentally for a deeper understanding of the biological mechanisms related to the phenomenon. In general, to date, this is the first mathematical model of Ca2+ signaling in ARPE-19 cells and one of the first in general to model epithelial Ca2+ dynamics presenting also predictions about the drug modified parameters.

Phosphorylation/inactivation of PTEN by Akt-independent PI3K signaling in retinal pigment epithelium

Retinal pigment epithelium (RPE) plays a critical role in vertebrate vision by providing functional and structural support to the retina. Degeneration of RPE by cumulative oxidative stresses or acute injury frequently results in retinal degenerative diseases, notably age-related macular degeneration (AMD). Moreover, it has been shown that phosphorylation-mediated inactivation of PTEN (phosphatase and tensin homolog) in RPE is closely linked to AMD-like retinal degeneration in mice [1]. In this study, we used AMD mouse models, in which chemokine (C–C motif) ligand 2 (Ccl2) or chemokine (C–C motif) receptor 2 (Ccr2) were genetically ablated, to examine mechanisms linking reactive oxygen species (ROS) to phosphorylation/inactivation of PTEN in RPE. We found that ROS levels were increased in these RPE cells in association with phosphorylation/inactivation of PTEN. Both PTEN phosphorylation/inactivation and consequent Akt activation in the RPE of AMD model mice were inhibited by antioxidant treatment, indicating a functional role for elevated intracellular ROS. We further discovered that PTEN phosphorylation in oxidatively stressed RPE was repressed by a phosphoinositide 3-kinase (PI3K) inhibitor, but not by an Akt inhibitor. Taken together, these results suggest that ROS-activated PI3K potentiates AMD-related RPE pathogenesis through phosphorylation/inactivation of PTEN.

Modulation of Migration and Ca2+ Signaling in Retinal Pigment Epithelium Cells by Recombinant Human CTGF

Purpose: The migration of retinal pigment epithelium (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). We investigated the expression of connective tissue growth factor (CTGF) in an in vitro model of wound healing and effects of recombinant human CTGF (rhCTGF) on modulating migration and Ca2+ signaling in RPE cells.Methods: Cultured human RPE monolayers were used to establish a wound-healing model. Western blot and in situ hybridization were used to detect the CTGF expression in RPE cells. Migration of RPE cells was measured under the stimulation of rhCTGF alone or in combination with dexamethasone (DEX) or 8-Br-cAMP. To determine the concentration of cytoplasmic-free Ca2+ ([Ca2+]i) responding to CTGF, the fluo-3/AM-loaded RPE cells were observed with a laser scanning confocal microscope.Results: The CTGF expression first increased after being wounded in RPE cells, then reached a peak and maintained at a high level. The positive expression was mainly at the edge of scrape and in motile RPE cells. rhCTGF-stimulated RPE cells migrated in a dose-dependent manner, and both DEX and 8-Br-cAMP could significantly inhibit the CTGF-induced migrations. CTGF induced a (Ca2+)i elevation in RPE cells in a concentration-dependent manner. Moreover, stimulation of RPE cells with CTGF and DEX or 8-Br-cAMP counteracted the elevation of (Ca2+)i induced by CTGF.Conclusions: The CTGF expression could be induced by an in vitro model of scrape wounding. rhCTGF stimulated the migration and Ca2+ signal pathway in RPE cells in a dose-dependent manner, and DEX and 8-Br-cAMP suppressed this effect. Our results indicate that CTGF is involved in the wound-healing process and plays an important role in the pathogenesis of intraocular proliferative diseases.