Chandrasekharam N. Nagineni

Transforming growth factor‐β induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: Involvement of mitogen‐activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age‐related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT‐PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF‐β1. TGF‐β1, β2, and β3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF‐α, and GM‐CSF had no effects. TGF‐β receptor type II antibody significantly reversed induction of VEGF secretion by TGF‐β. In contrast activin, inhibin and BMP, members of TGF‐β super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF‐β were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF‐κB pathway inhibitors, respectively. TGF‐β also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF‐β induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD. Published 2003 Wiley‐Liss, Inc. J. Cell. Physiol. 197: 453–462, 2003© 2003 Wiley‐Liss, Inc.

Inflammatory Cytokines Regulate MicroRNA-155 Expression in Human Retinal Pigment Epithelial Cells by Activating JAK/STAT Pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Toxoplasma gondii Infection Induces Gene Expression and Secretion of Interleukin 1 (IL-1), IL-6, Granulocyte-Macrophage Colony-Stimulating Factor, and Intercellular Adhesion Molecule 1 by Human Retinal Pigment Epithelial Cells

ABSTRACT We have used human retinal pigment epithelial (HRPE) cultures to investigate the primary cellular responses of retinal resident cells to intracellular Toxoplasma gondii replication. At 4 days postinoculation, when all of the cells were infected, the secretion of interleukin 1β (IL-1β), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular adhesion molecule 1 (ICAM-1) was augmented by 23-, 10-, 8-, and 5-fold, respectively, over the control. Northern and reverse transcriptase PCR analyses showed significant upregulation of steady-state levels of mRNA for IL-1β, IL-6, GM-CSF, and ICAM-1. The secretion of these molecules by HRPE cells may play a critical immunoregulatory role in the pathophysiological processes associated with T. gondii-induced retinochoroiditis.

Regulation of VEGF expression in human retinal cells by cytokines: Implications for the role of inflammation in age‐related macular degeneration

Chronic inflammation is implicated in the pathogenesis of age‐related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN‐γ + TNF‐α + IL‐1β) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10‐ and 9‐fold increase in VEGF‐A and VEGF‐C expression, respectively. The microarray results were validated by quantitative RT‐PCR and secretion of VEGFs proteins. IL‐1β is the most potent in inducing VEGFs secretion followed by IFN‐γ and TNF‐α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF‐κB and JAK‐STAT pathway, but not HIF‐1α, Sp‐1, Sp‐3, and STAT‐3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF‐A and VEGF‐C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF‐1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF‐A and VEGF‐C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD. J. Cell. Physiol. 227: 116–126, 2012.

Expression of PDGF and their receptors in human retinal pigment epithelial cells and fibroblasts: Regulation by TGF-β†

Platelet derived growth factors (PDGF) are known to be associated with vitreoretinal disorders such as proliferative vitreoretinopathy (PVR). We have studied the expression of PDGF and their receptors in human retinal pigment epithelial cells (HRPE) and choroid fibroblasts (HCHF), and the regulation of PDGF and its receptors by various cytokines and growth factors. RT‐PCR analyses showed enhanced expression of PDGF‐A and PDGF‐B mRNA in HRPE treated with TGF‐β, but not with other cytokines. A minimal increase was observed in PDGF‐A mRNA in TGF‐β treated HCHF cells. PDGF‐Rα mRNA, which was expressed prominently in HCHF and at very low levels in HRPE, was not affected by any of the agents. PDGF‐Rβ was not detectable in either HRPE or HCHF. HRPE secreted PDGF‐AA and AB constitutively, and this secretion was significantly enhanced by TGF‐β. In contrast, HCHF cultures did not secrete detectable levels of any of the three isoforms of PDGF (AA, AB, BB). All three human recombinant PDGF isoforms enhanced HCHF cell proliferation significantly, while only a minimal increase was observed in HRPE. PDGF isoforms also induced HCHF cell elongation and promoted migration of HCHF in an in vitro wound assay. The results presented in this study demonstrate that TGF‐β activated RPE cells produce PDGF that may act on fibroblasts and other mesenchyme derived cells which express PDGF receptors. These studies indicate that the promotion of the proliferation and migration of mesenchymal cells by RPE cell derived PDGF may facilitate the formation of fibrovascular tissues associated with PVR.

Transforming growth factor-β expression in human retinal pigment epithelial cells is enhanced by Toxoplasma gondii: a possible role in the immunopathogenesis of retinochoroiditis

Retinochoroiditis caused by Toxoplasma gondii infection results in inflammation and necrosis of the retina. We have used human retinal pigment epithelial cultures (HRPE) as an in vitro model to investigate the role of TGF‐β in T. gondii‐induced retinochoroiditis. RT‐PCR analyses showed enhanced steady state levels of TGF‐β1 and TGF‐β2 mRNA in T. gondii‐infected HRPE. Uninfected HRPE secrete TGF‐β1 in a latent form while 10–30% of the secreted TGF‐β2 was in the active form. T. gondii infection induced a significant increase (P < 0·01) in total TGF‐β1 and TGF‐β2 secretion by HRPE. In addition, soluble extracts of T. gondii (ST) stimulated secretion of both TGF‐β1 and TGF‐β2 significantly (P < 0·01). Interestingly, T. gondii infection as well as ST of the parasites completely inhibited secretion of the active form of TGF‐β2. Studies evaluating the effect of TGF‐β on T. gondii replication in HRPE revealed that TGF‐β enhanced parasite replication. The interactions between host retinal cells and T. gondii may play an active role in the pathogenesis of retinochoroiditis.

Inhibition of inflammatory cytokine production in human corneal cells by dexamethasone, but not cyclosporin.

Purpose: To evaluate the modulatory effects of anti-inflammatory agents, dexamethasone (Dex) and cyclosporin A (CsA), on the production of cytokines and chemokines by human corneal cells in vitro following stimulation by the pro-inflammatory cytokine after interleukin 1β (IL-1β). Methods: A human corneal epithelial (HCE) cell line and human corneal fibroblasts (HCFs) were stimulated in culture with IL-1β and treated with Dex or CsA. The gene expression for selected cytokines and chemokines was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The secretion of cytokines and chemokines was measured by enzyme-linked immunosorbent assay. Results: IL-1β enhanced the mRNA and/or protein levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, and monocyte chemotactic protein (MCP)-1 in HCE and IL-6, IL-8, MCP-3, and regulated on T-cell activation expressed secreted (RANTES) in HCFs. Treatment with CsA did not inhibit cytokine production in either HCE or HCFs. In contrast, Dex treatment inhibited the IL-1β-induced production of GM-CSF, IL-6, IL-8, MCP-3, and RANTES, but not MCP-1. Conclusion: These results show that Dex, but not CsA, has direct immunosuppressive effects on the resident corneal cells, HCE and HCFs. This suggests that the clinically observed immunosuppressive effects of topical CsA are mediated primarily through the immune cells.

Interferon-γ differentially regulates TGF-β1 and TGF-β2 expression in human retinal pigment epithelial cells through JAK-STAT pathway

Retinal pigment epithelium (RPE) and transforming growth factor‐β (TGF‐β) have been shown to be involved in various retinal diseases. We have studied the role of inflammatory cytokines on the expression and secretion of TGF‐β in human RPE cells (HRPE). Confluent cultures of HRPE derived from donor eyes were used. RT‐PCR analyses showed that TNF‐α and IL‐1β increased the mRNA levels of both TGF‐β1 and TGF‐β2. IFN‐γ enhanced constitutively expressed, as well as, TNF‐α‐and IL‐1β‐induced TGF‐β1 mRNA levels but decreased TGF‐β2 mRNA. The effects of these cytokines on TGF‐β1 and TGF‐β2 secretion correlated with the mRNA levels. TGF‐β1 was always produced as the latent form while 21–31% of TGF‐β2 was in the active form. IFN‐γ reduced the production of active form of TGF‐β2 to 4–9%. TGF‐β3 secretion was not detectable under any of the conditions. The Real‐Time PCR analysis of TGF‐β mRNAs confirmed the observed results. The TGF‐β1 and TGF‐β2 secretion was induced by TGF‐β2 and TGF‐β1, respectively. Under these conditions, the contrasting effects of IFN‐γ on TGF‐β1 and TGF‐β2 secretion were also observed. JAK inhibitor selectively inhibited IFN‐γ induced TGF‐β1 secretion and mRNA levels while reversing the inhibitory effects of IFN‐γ on TGF‐β2. Analyses of transcription factor activity strongly indicated the role of STAT‐1 but not NFκB, C‐Myc, C‐Jun, SP‐1, MEF‐2. Our data demonstrate that IFN‐γ differentially regulates constitutively expressed, as well as, cytokine‐induced TGF‐β1 and TGF‐β2 mRNA levels and secretion of TGF‐βs by HRPE. J. Cell. Physiol. 210: 192–200, 2007.

Conditional overexpression of TGF-β1 disrupts mouse salivary gland development and function

Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-β1 in the salivary gland, we created a transgenic mouse (β1glo) that conditionally overexpresses active TGF-β1 upon genomic recombination by Cre recombinase. β1glo mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (β1glo/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those β1glo/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-β signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-β1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-β in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögrens syndrome, or with radiation therapy given to head-and-neck cancer patients.

IFN-β Provides Immuno-Protection in the Retina by Inhibiting ICAM-1 and CXCL9 in Retinal Pigment Epithelial Cells

The retinal pigment epithelial (RPE) cell is a potent regulatory cell that facilitates normal physiologic processes and plays a critical role in a variety of retinal diseases. We evaluated IFN-β production in human RPE cells through TLR signaling and investigated the effects of IFN-β on RPE cells. RPE cells treated with poly(I:C) or infected with an RNA virus produce IFN-β. Kinetic studies revealed that IFN-β levels continue to increase over a 48-h period and this was associated with the up-regulation of IRF-7 gene expression, a known positive feedback molecule for IFN-β production. Microarray analysis revealed that in IFN-β treated cells, 480 genes of 22,283 genes were up or down-regulated by >2-fold. We hypothesize that IFN-β induction during TLR signaling in the retina is an immunosuppressive factor produced to limit immunopathologic damage. Cytokine activation of RPE cells results in the production of the chemokines, CXCL9 and CXCL10, and the adhesion molecule, ICAM-1. Pretreatment of RPE cells with IFN-β resulted in inhibition of ICAM-1 production and elimination of CXCL9 production. This treatment did not alter CXCL10 production. Anti-IFN-β Ab blocked the inhibitory action of IFN-β. Real time PCR analysis revealed that IFN-β treatment inhibited gene expression of sICAM-1 and CXCL9. The results indicate a critical role for RPE cell derived IFN-β in the down-regulation of CXCL9 and ICAM-1 expression in the retina and suggest that the inhibition of CXCL9 is an immuno-suppressive mechanism that protects the retina from excessive inflammation.