Aimin Sang

Roles of Wnt/β-catenin signaling in retinal neuron-like differentiation of bone marrow mesenchymal stem cells from nonobese diabetic mice.

Recent studies have shown that mesenchymal stem cells (MSCs) are expected to become promising therapeutic agents for the treatment of diabetic retinopathy (DR); moreover, we previously demonstrated that bone marrow (BM)-MSCs from nonobese diabetic (NOD) mice (an ideal DR model) had abnormal migration and adhesion. So, we hypothesized that NOD-MSCs also have abnormal retinal neuron-like differentiation potential. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. Western blot analysis and immunofluorescence both showed that the level of retinal neuron-like markers, such as glial fibrillary acidic protein, neuron-specific nuclear protein, tyrosine hydroxylase, Thy-1, glutamine synthetase, and rhodopsin was lower in NOD-MSCs than in imprinting control region MSCs. Furthermore, we explored the precise mechanisms controlling this change in NOD-MSCs. The expression levels of some important member proteins in Wnt/β-catenin signaling were determined and suggested the downregulation of Wnt/β-catenin signaling with retinal neuron-like differentiation of NOD-MSCs. Incubation of NOD-MSCs in medium supplemented with human recombinant Wnt1 resulted in a significant upregulation of retinal neuron-like markers, and the effects of Wnt1 were dose-dependent. Taken together, our study indicated that the inhibition of Wnt/β-catenin signaling in NOD-MSCs after induction could contribute to the abnormal retinal neuron-like differentiation. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for DR.Recent studies have shown that mesenchymal stem cells (MSCs) are expected to become promising therapeutic agents for the treatment of diabetic retinopathy (DR); moreover, we previously demonstrated that bone marrow (BM)-MSCs from nonobese diabetic (NOD) mice (an ideal DR model) had abnormal migration and adhesion. So, we hypothesized that NOD-MSCs also have abnormal retinal neuron-like differentiation potential. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. Western blot analysis and immunofluorescence both showed that the level of retinal neuron-like markers, such as glial fibrillary acidic protein, neuron-specific nuclear protein, tyrosine hydroxylase, Thy-1, glutamine synthetase, and rhodopsin was lower in NOD-MSCs than in imprinting control region MSCs. Furthermore, we explored the precise mechanisms controlling this change in NOD-MSCs. The expression levels of some important member proteins in Wnt/β-catenin signaling were determined and suggested the downregulation of Wnt/β-catenin signaling with retinal neuron-like differentiation of NOD-MSCs. Incubation of NOD-MSCs in medium supplemented with human recombinant Wnt1 resulted in a significant upregulation of retinal neuron-like markers, and the effects of Wnt1 were dose-dependent. Taken together, our study indicated that the inhibition of Wnt/β-catenin signaling in NOD-MSCs after induction could contribute to the abnormal retinal neuron-like differentiation. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for DR.

Spatiotemporal Changes in NFATc4 Expression of Retinal Ganglion Cells After Light-Induced Damage

Nuclear factor of activated T cells, cytoplasmic 4 (NFATc4) is one of the four members of the NFAT family, which were described first as essential components of T cells activation and lately as important regulators for the initiation and coordination of the immune response, including B cells and natural killer cells. Accumulating evidence has demonstrated that NFATc4 exerted a pro-apoptotic effect in the pathogenesis of various experimental central nervous system diseases by upregulating Fas ligand (FasL) levels. However, the function of NFATc4 in the retina is still with limited acquaintance. To investigate whether NFATc4 is involved in retinal neuron apoptosis, we performed a light-induced retinal damage model in adult rats. A significant upregulation of NFATc4 was detected in the retina after light-induced damage by using Western blotting and reverse transcriptase PCR (RT-PCR). Besides this, NFATc4 was observed to be localized mainly in the retinal ganglion cells (RGCs). In addition, the expression patterns of active caspase-3, active caspase-8, and FasL were parallel with that of NFATc4. We also found the co-localization of NFATc4 with active caspase-3 and FasL in RGCs after light exposure. Collectively, we hypothesized that NFATc4 might participate in RGCs apoptosis by upregulating FasL levels.

Involvement of Fra-1 in Retinal Ganglion Cell Apoptosis in Rat Light-Induced Retina Damage Model

Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

Müller Glia Cells Activation in Rat Retina After Optic Nerve Injury: Spatiotemporal Correlation with Transcription Initiation Factor IIB

Transcription initiation factor IIB (TFIIB) is an ideal factor to localize core promoters and plays a central role in the assembly of the pre-initiation complex. Previous studies showed that the assembly of TFIIB played an important role in rat ischemic brain injury. To elucidate the expression and possible functions of TFIIB in retina lesion and repair, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of TFIIB in retina after ONC. Immunofluorescent labeling indicated that TFIIB was localized mainly in the Müller glia cells (MGCs); colocalization of TFIIB and proliferating cell nuclear antigen (PCNA) in the injured retina suggested that TFIIB might participate in MGCs proliferation. In addition, we also examined the expression of the retinal progenitor markers (Nestin and Pax6) whose changes were correlated with the expression of TFIIB. In vitro, we induced MGCs differentiation with brain nerve growth factor (BNGF) and found that TFIIB expression was increased in the differentiated process, which was collected with the expression of PCNA, Nestin, and Pax6. Additionally, knocking TFIIB down with siRNA inhibited the expression of PCNA, Nestin, and Pax6. Collectively, we hypothesized ONC-induced upregulation of TFIIB in the retina was associated with MGCs activation and differentiation.

Up-Regulation of PKM2 Relates to Retinal Ganglion Cell Apoptosis After Light-Induced Retinal Damage in Adult Rats

Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a light-induced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant up-regulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism.

Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo.

Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV.

Upregulation of SYF2 Relates to Retinal Ganglion Cell Apoptosis and Retinal Glia Cell Proliferation After Light-Induced Retinal Damage

SYF2 (SYF2 homologue, RNA splicing factor), also known as CCNDBP1-interactor or p29, belongs to the SYF2 family, which are involved in pre-mRNA splicing and cell cycle progression. Accumulating evidences demonstrate that SYF2 exerted multiple effects including pro-apoptosis, cell differentiation, and glial activation in the pathogenesis of various experimental central nervous system (CNS) diseases. However, SYF2 expression and functions in the retina are still with limited acquaintance. To investigate whether SYF2 was involved in retinal degeneration, we performed a light-induced retinal damage model in adult rats. The SYF2 protein expression was dramatically upregulated after retinal damage. Besides that, SYF2 localized in the retinal ganglion cell (RGC) layer (GCL), inner unclear layer (INL), and outer nuclear layer (ONL) after light exposure. In addition, the expression of cyclin D1, CDK4, and active caspase-3 was parallel with SYF2. We also found the co-localization of SYF2 with active caspase-3, PCNA, and CD11b. Collectively, SYF2 might participate in RGC apoptosis and retinal glia cell proliferation after light-induced retinal damage.

YAP via interacting with STAT3 regulates VEGF-induced angiogenesis in human retinal microvascular endothelial cells

&NA; Endothelial dysfunction is a main feature of retinal neovascular diseases which are the leading cause of blindness in developed countries. Yes‐associated protein (YAP) and signal transducer and activator of transcription factor 3 (STAT3) participate in angiogenesis via vascular endothelial growth factor (VEGF) signaling. Additionally, YAP can bind STAT3 in endothelial cells. In the study, dimethyloxalylglycine (DMOG) stimulated human retinal microvascular endothelial cells (HRMECs) was used as retinal endothelial hypoxia model. The proliferation of HRMECs, as well as t‐YAP, p‐STAT3 (Tyr705) increased, while p‐YAP (Ser127), p‐YAP (Ser397) decreased following hypoxia. Meanwhile, YAP and STAT3 translocated to the nucleus. YAP knockdown inhibited the proliferation, migration and tube formation of HRMECs. YAP overexpression up‐regulated phosphorylation of STAT3. The YAP overexpression‐induced HRMECs proliferation, migration and tube formation were reversed by S3I‐201, a selective STAT3 inhibitor. YAP interacted with STAT3 to promote STAT3 nuclear translocation. Additionally, YAP and STAT3 promoted the transcription of VEGF synergistically. Finally, inhibition of YAP alleviated retinal pathological neovascularization in mouse oxygen‐induced retinopathy (OIR) model. In summary, activated YAP interacted with STAT3 to promote the activation and nuclear translocation of STAT3, hence boosted the proliferation, migration and tube formation of HRMECs via VEGF signaling following hypoxia. The data will further elucidate the mechanisms of retinal neovascular diseases.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine-threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.