Novel bispecific antibody-drug conjugate targeting PD-L1 and B7-H3 enhances antitumor efficacy and promotes immune-mediated antitumor responses.

BACKGROUND: Antibody-drug conjugates (ADCs) offer a promising approach, combining monoclonal antibodies with chemotherapeutic drugs to target cancer cells effectively while minimizing toxicity. METHODS: This study examined the therapeutic efficacy and potential mechanisms of a bispecific ADC (BsADC) in laryngeal squamous cell carcinoma. This BsADC selectively targets the immune checkpoints programmed cell death ligand-1 (PD-L1) and B7-H3, and the precise delivery of the small-molecule toxin monomethyl auristatin E. RESULTS: Our findings demonstrated that the BsADC outperformed its bispecific antibody and PD-L1 or B7-H3 ADC counterparts, particularly in terms of in vitro/in vivo tumor cytotoxicity, demonstrating remarkable immune cytotoxicity. Additionally, we observed potent activation of tumor-specific immunity and significant induction of markers of immunogenic cell death (ICD) and potential endoplasmic reticulum stress. CONCLUSION: In conclusion, this novel BsADC, through immune checkpoint inhibition and promotion of ICD, amplified durable tumor immune cytotoxicity, providing novel insights and potential avenues for future cancer treatments and overcoming resistance.

Systemic delivery of tannic acid-ferric-masked oncolytic adenovirus reprograms tumor microenvironment for improved therapeutic efficacy in glioblastoma.

Glioblastoma (GBM) represents the most aggressive primary brain tumor, and urgently requires effective treatments. Oncolytic adenovirus (OA) shows promise as a potential candidate for clinical antitumor therapy, including in the treatment of GBM. Nevertheless, the systemic delivery of OA continues to face challenges, leading to significantly compromised antitumor efficacy. In this study, we developed an innovative approach by encapsulating CXCL11-armed OA with tannic acid and Fe3+ (TA-Fe3+) to realize the systemic delivery of OA. The nanocarrier's ability to protect the OA from elimination by host immune response was evaluated in vitro and in vivo. We evaluated the antitumor effect and safety profile of OA@TA-Fe3+ in a GBM-bearing mice model. OA@TA-Fe3+ effectively safeguarded the virus from host immune clearance and extended its circulation in vivo. After targeting tumor sites, TA-Fe3+ could dissolve and release Fe3+ and OA. Fe3+-induced O2 production from H2O2 relieved the hypoxic state, and promoted OA replication, leading to a remarkable alteration of tumor immune microenvironment and enhancement in antitumor efficacy. Moreover, the systemic delivery of OA@TA-Fe3+ was safe without inflammation or organ damage. Our findings demonstrated the promising potential of systemically delivering the engineered OA for effective oncolytic virotherapy against GBM.

mRNA-Engineered CD5-CAR-γδTCD5- Cells for the Immunotherapy of T-Cell Acute Lymphoblastic Leukemia.

Clinical trials of Chimeric Antigen Receptor T-cell (CAR-T) therapy have demonstrated remarkable success in treating both solid tumors and hematological malignancies. Nanobodies (Nbs) have emerged as promising antigen-targeting domains for CARs, owing to their high specificity, robust stability, and strong affinity, leading to significant advancements in the field of Nb-CAR-T. In the realm of T-cell acute lymphoblastic leukemia (T-ALL) targets, CD5 stands out as a potentially excellent candidate for T-cell-based CAR therapy, due to its distinct expression on the surface of malignant T-ALL cells. To mitigate graft-versus-host disease associated with allogeneic CAR-T, γδT cells are selected and stimulated from peripheral blood mononuclear cells, and γδT cells are engineered via CRISPR/Cas9 to eliminate fratricide, enabling the creation of fratricide-resistant CAR-γδTCD5- cells. In vitro transcribed (IVT) mRNA is used to construct CAR-T, presenting a safer, faster, and cost-effective method compared to traditional viral vector approaches. In this study, a CD5-VHH library is constructed, and specific CD5-nanobodies are screened for subsequent use in CD5-CAR-γδTCD5- therapy. IVT-mRNA-CD5-CAR-γδTCD5- cells exhibited favorable functional characteristics and demonstrated antitumor efficacy against malignant T cell lines, underlining the potential for advancing mRNA-CD5-CAR-γδTCD5- therapy.

Systemic delivery of oncolytic herpes virus using CAR-T cells enhances targeting of antitumor immuno-virotherapy.

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.

A novel SLC3A2-targeting antibody-drug conjugate exerts potent antitumor efficacy in head and neck squamous cell cancer.

The development of innovative therapeutic strategies for head and neck squamous cell carcinoma (HNSCC) is a critical medical requirement. Antibody-drug conjugates (ADC) targeting tumor-specific surface antigens have demonstrated clinical effectiveness in treating hematologic and solid malignancies. Our investigation revealed high expression levels of SLC3A2 in HNSCC tissue and cell lines. This study aimed to develop a novel anti-SLC3A2 ADC and assess its antitumor effects on HNSCC both in vitro and in vivo. This study developed a potent anti-SLC3A2 ADC (19G4-MMAE) and systematically investigated its drug delivery potential and antitumor efficacy in preclinical models. This study revealed that 19G4-MMAE exhibited specific binding to SLC3A2 and effectively targeted lysosomes. Moreover, 19G4-MMAE induced a significant accumulation of reactive oxygen species (ROS) and apoptosis in SLC3A2-positive HNSCC cells. The compound demonstrated potent antitumor effects derived from MMAE against SLC3A2-expressing HNSCC in preclinical models, displaying a favorable safety profile. These findings suggest that targeting SLC3A2 with an anti-SLC3A2 ADC could be a promising therapeutic approach for treating HNSCC patients.

Improved antitumor effects elicited by an oncolytic HSV-1 expressing a novel B7H3nb/CD3 BsAb.

Oncolytic viruses have emerged as a promising modality for cancer treatment due to their unique abilities to directly destroy tumor cells and modulate the tumor microenvironment. Bispecific T-cell engagers (BsAbs) have been developed to activate and redirect cytotoxic T lymphocytes, enhancing the antitumor response. To take advantage of the specific infection capacity and carrying ability of exogenous genes, we generated a recombinant herpes simplex virus type 1 (HSV-1), HSV-1dko-B7H3nb/CD3 or HSV-1dko-B7H3nb/mCD3, carrying a B7H3nb/CD3 or B7H3nb/mCD3 BsAb that replicates and expresses BsAb in tumor cells in vitro and in vivo. The new generation of oncolytic viruses has been genetically modified using CRISPR/Cas9 technology and the cre-loxp system to increase the efficiency of HSV genome editing. Additionally, we used two fully immunocompetent models (GL261 and MC38) to assess the antitumor effect of HSV-1dko-B7H3nb/mCD3. Compared with the HSV-1dko control virus, HSV-1dko-B7H3nb/mCD3 induced enhanced anti-tumor immune responses and T-cell infiltration in both GL261 and MC38 models, resulting in improved treatment efficacy in the latter. Furthermore, flow cytometry analysis of the tumor microenvironment confirmed an increase in NK cells and effector CD8+ T cells, and a decrease in immunosuppressive cells, including FOXP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and CD206+ macrophages (M2). Overall, our study identified a novel camel B7H3 nanobody and described the genetic modification of the HSV-1 genome using CRISPR/Cas9 technology and the cre-loxp system. Our findings indicate that expressing B7H3nb/CD3 BsAb could improve the antitumor effects of HSV-1 based oncolytic virus.

Improved microalgae growth and lipid production in anaerobic digestate with ultraviolet radiation pretreatment.

Inappropriate sterilization strategies inhibit microalgal growth when culturing microalgae with anaerobic digestate. This study aimed to scientifically select a low-cost disinfection pretreatment of anaerobic digestate for large-scale microalgae cultivations. In this work, three different methods, including autoclaving, ultraviolet or NaClO treatments, were employed to sterilize the municipal anaerobic digestate. Scenedesmus quadricauda was then cultured in diluted liquid digestate for the simultaneous lipid production and nutrient removal. The results indicated that the growth of S. quadricauda was inhibited after NaClO treatment due to the residual free chlorine. The 15-min ultraviolet effectively mitigated microbial contamination and increasing nutrient availability, enhancing the electron transport of microalgal photosynthesis. After 6-days cultivation, the microalgal biomass concentration of the ultraviolet group was 1.09 g/L, comparable to that of the autoclaving group (1.15 g/L). High nutrient removal efficiency was observed: COD (93.30 %), NH4+-N (92.56 %), TN (85.82 %) and TP (95.12 %). Moreover, S. quadricauda outcompeted the indigenous microorganisms, contributing to its dominance in the culture system of ultraviolet group. The facultative anaerobe Comamonadaceae and aerobes Moraxellaceae, rather than strict anaerobe Paludibacteraceae and Bacteroidetes_vadinHA17, played vital roles in synergistic removal of contaminants by bacteria and algae. The potential competition for nitrogen and phosphorus by bacteria contributed to the ultraviolet group having the greatest lipid content (48.19 %). Therefore, this work suggested using 15-min ultraviolet treatment for anaerobic digestate in large-scale microalgae cultivation.

Delivery of CD47-SIRPα checkpoint blocker by BCMA-directed UCAR-T cells enhances antitumor efficacy in multiple myeloma.

In the treatment of relapsed or refractory multiple myeloma patients, BCMA-directed autologous CAR-T cells have showed excellent anti-tumor activity. However, their widespread application is limited due to the arguably cost and time-consuming. Multiple myeloma cells highly expressed CD47 molecule and interact with the SIRPα ligand on the surface of macrophages, in which evade the clearance of macrophages through the activation of "don't eat me" signal. In this study, a BCMA-directed universal CAR-T cells, BC404-UCART, secreting a CD47-SIRPα blocker was developed using CRISPR/Cas9 gene-editing system. BC404-UCART cells significantly inhibited tumor growth and prolonged the survival of mice in the xenograft model. The anti-tumor activity of BC404-UCART cells was achieved via two mechanisms, on the one hand, the UCAR-T cells directly killed tumor cells, on the other hand, the BC404-UCART cells enhanced the phagocytosis of macrophages by secreting anti-CD47 nanobody hu404-hfc fusion that blocked the "don't eat me" signal between macrophages and tumor cells, which provides a potential strategy for the development of novel "off-the-shelf" cellular immunotherapies for the treatment of multiple myeloma.

A drug screening to identify novel combinatorial strategies for boosting cancer immunotherapy efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cells and immune checkpoint blockades (ICBs) have made remarkable breakthroughs in cancer treatment, but the efficacy is still limited for solid tumors due to tumor antigen heterogeneity and the tumor immune microenvironment. The restrained treatment efficacy prompted us to seek new potential therapeutic methods. METHODS: In this study, we conducted a small molecule compound library screen in a human BC cell line to identify whether certain drugs contribute to CAR T cell killing. Signaling pathways of tumor cells and T cells affected by the screened drugs were predicted via RNA sequencing. Among them, the antitumor activities of JK184 in combination with CAR T cells or ICBs were evaluated in vitro and in vivo. RESULTS: We selected three small molecule drugs from a compound library, among which JK184 directly induces tumor cell apoptosis by inhibiting the Hedgehog signaling pathway, modulates B7-H3 CAR T cells to an effector memory phenotype, and promotes B7-H3 CAR T cells cytokine secretion in vitro. In addition, our data suggested that JK184 exerts antitumor activities and strongly synergizes with B7-H3 CAR T cells or ICBs in vivo. Mechanistically, JK184 enhances B7-H3 CAR T cells infiltrating in xenograft mouse models. Moreover, JK184 combined with ICB markedly reshaped the tumor immune microenvironment by increasing effector T cells infiltration and inflammation cytokine secretion, inhibiting the recruitment of MDSCs and the transition of M2-type macrophages in an immunocompetent mouse model. CONCLUSION: These data show that JK184 may be a potential adjutant in combination with CAR T cells or ICB therapy.

Renal artery thrombosis in SARS-CoV-2 infection: a case report.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is identified as the pneumonia and acute respiratory distress syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). The intravascular thrombotic phenomena related to the COVID-19 are emerging as an important complication that contribute to significant mortality. CASE PRESENTATION: We present a 62-year-old man with severe COVID-19 and type 2 diabetes. After symptomatic and supportive treatment, the respiratory function was gradually improved. However, the patient suddenly developed abdominal pain, and the enhanced CT scan revealed renal artery thrombosis. Given the risk of surgery and the duration of the disease, clopidogrel and heparin sodium were included in the subsequent treatment. The patient recovered and remained stable upon follow-up. CONCLUSIONS: Thrombosis is at a high risk in patients with severe COVID-19 pneumonia because of hypercoagulable state, blood stasis and endothelial injury. Thrombotic events caused by hypercoagulation status secondary to vascular endothelial injury deserves our attention. Because timely anticoagulation can reduce the risk of early complications, as illustrated in this case report.

Knockdown of lncRNA MEG8 inhibits cell proliferation and invasion, but promotes cell apoptosis in hemangioma, via miR­203­induced mediation of the Notch signaling pathway.

As a member of the long non­coding (lnc)RNA family, lncRNA maternally expressed 8, small nucleolar RNA host gene (MEG8), has been reported to serve an oncogenic role in several types of malignancies, including hepatocellular carcinoma, non­small cell lung cancer and pancreatic cancer. The current study aimed to investigate the effect of the knockdown of MEG8 on human hemangioma endothelial cell (HemEC) proliferation, apoptosis and invasion, in addition to determining the underlying molecular mechanism. The knockdown of lncRNA MEG8 was achieved by transfecting lncRNA MEG8 small interfering (si)RNA into HemECs, while the combined knockdown of lncRNA MEG8 knockdown and microRNA (miR)­203 was established by co­transfecting lncRNA MEG8 siRNA and a miR­203 inhibitor into HemECs. The cell proliferation, apoptosis and invasion and the expression levels of miR­34a, miR­200b, miR­200b and Notch signaling pathway­related factors were detected via CCK­8 Kit, flow cytometry, Transwell, reverse transcription­quantitative PCR and western blot assay, respectively. The knockdown of lncRNA MEG8 significantly inhibited proliferation (P<0.05) and invasion (P<0.05), but promoted apoptosis (P<0.01) in HemECs. Furthermore, lncRNA MEG8 knockdown upregulated miR­203 (P<0.01) expression, but did not alter miR­34a or miR­200b expression (both P>0.05). Subsequent experiments revealed that miR­203 silencing exerted no significant effect on the expression levels of lncRNA MEG8 (P>0.05) in HemECs. In addition, miR­203 silencing increased cell proliferation (P<0.05) and invasion (P<0.01), but suppressed apoptosis (P<0.05). miR­203 silencing also reversed the effect of lncRNA MEG8 knockdown on the proliferation (P<0.05), apoptosis (P<0.001) and invasion (P<0.01) of HemECs. Moreover, lncRNA MEG8 knockdown downregulated jagged canonical notch ligand 1 (JAG1; P<0.05) and Notch1 (P<0.05) expression levels, while miR­203 silencing upregulated JAG1 (P<0.01) and Notch1 (P<0.01) expression levels and reversed the effects of lncRNA MEG8 knockdown on JAG1 (P<0.01) and Notch1 (P<0.01) expression in HemECs. In conclusion, the findings of the present study suggested that lncRNA MEG8 knockdown may inhibit cell proliferation and invasion, but promote cell apoptosis in hemangioma via miR­203­induced mediation of the Notch signaling pathway.

Meta-analysis: combination of meropenem vs ceftazidime and amikacin for empirical treatment of cancer patients with febrile neutropenia.

BACKGROUND: Meropenem monotherapy vs ceftazidime plus amikacin have been approved for use against febrile neutropenia. To assess the effectiveness and safety of them for empirical treatment of cancer patients with febrile neutropenia, we conducted a meta-analysis of randomized controlled trial. METHODS: Randomized controlled trials on ceftazidime plus amikacin, or/and monotherapy with meropenem for the treatment of cancer patients with febrile neutropenia were identified by searching Cochrane Library, PubMed, Science Direct, Wiley Online, Science Citation Index, Google (scholar), National Center for Biotechnology Information, and China National Knowledge Infrastructure. Data on interventions, participants' characteristics and the outcomes of therapy, were extracted for statistical analysis. Seven trials fulfilled the inclusion criteria. RESULT: The treatment with ceftazidime plus amikacin was more effective than meropenem (OR = 1.17; 95% CI 0.93-1.46; 1270 participants). However, the treatment effects of the 2 therapy methods were almost parallel in adults (OR = 1.15; 95% CI 0.91-1.46; 1130 participants older than 16). Drug-related adverse effects afflicted more patients treated with ceftazidime plus amikacin (OR = 0.78; 95% CI 0.52-1.15; 1445 participants). The common responses were nausea, diarrhea, rash, and increased in serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and bilirubin. CONCLUSION: Ceftazidime plus amikacin should be the first choice for empirical treatment of cancer patients with febrile neutropenia, and meropenem may be chosen as a last defense against pathogenic bacteria.

Effects of green waste addition on waste activated sludge and fat, oil and grease co-digestion in mesophilic batch digester.

Anaerobic digestion (AD) is regarded as an effective method to treat waste activated sludge (WAS) and fat, oil and grease (FOG). Co-digestion of WAS/FOG could promote the methane yield but it will cause acid and salinity inhibition. Green waste (GW) was added into the digesters, and its effects on co-digestion of WAS and FOG in the mesophilic batch digester were investigated. Digestive performances (such as hydrolysis, acidogenesis and methanogenesis) were studied emphatically. The results showed that digester L6 (WAS:FOG:GW = 1:2:1, VS basis) presented the highest specific methane yield (SMY, 341.5 mL/g VS). The results of kinetics study verified that there was a slower hydrolysis rate when GW was applied as a co-substrate, which could reduce the potential of acid inhibition. Volatile fatty acid (VFA) and electrical conductivity analysis showed that GW addition could keep moderate VFA concentrations and alleviate the negative effects of high-salinity substrates on the digestive systems. The microbial community and diversity analysis proved that GW addition was beneficial to keep the balance of hydrolytic bacteria, acidogens and acetogens. The results of this study indicated that GW addition could enhance the energy recovery and system stability in the WAS/FOG co-digestive system.

Effects of hydrothermal pretreatment on the mono- and co-digestion of waste activated sludge and wheat straw.

The refractory properties of waste activated sludge and wheat straw inhibit their bioenergy recovery by anaerobic digestion. This paper attempted to estimate the digestive performance, energy conversion efficiency and economic feasibility of wheat straw mono-digestion and its co-digestion with sludge by hydrothermal pretreatment at different temperature gradients (125, 150 and 175 °C). The results illustrated that the hydrolysis of both wheat straw and sludge were improved with the temperature increasing. It is noted that after pretreatment at 175 °C, wheat straw mono-digestion obtained the cumulative specific methane yield of 168.8 mL/g·VS, 6.9% reduction compared to the unpretreated straw (181.4 mL/g·VS) due to the inhibition by by-products (furfural and 5-hydroxymethylfurfural, 5-HMF) formed at high temperatures. The highest cumulative specific methane yield of 225.7 mL/g·VS was achieved by the co-digestion of pretreated wheat straw and pretreated sludge under 175 °C, indicating that the participation of sludge in co-digestion improved the buffer capacity of the system to relieve the inhibition. In addition, the co-digestion of sludge and wheat straw both pretreated at 175 °C obtained the maximum energy production of 7901.1 MJ/t, 52% promotion compared to the mono-digestion without pretreatment. The results of economic analysis showed that the mono-digestion of wheat straw obtained relatively low net profits and the mono-digestion of sludge pretreated at 175 °C achieved the highest net profit of 31.44 US$/t. These results suggest that the co-digestion of both pretreated wheat straw and sludge can achieve the highest biogas production and energy conversion efficiency.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy.

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy (DR). Our previous studies demonstrated that G protein-coupled receptor 91(GPR91) participated in the regulation of vascular endothelial growth factor (VEGF) secretion in DR. The present study induced OIR model in newborn rats using exposure to alternating 24-hour episodes of 50% and 12% oxygen for 14 days. Treatment with GPR91 shRNA attenuated the retinal avascular area, abnormal neovascularization and pericyte loss. Western blot and qRT-PCR demonstrated that CoCl2 exposure promoted VEGF expression and secretion, activated the ERK1/2 signaling pathways and upregulated C/EBP and AP-1. Knockdown of GPR91 inhibited ERK1/2 activity. GPR91 siRNA transduction and the ERK1/2 inhibitor U0126 inhibited the increases in C/EBP ß, C/EBP δ, c-Fos and HIF-1α. Luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay demonstrated that C/EBP ß and c-Fos bound the functional transcriptional factor binding site in the region of the VEGF promoter, but not C/EBP δ. Knockdown of C/EBP ß and c-Fos using RNAi reduced VEGF expression. Our data suggest that activation of the GPR91-ERK1/2-C/EBP ß (c-Fos, HIF-1α) signaling pathway plays a tonic role in regulating VEGF transcription in rat retinal ganglion cells.

Population-based survey of prevalence, causes, and risk factors for blindness and visual impairment in an aging Chinese metropolitan population.

AIM: To assess the prevalence, causes, and risk factors for blindness and visual impairment among elderly (≥60 years of age) Chinese people in a metropolitan area of Shanghai, China. METHODS: Random cluster sampling was conducted to identify participants among residents ≥60 years of age living in the Xietu Block, Xuhui District, Shanghai, China. Presenting visual acuity (PVA) and best-corrected visual acuity (BCVA) were checked by the Early Treatment Diabetic Retinopathy Study (ETDRS) visual chart. All eligible participants underwent a comprehensive eye examination. Blindness and visual impairment were defined according to World Health Organization (WHO) criteria. RESULTS: A total of 4190 persons (1688 men and 2502 women) participated in the study, and the response rate was 91.1%. Based on PVA, the prevalence of blindness was 1.1% and that of visual impairment was 7.6%. Based on BCVA, the prevalence of blindness and visual impairment decreased to 0.9% and 3.9%, respectively. Older (≥80 years of age) women, with low educational levels and smoking habits, exhibited a significantly greater chance for blindness and visual impairment than did those with high educational levels and no smoking habits (P<0.05). Based on PVA and BCVA, the main causes of blindness were cataract, myopic maculopathy, and age-related macular degeneration (AMD). CONCLUSION: Our findings help to identify the population in need of intervention, to highlight the need for additional eye healthcare services in urban China.

The MAPK signaling pathway mediates the GPR91-dependent release of VEGF from RGC-5 cells.

Vascular endothelial growth factor (VEGF) is one of the major regulatory molecules in diabetic retinopathy (DR). In our previous study, we demonstrated that succinate levels were elevated in the retinas of diabetic rats and that the knockdown of the succinate receptor, G-protein-coupled receptor 91 (GPR91), inhibited the release of VEGF and attenuated retinal vascular disorder in the early stages of DR. In the present study, we examined the signaling pathways involved in the GPR91-dependent release of VEGF in the retinal ganglion cell line, RGC-5. The cells were infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91). Immunofluorescence staining revealed that GPR91 was predominantly localized in the cell bodies of the RGC-5 cells. RT-qPCR, western blot analysis and ELISA indicated that succinate exposure upregulated VEGF expression, activated the extracellular signal-regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways and led to the release of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The knockdown of GPR91 inhibited ERK1/2 and JNK activity, but did not inhibit the activation of the p38 MAPK pathway. The increase in COX-2 expression and the release of PGE2 were inhibited by transduction with LV.shGPR91 and ERK1/2, JNK and COX-2 inhibitors. The expression and release of VEGF showed similar results. Cell Counting Kit-8 (CCK-8) assays revealed that the shRNA-mediated knockdown of GPR91 decreased the proliferation of RF/6A cells cultured in succinate-conditioned medium. Our data suggest that GPR91 modulates the succinate-induced release of VEGF through the MAPK/COX-2/PGE2 signaling pathway.

ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes.

PURPOSE: Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes. METHODS: Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT-PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay. RESULTS: Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398. CONCLUSIONS: Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR.

Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91.

Recent research using a rat oxygen-induced retinopathy model has demonstrated that the G protein-coupled receptor 91 (GPR91) of retinal ganglion neurons is the principal respondent to succinate and consequently induces the release of angiogenic factor vascular endothelial growth factor (VEGF). The aim of this study was to determine whether GPR91 modulate the release of VEGF from retinal ganglion cells in a high-glucose model in vitro and to dissect the role of GPR91 in the pathogenesis of diabetic retinopathy. We constructed a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91) and infected the retinal ganglion cell line RGC-5 to obtain stably transduction system. The knockdown effect of GPR91 was detected by Western blotting. After incubation with succinate and various concentrations of glucose, the expression of VEGF in RGC-5 cells was evaluated by real-time PCR and Western blotting, and the release of VEGF protein was measured using an ELISA assay. Conditioned media were also collected, and the effects of proliferation and migration of RF/6A cells, a vascular endothelial cell line, were evaluated by CCK-8 and Transwell assays. The phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK) in RGC-5 cells after exposure to high glucose were evaluated by Western blotting. Following a single exposure of RGC-5 cells to the encoding lentivirus, more than 80% of infected cells expressed GFP at 72 h, and the level of GPR91 protein was significantly downregulated. GPR91 shRNA inhibited the cell survival rates of RGC-5 cells incubated with high glucose (F = 21.36, P = 0.002). The mRNA and protein expression of VEGF in LV.shGPR91 RGC-5 cells decreased markedly compared to that of LV.shScrambled or untransduced control cells incubated with different concentrations of glucose or succinate (P < 0.01). The VEGF protein level in medium from RGC-5 cells treated with high glucose (F = 57.43, P = 0.000) or succinate (F = 241.91, P = 0.000) was also downregulated when transduced with GPR91 shRNA. The siRNA-mediated knockdown of GPR91 was also found to inhibit the proliferation of RF/6A cells in high glucose-stimulated (t = 8.21, P = 0.001) or succinate-stimulated (t = 3.36, P = 0.028) conditioned media. However, the siRNA-mediated knockdown of GPR91 suppressed the migration of RF/6A cells incubated with moderate levels of glucose (t = 2.97, P = 0.018). The exposure of RGC-5 cells to high glucose activated ERK1/2 and JNK MAPK signaling blocking by GPR91 shRNA (P < 0.01). These results indicate that GPR91 modulates the high glucose-induced VEGF release of RGC-5 cells, possibly by inhibiting ERK1/2 and JNK MAPK signaling.

Inhibition of JAK2/STAT3-mediated VEGF upregulation under high glucose conditions by PEDF through a mitochondrial ROS pathway in vitro.

PURPOSE: Hyperglycemia-induced mitochondrial reactive oxygen species (ROS) production plays an important role in the development of complications of diabetes such as retinopathy. However, whether pigment epithelium-derived factor (PEDF) can decrease ROS production remains uncertain. The aim of this study was to clarify whether PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate vascular endothelial growth factor (VEGF) expression; the authors also investigated the involvement of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) in the process. METHODS: Bovine retinal capillary endothelial cells (BRECs) were exposed to normal glucose (NG), H(2)O(2), or high glucose (HG) in the presence or absence of PEDF. Expression of JAK2/STAT3, VEGF, uncoupling protein (UCP)-2, and proliferator-activated receptor gamma (PPARgamma) in the BRECs was examined by Western blot analysis assay; VEGF and UCP-2 mRNA were determined by real-time RT-PCR. Mitochondrial membrane potential (Deltapsim) and ROS production were assayed using JC-1 and CM-H2DCFDA, respectively. RESULTS: HG exposure caused hyperpolarization of Deltapsim and increased ROS generation in BRECs; meanwhile, like H(2)O(2), it also induced the phosphorylation of JAK2/STAT3 and increased VEGF expression; these changes were inhibited by PEDF. The authors also found that PEDF-induced ROS inhibition was a result of decreased Deltapsim, which was caused by the upregulation of PPARgamma and UCP-2 expression. CONCLUSIONS: For the first time it has been demonstrated that PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate VEGF expression, possibly through inhibiting HG-induced JAK2/STAT3 activation, which may offer a promising strategy for halting the development of complications of diabetes.