Associations between psycho-behavioral risk factors and diabetic retinopathy: NHANES (2005-2018).

Introduction: Diabetes mellitus (DM) and diabetic retinopathy (DR) increase the global burden. Since their pathogenesis is complex, it is necessary to use the biopsychosocial model to discover the most effective strategies. The study is aimed to investigate the psycho-behavioral factors of DR and confirm the discrepancies from previous studies. Research design and methods: The study comprised seven cycles of cross-sectional data of the National Health and Nutrition Examination Survey (NHANES) from 2005-2006 to 2017-2018. Samples of DM were selected from this complex multi-stage probability sample and divided into the non-DR and DR groups, where 4,426 samples represented 18,990,825 individuals after weighting. This study comprehensively explored the biological, social, and psychological risk factors of DR, among which the biological factors included blood pressure, blood routine, HbA1c%, blood glucose, the duration of DM, family history, comorbidities, and treatment methods. Social aspects include gender, education, income, insurance, smoking, drinking, sleep habits, and recreational activities. The Patient Health Questionnaire-9 (PHQ-9) was used to assess the psychological state. Taylor series regression was used to examine the connection between factors and DR. Results: Men accounted for 55.5% of the DR group (P = 0.0174). Lymphocyte count, insulin treatment, heart failure, stroke, liver condition, and renal failure showed significant differences in DR (P < 0.05). The incidence of depression in DR was 40.5%. Mild to moderate depression [odds ratio was associated with DR [(OR) = 1.37, 95% confidence interval (CI): 1.06-1.79], but there was no statistical difference in severe depression (OR = 1.34, 95% CI: 0.83-2.17). Although ≤ 6 h of sleep was associated with DR (OR = 1.38, 95% CI: 1.01-1.88), we found no statistical differences in alcohol consumption, recreational activities, or sedentary time between the two groups in our current study (P > 0.05). Conclusions: The biological risk factors of DR are significant. It showed that stroke is associated with DR, and retinal exams have the potential value as a screening tool for the brain. Besides, psycho-behavioral risk factors of DR should also be paid attention. Our study highlights that mild and moderate depression and ≤6 h of sleep are distinguishably associated with DM complicated with DR. It indicates that psycho-behavioral risk factors confer a vital influence on diabetic health care and DR.

The Significance of the Ocular Adverse Effect Induced by Systemic Taxane Application.

In recent years, in-depth research on anti-tumor therapy has brought the emergence of new active chemotherapeutic agents and combination regimens. However, as one of them, taxane drugs are widely used in clinical practice, but it should be noted that many side reactions caused by their application bring some difficulties to routine management. Among the side reactions related to taxane anti-tumor therapy, ocular adverse reactions are occasionally reported and are not life-threatening but may seriously affect patients' life quality. Thus, the continuation, reduction and cessation of taxane chemotherapy still need to be further evaluated by ophthalmologists and oncologists once the side effects show up. To prevent ocular side reactions, close attention should be paid to complications during medication. To facilitate the oncology department and ophthalmologists to comprehensively understand the ophthalmic adverse reactions of taxane drugs and their possible mechanisms and improve drug use efficiency, we collected relevant literature and reviewed and provided some suggestions for the monitoring and managing of ophthalmic toxicity.

Notch-Regulated c-Kit-Positive Liver Sinusoidal Endothelial Cells Contribute to Liver Zonation and Regeneration.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (SECs) promote the proliferation of hepatocytes during liver regeneration. However, the specific subset of SECs and its mechanisms during the process remain unclear. In this study, we investigated the potential role of c-kit+ SECs, a newly identified subset of SECs in liver regeneration. METHODS: Partial hepatectomy mice models were established to induce liver regeneration. Hepatic c-kit expression was detected by quantitative reverse-transcription polymerase chain reaction, immunofluorescent staining, and fluorescence-activated cell sorting. VE-cadherin-cyclization recombinase-estrogen receptor (Cdh5-Cre-ERT) Notch intracellular domain and Cdh5-Cre recombination signal binding protein Jκfloxp mice were introduced to mutate Notch signaling. c-Kit+ SECs were isolated by magnetic beads. Single-cell RNA sequencing was performed on isolated SECs. Liver injuries were induced by CCl4 or quantitative polymerase chain reaction injection. RESULTS: Hepatic c-kit is expressed predominantly in SECs. Liver resident SECs contribute to the increase of c-kit during partial hepatectomy-induced liver regeneration. Isolated c-kit+ SECs promote hepatocyte proliferation in vivo and in vitro by facilitating angiocrine. The distribution of c-kit shows distinct spatial differences that are highly coincident with the liver zonation marker wingless-type MMTV integration site family, member2 (Wnt2). Notch mutation reshapes the c-kit distribution and liver zonation, resulting in altered hepatocyte proliferation. c-Kit+ SECs were shown to regulate hepatocyte regeneration through angiocrine in a Wnt2-dependent manner. Activation of the Notch signaling pathway weakens liver regeneration by inhibiting positive regulatory effects of c-kit+ SECs on hepatocytes. Furthermore, c-kit+ SEC infusion attenuates toxin-induced liver injuries in mice. CONCLUSIONS: Our results suggest that c-kit+ SECs contributes to liver zonation and regeneration through Wnt2 and is regulated by Notch signaling, providing opportunities for novel therapeutic approaches to liver injury in the future. Transcript profiling: GEO (accession number: GSE134037).

Notch-triggered maladaptation of liver sinusoidal endothelium aggravates nonalcoholic steatohepatitis through endothelial nitric oxide synthase.

BACKGROUND AND AIMS: Although NASH can lead to severe clinical consequences, including cirrhosis and hepatocellular carcinoma, no effective treatment is currently available for this disease. Increasing evidence indicates that LSECs play a critical role in NASH pathogenesis; however, the mechanisms involved in LSEC-mediated NASH remain to be fully elucidated. APPROACH AND RESULTS: In the current study, we found that LSEC homeostasis was disrupted and LSEC-specific gene profiles were altered in methionine-choline-deficient (MCD) diet-induced NASH mouse models. Importantly, Notch signaling was found to be activated in LSECs of NASH mice. To then investigate the role of endothelial Notch in NASH progression, we generated mouse lines with endothelial-specific Notch intracellular domain (NICD) overexpression or RBP-J knockout to respectively activate or inhibit Notch signaling in endothelial cells. Notably, endothelial-specific overexpression of the NICD accelerated LSEC maladaptation and aggravated NASH, whereas endothelial cell-specific inhibition of Notch signaling restored LSEC homeostasis and improved NASH phenotypes. Furthermore, we demonstrated that endothelial-specific Notch activation exacerbated NASH by inhibiting endothelial nitric oxide synthase (eNOS) transcription, whereas administration of the pharmacological eNOS activator YC-1 alleviated hepatic steatosis and lipid accumulation resulting from Notch activation. Finally, to explore the therapeutic potential of using Notch inhibitors in NASH treatment, we applied two gamma-secretase inhibitors-DAPT and LY3039478-in an MCD diet-induced mouse model of NASH, and found that both inhibitors effectively ameliorated hepatic steatosis, inflammation, and liver fibrosis. CONCLUSIONS: Endothelial-specific Notch activation triggered LSEC maladaptation and exacerbated NASH phenotypes in an eNOS-dependent manner. Genetic and pharmacological inhibition of Notch signaling effectively restored LSEC homeostasis and ameliorated NASH progression.

Shear stress-induced cellular senescence blunts liver regeneration through Notch-sirtuin 1-P21/P16 axis.

BACKGROUND AND AIMS: The mechanisms involved in liver regeneration after partial hepatectomy (pHx) are complicated. Cellular senescence, once linked to aging, plays a pivotal role in wound repair. However, the regulatory effects of cellular senescence on liver regeneration have not been fully elucidated. APPROACH AND RESULTS: Mice subjected to pHx were analyzed 14 days after surgery. The incomplete remodeling of liver sinusoids affected shear stress-induced endothelial nitric oxide synthase (eNOS) signaling on day 14, resulting in the accumulation of senescent LSECs. Removing macrophages to augment LSEC senescence led to a malfunction of the regenerating liver. A dynamic fluctuation in Notch activity accompanied senescent LSEC accumulation during liver regeneration. Endothelial Notch activation by using Cdh5-CreERT NICeCA mice triggered LSEC senescence and senescence-associated secretory phenotype, which disrupted liver regeneration. Blocking the Notch by γ-secretase inhibitor (GSI) diminished senescence and promoted LSEC expansion. Mechanically, Notch-hairy and enhancer of split 1 signaling inhibited sirtuin 1 (Sirt1) transcription by binding to its promoter region. Activation of Sirt1 by SRT1720 neutralized the up-regulation of P53, P21, and P16 caused by Notch activation and eliminated Notch-driven LSEC senescence. Finally, Sirt1 activator promoted liver regeneration by abrogating LSEC senescence and improving sinusoid remodeling. CONCLUSIONS: Shear stress-induced LSEC senescence driven by Notch interferes with liver regeneration after pHx. Sirt1 inhibition accelerates liver regeneration by abrogating Notch-driven senescence, providing a potential opportunity to target senescent cells and facilitate liver repair after injury.

Notch activation promotes endothelial quiescence by repressing MYC expression via miR-218.

After angiogenesis-activated embryonic and early postnatal vascularization, endothelial cells (ECs) in most tissues enter a quiescent state necessary for proper tissue perfusion and EC functions. Notch signaling is essential for maintaining EC quiescence, but the mechanisms of action remain elusive. Here, we show that microRNA-218 (miR-218) is a downstream effector of Notch in quiescent ECs. Notch activation upregulated, while Notch blockade downregulated, miR-218 and its host gene Slit2, likely via transactivation of the Slit2 promoter. Overexpressing miR-218 in human umbilical vein ECs (HUVECs) significantly repressed cell proliferation and sprouting in vitro. Transcriptomics showed that miR-218 overexpression attenuated the MYC proto-oncogene, bHLH transcription factor (MYC, also known as c-myc) signature. MYC overexpression rescued miR-218-mediated proliferation and sprouting defects in HUVECs. MYC was repressed by miR-218 via multiple mechanisms, including reduction of MYC mRNA, repression of MYC translation by targeting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and promoting MYC degradation by targeting EYA3. Inhibition of miR-218 partially reversed Notch-induced repression of HUVEC proliferation and sprouting. In vivo, intravitreal injection of miR-218 reduced retinal EC proliferation accompanied by MYC repression, attenuated pathological choroidal neovascularization, and rescued retinal EC hyper-sprouting induced by Notch blockade. In summary, miR-218 mediates the effect of Notch activation of EC quiescence via MYC and is a potential treatment for angiogenesis-related diseases.

Integrin α5ß1 promotes BMCs mobilization and differentiation to exacerbate choroidal neovascularization.

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-ß and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV.

Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice.

Macrophages have been recognized as an important inflammatory component in choroidal neovascularization (CNV). However, it is unclear how these cells are activated and polarized, how they affect angiogenesis and what the underlining mechanisms are during CNV. Notch signaling has been implicated in macrophage activation. Previously we have shown that inducible disruption of RBP-J, the critical transcription factor of Notch signaling, in adult mice results in enhanced CNV, but it is unclear what is the role of macrophage-specific Notch signaling in the development of CNV. In the current study, by using the myeloid specific RBP-J knockout mouse model combined with the laser-induced CNV model, we show that disruption of Notch signaling in macrophages displayed attenuated CNV growth, reduced macrophage infiltration and activation, and alleviated angiogenic response after laser induction. The inhibition of CNV occurred with reduced expression of VEGF and TNF-α in infiltrating inflammatory macrophages in myeloid specific RBP-J knockout mice. These changes might result in direct inhibition of EC lumen formation, as shown in an in vitro study. Therefore, clinical intervention of Notch signaling in CNV needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

miR-342-5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor ß Signaling.

BACKGROUND: Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor ß, and other signals, but the detailed molecular mechanisms remain unclear. METHODS AND RESULTS: Small RNA sequencing initially identified miR-342-5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR-342-5p targeted endoglin and modulated transforming growth factor ß signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR-342-5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads-based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR-342-5p agomir in P3 pups. Moreover, miR-342-5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial-mesenchymal transition. Transfection of endoglin at least partially reversed endothelial-mesenchymal transition induced by miR-342-5p. The expression of miR-342-5p was upregulated by transforming growth factor ß, and inhibition of miR-342-5p attenuated the inhibitory effects of transforming growth factor ß on lumen formation and sprouting by ECs. In addition, VEGF repressed miR-342-5p expression, and transfection of miR-342-5p repressed VEGFR2 and VEGFR3 expression and VEGF-triggered Akt phosphorylation in ECs. miR-342-5p repressed angiogenesis in a laser-induced choroidal neovascularization model in mice, highlighting its clinical potential. CONCLUSIONS: miR-342-5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways.

Hyperglycemia promotes vasculogenesis in choroidal neovascularization in diabetic mice by stimulating VEGF and SDF-1 expression in retinal pigment epithelial cells.

To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV) in diabetic mice, especially the involvement of bone marrow-derived cells (BMCs) and underlying molecular mechanisms. The mice were randomly divided into control group, diabetes group and diabetes treated with insulin group, which were laser treated to induce CNV. The CNV severity was evaluated by fundus fluorescein angiography, HE staining and choroidal flatmount. The BMCs recruitment and differentiation in CNV were examined in GFP chimeric mice by choroidal flatmount and immunofluorescence. The bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and migration were tested in vivo and in vitro. VEGF and SDF-1 production in vivo and in vitro were tested by realtime PCR and ELISA. The CNV severity and expression of VEGF and SDF-1 were enhanced in DM mice compared with control mice and that insulin treatment decreased CNV severity in DM mice. The DM mice demonstrated more BMCs and bone marrow-derived mesenchymal stem cells (BMSCs) recruited and incorporated into CNV, increased ratio of BMCs expressing endothelial cell marker or macrophage marker, and up-regulated expression of VEGF and SDF-1 in CNV. Human BMSCs migration and expression of VEGF and SDF-1 in retinal pigment epithelial (RPE) cells increased when cultured under high glucose. This study suggested that hyperglycemia enhanced the expression of VEGF and SDF-1 in RPE cells, and promoted recruitment and incorporation of BMCs and affected differentiation of BMCs in CNV, which led to more severe CNV in diabetic mice.

Inhibition of tumor angiogenesis and tumor growth by the DSL domain of human Delta-like 1 targeted to vascular endothelial cells.

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy.

Preliminary study of retinal pathological features in preterm birth pups exposed to an animal model of oxygen-induced retinopathy in mice.

BACKGROUND: The main risk factors of retinopathy of prematurity (ROP) are low gestational age and low birth weight, which are mainly caused by preterm birth. Currently, the animal model of oxygen-induced retinopathy (OIR) in mice is the most widely used model in ROP-associated studies. However, the experimental mice are normal-term pups, and may not mimic the pathogenic status of human ROP patients. In this study, we investigated the retinal pathological features in preterm birth pups exposed to an animal model of oxygen-induced retinopathy in mice. METHODS: Preterm-birth mice were obtained from pregnant C57BL/6J mice that were induced by an intraperitoneal injection of lipopolysaccharide (LPS). The preterm and control mice were treated with high oxygen (75%) from postnatal day 7 (P7) to P12. The mice were perfused with high-molecular-weight FITC-dextran on P12, P15 and P17, and the retinas were whole-mounted and imaged. Vascular endothelial growth factor (VEGF) mRNA was also detected. Cross-sections of the retina were stained with hematoxylin and eosin (H&E) to identify preretinal neovascular tufts. For general observation, whole retinal images were also obtained using a microscope. RESULTS: Leakage of the retinal blood vessels was aggravated in the preterm mice, particularly on P12 and P15. The non-perfused areas of the retina (pixel value, 183,673 ± 28,148 vs 132,110 ± 23,732, P = 0.009) and the number of preretinal endothelial cell nuclei were smaller (30.17 ± 8.33 vs 22.17 ± 6.74, P < 0.0001) on P17. The VEGF mRNA levels in the retinas were higher on P12 and P15 but lower on P17, compared with the control mice. Retinal hemorrhage was observed in the preterm mouse group (five out of six examined eyes). CONCLUSIONS: Preterm-birth mice that were subject to OIR exhibited several pathological features, such as retinal hemorrhage, severe retinal leakage and moderate retinal neovascularization, which were similar to the clinical manifestations in ROP patients.

αA crystallin may protect against geographic atrophy-meta-analysis of cataract vs. cataract surgery for geographic atrophy and experimental studies.

BACKGROUND: Cataract and geographic atrophy (GA, also called advanced "dry" age-related macular degeneration) are the two major causes of visual impairment in the developed world. The association between cataract surgery and the development of GA was controversial in previous studies. METHODS/PRINCIPAL FINDINGS: We performed a meta-analysis by pooling the current evidence in literature and found that cataract is associated with an increased risk of geographic atrophy with a summary odds ratio (OR) of 3.75 (95% CI: 95% CI: 1.84-7.62). However, cataract surgery is not associated with the risk of geographic atrophy (polled OR=3.23, 95% CI: 0.63-16.47). Further experiments were performed to analyze how the αA-crystallin, the major component of the lens, influences the development of GA in a mouse model. We found that theαA-crystallin mRNA and protein expression increased after oxidative stress induced by NaIO(3) in immunohistochemistry of retinal section and western blot of posterior eyecups. Both functional and histopathological evidence confirmed that GA is more severe in αA-crystallin knockout mice compared to wild-type mice. CONCLUSIONS: Therefore, αA-crystallin may protect against geographic atrophy. This study provides a better understanding of the relationship between cataract, cataract surgery, and GA.

Hypoxia specific SDF-1 expression by retinal pigment epithelium initiates bone marrow-derived cells to participate in Choroidal neovascularization in a laser-induced mouse model.

PURPOSE: Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age-related macular degeneration (AMD). Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) plays a critical role in homing of bone marrow-derived cells (BMCs) to choroidal neovascularization (CNV). In this study, we investigated the contribution of hypoxia specific HIF-1α-induced SDF-1 expression in retinal pigment epithelium (RPE) cells and the potential role of SDF-1 in CNV formation. MATERIALS AND METHODS: Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells of gfp(+/+) transgenic mice to sublethally irradiated C57BL/6J mice. CNV was induced by laser photocoagulation. Ocular tissue was processed for immunofluorescence to detect HIF-1α and SDF-1 expression, and cell surface markers such as CXCR4, CD34 and CD31 and so on during CNV formation. In vitro, adult human RPE (hRPE) cells were cultured under conditions of chemical hypoxia using CoCl2 administration. And RNAi technique was used to knock down HIF-1α gene to observe the expression of HIF-1α and SDF-1 in hRPE cells. RESULTS: BMCs trafficked around laser lesion adjacent to RPE layer 4 h after laser photocoagulation, where SDF-1 expression was relatively higher. With increasing expression of SDF-1, more BMCs were infiltrated into laser lesion to participate in CNV, and both reached peak at 3 d (p < 0.05). About 81% BMCs involved in CNV were CXCR4+. Many of them acquired the surface marker of endothelial precursor cells (CD34+) and endothelial cells (CD31+). The constituent ratio of CD34+ and CD31+ BMCs increased with SDF-1 expression. In vitro, we proved that hypoxia specific-HIF-1α influenced SDF-1 expression in hRPE cells. CONCLUSIONS: These findings suggested that hypoxia-induced SDF-1 expression in RPE might be a critical initiator for recruitment of BMCs in CNV. SDF-1 might be another important factor in BMCs' differentiation into endothelial cells to participate in the CNV.

Influence of Dll4 via HIF-1α-VEGF signaling on the angiogenesis of choroidal neovascularization under hypoxic conditions.

Choroidal neovascularization (CNV) is the common pathological basis of irreversible visual impairment encountered in a variety of chorioretinal diseases; the pathogenesis of its development is complicated and still imperfectly understood. Recent studies indicated that delta-like ligand 4 (Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF pathway to regulate CNV angiogenesis. But little is known about the influence and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time RT-PCR, Western blotting were used to analyze the expression alteration of Dll4, VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a laser-induced rat CNV model and intravitreal injection techniques were used to confirm the relationships among these molecules in vitro and in vivo. RPE-RF/6A cell co-culture systems were used to investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the co-culture system showed that the enhancement of Dll4 expression in RF/6A cells led to the significantly faster proliferation and stronger tube forming ability, but inhibited cells migration and invasion across a monolayer of RPE cells in hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite effects. Pharmacological disruption of Notch signaling using gamma-secretase inhibitor (GSI) produced similar, but not identical effects, to that caused by the Dll4 siRNA. In addition, the expression of several key molecules involved in the angiogenesis of CNV was altered in RF/6A cells showing constitutively active Dll4 expression. These results suggest that Dll4 play an important role in CNV angiogenesis, which appears to be regulated by HIF-1α and VEGF during the progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may facilitate further understanding of the mechanisms that underlie CNV angiogenesis.

The transcription factor RBP-J-mediated signaling is essential for dendritic cells to evoke efficient anti-tumor immune responses in mice.

BACKGROUND: Dendritic cells (DCs) are professional antigen presenting cells that initiate specific immune responses against tumor cells. Transcription factor RBP-J-mediated Notch signaling regulates DC genesis, but whether this pathway regulates DC function in anti-tumor immunity remains unclear. In the present work we attempted to identify the role of Notch signaling in DC-mediated anti-tumor immune response. RESULTS: When DCs were co-inoculated together with tumor cells, while the control DCs repressed tumor growth, the RBP-J deficient DCs had lost tumor repression activity. This was most likely due to that DCs with the conditionally ablated RBP-J were unable to evoke anti-tumor immune responses in the solid tumors. Indeed, tumors containing the RBP-J deficient DCs had fewer infiltrating T-cells, B-cells and NK-cells. Similarly, the draining lymph nodes of the tumors with RBP-J-/- DCs were smaller in size, and contained fewer cells of the T, B and NK lineages, as compared with the controls. At the molecular level, the RBP-J deficient DCs expressed lower MHC II, CD80, CD86, and CCR7, resulting in inefficient DC migration and T-cell activation in vitro and in vivo. T-cells stimulated by the RBP-J deficient DCs did not possess efficient cytotoxicity against tumor cells, in contrast to the control DCs. CONCLUSION: The RBP-J-mediated Notch signaling is essential for DC-dependent anti-tumor immune responses. The deficiency of RBP-J impairs the DC-based anti-tumor immunity through affecting series of processes including maturation, migration, antigen presentation and T-cell activation. The Notch signaling pathway might be a target for the establishment of the DC-based anti-tumor immunotherapies.

Notch-RBP-J signaling regulates the mobilization and function of endothelial progenitor cells by dynamic modulation of CXCR4 expression in mice.

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx) model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

Blockade of Notch signaling in tumor-bearing mice may lead to tumor regression, progression, or metastasis, depending on tumor cell types.

It has been reported that blocking Notch signaling in tumor-bearing mice results in abortive angiogenesis and tumor regression. However, given that Notch signaling influences numerous cellular processes in vivo, a comprehensive evaluation of the effect of Notch inactivation on tumor growth would be favorable. In this study, we inoculated four cancer cell lines in mice with the conditional inactivation of recombination signal-binding protein-Jkappa (RBP-J), which mediates signaling from all four mammalian Notch receptors. We found that whereas three tumors including hepatocarcinoma, lung cancer, and osteogenic sarcoma grew slower in the RBP-J-deficient mice, at least a melanoma, B16, grew significantly faster in the RBP-J-deficient mice than in the controls, suggesting that the RBP-J-deficient hosts could provide permissive cues for tumor growth. All these tumors showed increased microvessels and up-regulated hypoxia-inducible factor 1alpha, suggesting that whereas defective angiogenesis resulted in hypoxia, different tumors might grow differentially in the RBP-J-deleted mice. Similarly, increased infiltration of Gr1(+)/Mac1(+) cells were noticed in tumors grown in the RBP-J-inactivated mice. Moreover, we found that when inoculated in the RBP-J knockout hosts, the H22 hepatoma cells had a high frequency of metastasis and lethality, suggesting that at least for H22, deficiency of environmental Notch signaling favored tumor metastasis. Our findings suggested that the general blockade of Notch signaling in tumor-bearing mice could lead to defective angiogenesis in tumors, but depending on tumor cell types, general inhibition of Notch signaling might result in tumor regression, progression, or metastasis.

Disruption of the transcription factor recombination signal-binding protein-Jkappa (RBP-J) leads to veno-occlusive disease and interfered liver regeneration in mice.

UNLABELLED: Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal-binding protein-Jkappa (RBP-J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP-J resulted in LSEC proliferation and a veno-occlusive disease-like phenotype in the liver, as manifested by liver congestion, deposition of fibrin-like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP-J-deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma-membrane of LSs to hamper the microcirculation and intensify veno-occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules-such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin-6, and hepatocyte growth factor-was disturbed. CONCLUSION: Notch/RBP-J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation.

Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1alpha with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system.

BACKGROUND: Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia. METHODS: RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively. RESULTS: Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h. CONCLUSION: RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.