Hypoxia-induced AFAP1L1 regulates pathological neovascularization via the YAP-DLL4-NOTCH axis.

BACKGROUND: Pathological neovascularization plays a pivotal role in the onset and progression of tumors and neovascular eye diseases. Despite notable advancements in the development of anti-angiogenic medications that target vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), the occurrence of adverse reactions and drug resistance has somewhat impeded the widespread application of these drugs. Therefore, additional investigations are warranted to explore alternative therapeutic targets. In recent years, owing to the swift advancement of high-throughput sequencing technology, pan-cancer analysis and single-cell sequencing analysis have emerged as pivotal methodologies and focal areas within the domain of omics research, which is of great significance for us to find potential targets related to the regulation of pathological neovascularization. METHODS: Pan-cancer analysis and scRNA-seq data analysis were employed to forecast the association between Actin filament-associated protein 1 like 1 (AFAP1L1) and the development of tumors and endothelial cells. Tumor xenograft model and ocular pathological neovascularization model were constructed as well as Isolectin B4 (IsoB4) staining and immunofluorescence staining were used to assess the effects of AFAP1L1 on the progression of neoplasms and neovascular eye diseases in vivo. Transwell assay, wound scratch assay, tube forming assay, three-dimensional germination assay, and rhodamine-phalloidin staining were used to evaluate the impact of AFAP1L1 on human umbilical vein endothelial cells (HUVECs) function in vitro; Dual luciferase reporting, qRT-PCR and western blot were used to investigate the upstream and downstream mechanisms of pathological neovascularization mediated by AFAP1L1. RESULTS: Our investigation revealed that AFAP1L1 plays a crucial role in promoting the development of various tumors and demonstrates a strong correlation with endothelial cells. Targeted suppression of AFAP1L1 specifically in endothelial cells in vivo proves effective in inhibiting tumor formation and ocular pathological neovascularization. Mechanistically, AFAP1L1 functions as a hypoxia-related regulatory protein that can be activated by HIF-1α. In vitro experiments demonstrated that reducing AFAP1L1 levels can reverse hypoxia-induced excessive angiogenic capacity in HUVECs. The principal mechanism of angiogenesis inhibition entails the regulation of tip cell behavior through the YAP-DLL4-NOTCH axis. CONCLUSION: In conclusion, AFAP1L1, a newly identified hypoxia-related regulatory protein, can be activated by HIF-1α. Inhibiting AFAP1L1 results in the inhibition of angiogenesis by suppressing the germination of endothelial tip cells through the YAP-DLL4-NOTCH axis. This presents a promising therapeutic target to halt the progression of tumors and neovascular eye disease.

Targeting FSCN1 with an oral small-molecule inhibitor for treating ocular neovascularization.

BACKGROUND: Ocular neovascularization is a leading cause of blindness and visual impairment. While intravitreal anti-VEGF agents can be effective, they do have several drawbacks, such as endophthalmitis and drug resistance. Additional studies are necessary to explore alternative therapeutic targets. METHODS: Bioinformatics analysis and quantitative RT-PCR were used to detect and verify the FSCN1 expression levels in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) mice model. Transwell, wound scratching, tube formation, three-dimensional bead sprouting assay, rhodamine-phalloidin staining, Isolectin B4 staining and immunofluorescent staining were conducted to detect the role of FSCN1 and its oral inhibitor NP-G2-044 in vivo and vitro. HPLC-MS/MS analysis, cell apoptosis assay, MTT assay, H&E and tunnel staining, visual electrophysiology testing, visual cliff test and light/dark transition test were conducted to assess the pharmacokinetic and security of NP-G2-044 in vivo and vitro. Co-Immunoprecipitation, qRT-PCR and western blot were conducted to reveal the mechanism of FSCN1 and NP-G2-044 mediated pathological ocular neovascularization. RESULTS: We discovered that Fascin homologue 1 (FSCN1) is vital for angiogenesis both in vitro and in vivo, and that it is highly expressed in oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV). We found that NP-G2-044, a small-molecule inhibitor of FSCN1 with oral activity, can impede the sprouting, migration, and filopodia formation of cultured endothelial cells. Oral NP-G2-044 can effectively and safely curb the development of OIR and CNV, and increase efficacy while overcoming anti-VEGF resistance in combination with intravitreal aflibercept (Eylea) injection. CONCLUSION: Collectively, FSCN1 inhibition could serve as a promising therapeutic approach to block ocular neovascularization.

The role of PIWIL4 and piRNAs in the development of choroidal neovascularization.

Wet age-related macular degeneration (wAMD) is the leading cause of blindness among the elderly in industrialized nations. Anti-vascular epidermal growth factor (VEGF) therapy via intravitreal injection is the most effective clinical treatment for wAMD due to high concentrations of VEGF that promote choroidal neovascularization. While PIWI proteins participate in various biological processes, their function in AMD remains unclear. In this study, we discovered that PIWIL4 expression is elevated in a laser-induced choroidal neovascularization (CNV) model and that it regulates angiogenesis in vitro and in vivo. Differentially expressed piwi-interacting RNAs (piRNAs) were identified in a CNV model and were shown to potentially regulate angiogenesis via bioinformatics analysis. PIWIL4 knockdown inhibits VEGF secretion and VEGFR2 phosphorylation. Overall, PIWIL4 may serve as a novel target to block pathological choroidal neovascularization, and the study of the PIWI-piRNAs pathway in wAMD highlights its broad function in somatic cells.

Suppression of Pathological Ocular Neovascularization by a Small Molecular Multi-Targeting Kinase Inhibitor, DCZ19903.

Purpose: The administration of anti-vascular endothelial growth factor agents is the standard firs-line therapy for ocular vascular diseases, but some patients still have poor outcomes and drug resistance. This study investigated the role of DCZ19903, a small molecule multitarget kinase inhibitor, in ocular angiogenesis. Methods: The toxicity of DCZ19903 was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays, flow cytometry, Calcein-AM/PI staining, and terminal uridine nick-end labeling staining. Oxygen-induced retinopathy and laser-induced choroidal neovascularization models were adopted to assess the antiangiogenic effects of DCZ19903 by Isolectin B4 (GS-IB4) and hematoxylin-eosin staining. EdU assays, transwell migration assays, tube formation, and choroid sprouting assays were performed to determine the antiangiogenic effects of DCZ19903. The antiangiogenic mechanism of DCZ19903 was determined using network pharmacology approach and western blots. Results: There was no obvious cytotoxicity or tissue toxicity after DCZ19903 treatment. DCZ19903 exerted the antiangiogenic effects in OIR model and choroidal neovascularization model. DCZ19903 inhibited the proliferation, tube formation, migration ability of endothelial cells, and choroidal explant sprouting. DCZ19903 plus ranibizumab achieved greater antiangiogenetic effects than DCZ19903 or ranibizumab alone. DCZ19903 exerted its antiangiogenic effects via affecting the activation of ERK1/2 and p38 signaling. Conclusions: DCZ19903 is a promising drug for antiangiogenic treatment in ocular vascular diseases. Translational Relevance: These findings suggest that DCZ19903 possesses great antiangiogenic potential for treating ocular vascular diseases.

DCZ19931, a novel multi-targeting kinase inhibitor, inhibits ocular neovascularization.

Neovascularization is a prominent cause of irreversible blindness in a variety of ocular diseases. Current therapies for pathological neovascularization are concentrated on the suppression of vascular endothelial growth factors (VEGF). Despite the remarkable efficacy of anti-VEGF drugs, several problems still exist, including ocular complications and drug resistance. Thus, it is still required to design novel drugs for anti-angiogenic treatment. This study aimed to investigate the anti-angiogenic effects of a small molecule multi-target tyrosine kinase inhibitor, DCZ19931, on ocular neovascularization. The results showed that administration of DCZ19931 at the tested concentrations did not cause obvious cytotoxicity and tissue toxicity. DCZ19931 could reduce the size of choroidal neovascularization (CNV) lesions in laser-induced CNV model and suppress ocular neovascularization in oxygen-induced retinopathy (OIR) model. DCZ19931 could suppress VEGF-induced proliferation, migration, and tube formation ability of endothelial cells, exhibiting similar anti-angiogenic effects as Ranibizumab. DCZ19931 could reduce the levels of intercellular cell adhesion molecule-1 (ICAM-1) expression in vivo and in vitro. Network pharmacology prediction and western blots revealed that DCZ19931 exerted its anti-angiogenic effects through the inactivation of ERK1/2-MAPK signaling and p38-MAPK signaling. In conclusion, this study indicates that DCZ19931 is a promising drug for anti-angiogenic therapy for ocular diseases.

NKG2D ligand RAE1ε induces generation and enhances the inhibitor function of myeloid-derived suppressor cells in mice.

Expression of surface NKG2D ligands on tumour cells, which activates nature killer (NK) cells and CD8+ T cells, is crucial in antitumour immunity. Some types of tumours have evolved mechanisms to suppress NKG2D-mediated immune cell activation, such as tumour-derived soluble NKG2D ligands or sustained NKG2D ligands produced by tumours down-regulate the expression of NKG2D on NK cells and CD8+ T cells. Here, we report that surface NKG2D ligand RAE1ε on tumour cells induces CD11b+ Gr-1+ myeloid-derived suppressor cell (MDSC) via NKG2D in vitro and in vivo. MDSCs induced by RAE1ε display a robust induction of IL-10 and arginase, and these MDSCs show greater suppressive activity by inhibiting antigen-non-specific CD8+ T-cell proliferation. Consistently, upon adoptive transfer, MDSCs induced by RAE1ε significantly promote CT26 tumour growth in IL-10- and arginase-dependent manners. RAE1ε moves cytokine balance towards Th2 but not Th1 in vivo. Furthermore, RAE1ε enhances inhibitory function of CT26-derived MDSCs and promotes IL-4 rather than IFN-γ production from CT26-derived MDSCs through NKG2D in vitro. Our study has demonstrated a novel mechanism for NKG2D ligand+ tumour cells escaping from immunosurveillance by facilitating the proliferation and the inhibitory function of MDSCs.

[A study of hepatitis B virus subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou].

OBJECTIVE: To investigate hepatitis B virus (HBV) subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou and to study the relationship between the subtypes and the progression of their liver diseases. METHODS: Using PCR, 309 bp gene fragments in the HBV p region were amplified. The products of PCR were digested by VspI, NciI, BstEII and subjected to agarose gel electrophoresis. The subtypes of C1 and C2 were detected by restriction fragment length polymorphism (RFLP). B subtype was determined by direct sequencing of PCR product. One hundred seventy-eight patients with genotype B or C HBV infection in Guizhou, including 50 asymptomatic carriers (ASC), 100 chronic hepatitis (CHB), 14 liver cirrhosis (LC), and 14 hepatocellular carcinoma (HCC) patients were examined. The relationship between HBV C subtypes and the progression of their liver diseases was studied by analyzing the HBeAg positivity, HBV DNA loads and ALT levels of the patients. RESULTS: Of 84 patients with HBV genotype C, 27 (32.14%) and 56 (66.67%) were subtype C1 and C2, respectively. In 94 genotype B, 93 (98.94%) were subtype Ba and only one was subtype Bj. Subtype C1 showed a trend of gradual decrease from ASC and CHB to LC/HCC groups. In contrast, subtype C2 showed a gradual increase (trend) in the same order. The HBeAg positivity was significantly lower in subtype C1 than that in subtype C2. The ALT levels and HBV DNA loads were higher in patients with subtype C2 than those in subtype C1, however no statistical significance was found in these primes (t=0.95, 0.79). CONCLUSION: Subtype Ba is major and subtype C2 is more common in Guizhou. The distribution of subtype C1 and C2 are different in various stages of liver disease. The PCR-RFLP method is simple and accurate and can be used in a large-scale survey.

[Study on the distribution of hepatitis B virus precore and basic core promoter mutations in Guizhou area].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) precore A1896 and basic core promoter (BCP)T1762/A1764 mutations in Guizhou area. METHODS: 482 patients with chronic HBV infection, belonging to 4 nationalities, including 225 asymptomatic carriers (ASC), 158 chronic hepatitis (CH), 57 liver cirrhosis (LC), 42 hepatocellular carcinoma (HCC), from 4 areas of Guizhou province were examined. HBV A1896 and T1762/A1764 mutations were determined by direct sequencing and restriction fragment length polymorphism (RFLP). HBV genotypes were determined by PCR-RFLP based on S gene. The relationship among these mutations and genotype and the progression of liver disease were studied by multi-normal logistic regression analysis. RESULTS: A1896 and T1762/A1764 mutations were detected 23.03% and 29.67% among 482 patients. These mutations were more prevalent in Hans than in Dong, Miao and Buyi minorities (P < 0.01, respectively). The mutations of A1896 and T1762/ A1764 were more commonly seen in HBeAg negative than in HBeAg positive patients (P < 0.01, respectively). The mutation of T1762/A1764 was significantly higher in genotype C than in genotype B (P < 0.01). There were significantly statistical differences in the detective rate of A1896 and T1762/ A1764 mutations between patients with HCC, LC and CH, ASC. The distribution of these mutations in Guiyang (31.79% and 41.06%) was higher than in Zunye (10.94%, 14.06%), Duyun (8.64%, 11.11%) or Kaili (2.86%, 2.86%). However, there was no statistical difference by multi-normal logistic regression analysis after controlling the influence of HBeAg statu, genotype and clinical types. CONCLUSION: The distributions of A1896 and T1762/A1764 mutations were different in some nationalities of Guizhou province. The mutation of T1762/A1764 was more commonly seen in genotype C than in genotypr B. These mutations were closely related to progression of chronic liver diseases. Hepatitis B virus; Genotype; Restriction fragment length polymorphism

[Comparative study on detection of hepatitis B virus mutants in precore region with two methods].

OBJECTIVE: To establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detection of hepatitis B virus (HBV) mutation in precore A1896, and compare with direct sequencing for evaluating its applicability. METHODS: According to the principle of mPCR, 194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis. A method for detecting procore A1896 mutation was established by restricted fragment length polymorphism. Totally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with precore wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing. RESULTS: From 134 sera, 117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method, 109 could be analyzed by sequencing. In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains. Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning. CONCLUSION: Compared with sequencing, the mPCR-RFLP method is simple, accurate and can be used in large-scale surveys and clinical research.

[A study in the Guizhou area on pre-S region mutations of hepatitis B virus].

OBJECTIVE: To study the relationships between hepatitis B virus (HBV ) pre-S region mutations and their genotypes and the stages of liver disease of the patients. METHODS: The entire HBV pre-S1 and pre-S2 genes were amplified by polymerase chain reaction (PCR). The amplified products were digested by NlaIII restriction enzyme. A detecting method for pre-S2 start codon mutation was established according to the restriction fragment length polymorphism (RFLP) analysis. Pre-S region deletion was revealed by polyacrylamide gel electrophoresis (PAGE). Fourteen sera having pre-S deletions or pre-S2 start codon mutations and wild strains were directly sequenced. HBV genotypes were determined by RFLP based on S-gene PCR products. One hundred sixty serum samples were collected from patients with HBV related diseases and they were determined by the above methods. The relationships between HBV pre-S region mutations and their genotypes and the stages of liver disease of the patients were analysed. RESULTS: Of the 160 sera, genotype B and C were identified in 81 and 79 respectively. The detected ratios of pre-S2 start codon and pre-S deletion mutations were significantly higher in genotype C than in genotype B (43.04% vs 1.23%, 36.71% vs 19.75%, P<0.05, respectively). The detection rates of pre-S2 start codon mutation were significantly different in different groups: from 50.00% (HCC), 39.47% (LC), 8.00% (CH), to ASC (0). The detection rates of pre-S deletion mutations among patients with HCC (53.13%), LC (42.11%), CH (18.00%) and ASC (7.50%) also varied significantly. The results obtained from sequencing and PCR-RFLP/PAGE were completely compatible. Multivariate analysis indicated that genotype C (OR=6.26, P<0.01) and advanced liver disease (OR=11.99, P<0.01) were significant variables for pre-S mutations development. CONCLUSION: The pre-S2 start codon and pre-S deletion mutations are more common in genotype C than in genotype B. These mutations are closely related to the progression of liver disease.

[A study on detection method of lamivudine related mutations in hepatitis B virus polymerase gene].

OBJECTIVE: To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene. METHODS: HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing. RESULTS: The nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases. CONCLUSION: The nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

[Establishing a new genotyping method of hepatitis B virus by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) to analysis on S region and its application].

OBJECTIVE: To establish a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method of genotyping HBV using Mbo I, BsTN I, BsmA I, Hpa II and investigate the relationship between genotype and clinical spectrum of hepatitis B. METHODS: 124 full-genomic HBV sequences and 13 S-genomic sequences were analyzed, genotype specific regions were identified by the restriction enzymes Mbo I, BsTN I, BsmA I, Hpa II. And 176 samples from different kinds of hepatitis B were genotyped by this method. Five samples had been randomly selected and directly sequenced their S gene, to assess the accuracy. RESULTS: In 176 serum samples of patients with hepatitis B from Guizhou area, genotype B and C were found in 56.8% and 43.2% respectively. The proportions of genotype B and C in ASC were 40.0% and 15.7% (chi-square = 12.16, P < 0.005); and they were 31.6% and 14.0% in CHB (chi-square = 7.88, P < 0.005). CONCLUSION: Genotyping HBV, based on S gene RFLP seems to be highly sensitive, differential and accurate and could be used in large-scale surveys. HBV genotype B and C are existed in Guizhou area.

[Distribution of hepatitis B virus genotype among population of Dong, Miao minority and Han in Guizhou].

OBJECTIVE: To study the distribution of hepatitis B virus (HBV) genotype in population of Dong, Miao minority and Han in Guizhou. METHODS: S region nucleotides were compared in 127 strains whole sequence of HBV and three restriction enzymes which can be used for genotyping were found by DNA software analysis system. The partial gene fragment of HBV S region was amplified by nested polymerase chain reaction (nPCR). The products were digested with Mbo I, BstN I or BsmA I and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were analysed. The genotypes were determined by nPCR-RFLP in 166 asymptomatic HBV carriers (ASC), including 48 Dong minority, 52 Miao minority and 66 Han subjects. Some of the ASC were also analysed by direct sequencing of PCR products. RESULTS: The nPCR-RFLP method was simple and accurate. Of the 166 ASC, 138 (83.13%) were genotype B, and 28 (16.87%) were genotype C. Of the 48 Dong minority subjects, 47 (97.2%) were genotype B and 1 (2.08%) was type C. Of the 52 Miao minority subjects, 49 (94.23%) were genotype B and 3 (5.77%) were genotype C. Of the 66 Han subjects, 42 (63.64%) were genotype B and 24 (36.36%) were genotype C. There was a statistical significance in the distribution of genotype C between Dong, Miao minority and Han (7.85% vs 36.36% P less than 0.005). CONCLUSION: Genotype B and C exist in Guizhou and genotype B is the major genotype. Genotype C is found more frequently in Han than in Dong and Miao minority.

[Quantitative analysis of HBV DNA amplified products with microtiter hybridization].

BACKGROUND: To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA). METHODS: The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length. RESULTS: Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively. CONCLUSIONS: The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

[Detection of TTV DNA and analysis of partial TTV gene sequence in different population of Guizhou area].

OBJECTIVE: To investigate the prevalence of transfusion-transmitted virus (TTV) infection and analyze partial nucleotide sequence of TTV isolated from Guizhou area. METHODS: A nested polymerase chain reaction (nPCR) assay with two-set of primers deduced from the first read frame of TTV genome was established, the prevalence of TTV DNA were detected in 395 sera collected from different population in Guizhou area. The partial genes of TTV DNA fragments amplified from two liver disease patients and one hemodialysis patient were analysed and then compared with gene from Japan strain. RESULTS: The positive rates of TTV DNA in 62 normal adults, 37 blood donors, 50 hemodialysis patients, 107 intravenous drug users (IVDUs), 139 patients with liver diseases were 6.45% (4/62), 8.10%(3/37), 26.00% (13/50), 25.23% (27/107) and 16.54% (23/139), respectively. The homology of TTV DNA sequence was from 99% to 100% within three Guizhou strains. As compared with published sequence, the nucleotide homology is 98% between all three Guizhou strains and Japan strain. CONCLUSIONS: There are TT virus infection in Guizhou area, the infection rates were higher in hemodialysis patients and IVDUs. The nucleotide homology was high between Guizhou strain and Japan strain.