Single-cell communication patterns and their intracellular information flow in synovial fibroblastic osteoarthritis and rheumatoid arthritis.

BACKGROUND: Synovial fibroblasts are critical for maintaining homeostasis in major autoimmune diseases involving joint inflammation, including osteoarthritis and rheumatoid arthritis. However, little is known about the interactions among different cell subtypes and the specific sets of signaling pathways and activities that they trigger. METHODS: Using social network analysis, pattern recognition, and manifold learning approaches, we identified patterns of single-cell communication in OA (osteoarthritis) and RA (rheumatoid arthritis). RESULTS: Our results suggest that OA and RA have distinct cellular communication patterns and signaling pathways. The LAMININ (Laminin) and COLLAGEN (Collagen) pathways predominate in osteoarthritis, while the EGF (Epidermal growth factor), NT (Neurotrophin) and CDH5 (Cadherin 5) pathways predominate in rheumatoid arthritis, with a central role for THY1 (Thy-1 cell surface antigen) +CDH11 (Cadherin 11) + cells. The OA opens the PDGF (Platelet-derived growth factors) pathway (driver of bone angiogenesis), the RA opens the EGF pathway (bone formation) and the SEMA3 (Semaphorin 3A) pathway (involved in immune regulation). Interestingly, we found that OA no longer has cell types involved in the MHC complex (Major histocompatibility complex) and their activity, whereas the MHC complex functions primarily in RA in the presentation of inflammatory antigens, and that the complement system in OA has the potential to displace the function of the MHC complex. The specific signaling patterns of THY1+CDH11+ cells and their secreted ligand receptors are more conducive to cell migration and lay the foundation for promoting osteoclastogenesis. This subpopulation may also be involved in the accumulation of lymphocytes, affecting the recruitment of immune cells. Members of the collagen family (COL1A1 (Collagen Type I Alpha 1 Chain), COL6A2 (Collagen Type VI Alpha 2 Chain) and COL6A1 (Collagen Type VI Alpha 1 Chain)) and transforming growth factor (TGFB3) maintain the extracellular matrix in osteoarthritis and mediate cell migration and adhesion in rheumatoid arthritis, including the PTN (Pleiotrophin) / THBS1 (Thrombospondin 1) interaction. CONCLUSION: Increased understanding of the interaction networks between synovial fibroblast subtypes, particularly the shared and unique cellular communication features between osteoarthritis and rheumatoid arthritis and their hub cells, should help inform the design of therapeutic agents for inflammatory joint disease.

Targeting mutant p53 stabilization for cancer therapy.

Over 50% cancer bears TP53 mutation, the highly stabilized mutant p53 protein drives the tumorigenesis and progression. Mutation of p53 not only cause loss-of-function and dominant-negative effects (DNE), but also results in the abnormal stability by the regulation of the ubiquitin-proteasome system and molecular chaperones that promote tumorigenesis through gain-of-function effects. The accumulation of mutant p53 is mainly regulated by molecular chaperones, including Hsp40, Hsp70, Hsp90 and other biomolecules such as TRIM21, BAG2 and Stat3. In addition, mutant p53 forms prion-like aggregates or complexes with other protein molecules and result in the accumulation of mutant p53 in tumor cells. Depleting mutant p53 has become one of the strategies to target mutant p53. This review will focus on the mechanism of mutant p53 stabilization and discuss how the strategies to manipulate these interconnected processes for cancer therapy.

Butein inhibits cancer cell growth by rescuing the wild-type thermal stability of mutant p53.

p53 is a transcription factor that activates the expression of various genes involved in the maintenance of genomic stability, and more than 50% of cancers harbor inactivating p53 mutations, which are indicative of highly aggressive cancer and poor prognosis. Pharmacological targeting of mutant p53 to restore the wild-type p53 tumor-suppressing function is a promising strategy for cancer therapy. In this study, we identified a small molecule, Butein, that reactivates mutant p53 activity in tumor cells harboring the R175H or R273H mutation. Butein restored wild-type-like conformation and DNA-binding ability in HT29 and SK-BR-3 cells harboring mutant p53-R175H and mutant p53-R273H, respectively. Moreover, Butein enabled the transactivation of p53 target genes and decreased the interactions of Hsp90 with mutant p53-R175H and mutant p53-R273H proteins, while Hsp90 overexpression reversed targeted p53 gene activation. In addition, Butein induced thermal stabilization of wild-type p53, mutant p53-R273H and mutant p53-R175H, as determined via CETSA. From docking study, we further proved that Butein binding to p53 stabilized the DNA-binding loop-sheet-helix motif of mutant p53-R175H and regulated its DNA-binding activity via an allosteric mechanism, conferring wild-type-like the DNA-binding activity of mutant p53. Collectively, the data suggest that Butein is a potential antitumor agent that restores p53 function in cancers harboring mutant p53-R273H or mutant p53-R175H. SIGNIFICANCE: Butein restores the ability of mutant p53 to bind DNA by reversing its transition to the Loop3 (L3) state, endows p53 mutants with thermal stability and re-establishes their transcriptional activity to induce cancer cell death.

Outcomes of combined phacoemulsification, anterior vitrectomy, and sclerectomy in nanophthalmic eyes with glaucoma.

OBJECTIVES: To compare the long-term efficacy and safety of combined phacoemulsification, anterior vitrectomy, and sclerectomy (triple procedure surgery, TS); combined phacoemulsification and anterior vitrectomy (double procedure surgery, DS); and filtering surgery (FS) in nanophthalmos with angle-closure glaucoma (NACG). METHODS: Retrospective cohort study. Forty patients (44 eyes) diagnosed with NACG who underwent TS, DS, and FS were included. All eyes in the TS group and seven (47%) eyes in the DS group also underwent goniosynechialysis during the surgery. The main outcome measures (intraocular pressure [IOP], best-corrected visual acuity, complications, and second surgeries) were recorded at the early- (within 1 week) and late-stage (>3 months) follow-up. RESULTS: The late-stage IOP was significantly lower in the TS (mean ± standard deviation: 13.29 ± 2.49 mm Hg) than in the DS (19.69 ± 6.97 mm Hg) and FS groups (27.57 ± 12.26 mm Hg, p < 0.001). More visual improvements were observed in the TS and DS groups than in the FS group at late-stage follow-up (p = 0.04). The complication rates in the TS, DS, and FS groups were 26%, 33%, and 70%, respectively (p = 0.046); the second surgery rates were 0%, 33%, and 60%, respectively (p < 0.001). In total, one, three, and six severe complications were observed in the TS, DS, and FS groups, respectively. The mean follow-up durations in the TS, DS, and FS groups were 18.89, 20.02, and 25.75 months, respectively. CONCLUSIONS: NACG management remains challenging. TS presented relatively good clinical efficacy and safety with better postoperative IOP outcomes, lower complications, and second surgery rates among the three groups in eyes with NACG.

A score of DNA damage repair pathway with the predictive ability for chemotherapy and immunotherapy is strongly associated with immune signaling pathway in pan-cancer.

DNA damage repair (DDR) is critical in maintaining normal cellular function and genome integrity and is associated with cancer risk, progression, and therapeutic response. However, there is still a lack of a thorough understanding of the effects of DDR genes' expression level in cancer progression and therapeutic resistance. Therefore, we defined a tumor-related DDR score (TR-DDR score), utilizing the expression levels of 20 genes, to quantify the tumor signature of DNA damage repair pathways in tumors and explore the possible function and mechanism for the score among different cancers. The TR-DDR score has remarkably predictive power for tumor tissues. It is a more accurate indicator for the response of chemotherapy or immunotherapy combined with the tumor-infiltrating lymphocyte (TIL) and G2M checkpoint score than the pre-existing predictors (CD8 or PD-L1). This study points out that the TR-DDR score generally has positive correlations with patients of advanced-stage, genome-instability, and cell proliferation signature, while negative correlations with inflammatory response, apoptosis, and p53 pathway signature. In the context of tumor immune response, the TR-DDR score strongly positively correlates with the number of T cells (CD4+ activated memory cells, CD8+ cells, T regs, Tfh) and macrophages M1 polarization. In addition, by difference analysis and correlation analysis, COL2A1, MAGEA4, FCRL4, and ZIC1 are screened out as the potential modulating factors for the TR-DDR score. In summary, we light on a new biomarker for DNA damage repair pathways and explore its possible mechanism to guide therapeutic strategies and drug response prediction.

Performance of human papillomavirus (HPV) mRNA testing and HPV 16 and 18/45 genotyping combined with age stratification in the triaging of women with ASC-US cytology.

OBJECTIVES: This study aimed to assess the clinical performance of an HPV E6/E7 mRNA assay (Aptima HPV, AHPV) and AHPV 16 18/45 genotype assay (AHPV-GT) combined with age stratification for triaging women with atypical squamous cells of undetermined significance (ASC-US) cytology. METHODS: In total, 3052 women >21 years old with ASC-US cytology underwent AHPV testing, and AHPV-positive samples were reflex-tested with the AHPV-GT test. All women were referred for colposcopy and then biopsy if indicated. The AHPV and AHPV-GT test performances and risk estimates by hrHPV status with age stratification were calculated. RESULTS: Overall, 1599 women (52.4%) tested AHPV positive; of these women, 225 (7.4%), 101 (3.3%) and 1273 (41.7%) tested HPV 16+, HPV 18/45+ and other hrHPV-genotype-positive. When identifying CIN3+, the AHPV test had a 93.2% sensitivity and achieved a higher NPV (99.7% vs. 98.5%, P < 0.001) but a lower PPV (4.3% vs. 10.4%, P < 0.001) than the AHPV-GT test. The immediate risks of CIN3+ in AHPV+, other hrHPV+, and AHPV-GT+ women were 4.3%, 2.7%, and 10.4%, respectively. In the 21-24-year-old group, the immediate risks were 1.6%, 2.0% and 0.0%, which were below the 4.0% threshold for immediate colposcopy. The immediate colposcopy referral rate for AHPV-positive/ASC-US women 25 years or older was reduced from 51.7% to 10.5% by the AHPV-GT risk stratification method. CONCLUSIONS: AHPV testing with age stratification is effective for triaging women with ASC-US cytology. AHPV-GT testing may be a proper risk stratification method for women with AHPV-positive ASC-US cytology.

Etiologies and clinical characteristics of young patients with angle-closure glaucoma: a 15-year single-center retrospective study.

PURPOSE: To investigate the etiologies and the clinical characteristics of angle-closure glaucoma (ACG) patients younger than 40 years old in Chinese. METHODS: Inpatients with diagnosis of ACG and diagnosed age younger than or equal to 40 years old, who were admitted in Eye, Ear, Nose, and Throat Hospital Fudan University from 2002 to 2017, were included in this retrospective non-comparative case series. The underlying causes and clinical features for all the patients were analyzed by comprehensive review of medical charts. RESULTS: A total of 298 patients (463 eyes) met the criteria, including 153 females (51.3%) and 145 males (48.7%); the mean age was 25.6 ± 13.0 years. Primary angle-closure glaucoma (PACG), uveitis, and anterior segment dysgenesis (ASD) were the top three etiologies in our patients, which accounted for 32.6%, 20.3%, and 15.1% of the total patients respectively. PACG mainly occurs after 30 years of age and ASD is the top reason of ACG in patients younger than 20 years old. Other known etiologies include iridocorneal endothelial syndrome, neovascular glaucoma, nanophthalmos, retinitis pigmentosa, spherophakia, bestrophinopathy, persistent fetal vasculature, iridociliary cysts, congenital retinoschisis, Marfan's syndrome, retinopathy of prematurity, familial exudative vitreoretinopathy, congenital retinal folds, Coat's disease, and neurofibromatosis. CONCLUSIONS: We described the uncommon presentation of ACG in Chinese young patients. Although unusual, most of the etiologies could be identified. Therefore, more careful and comprehensive examinations are needed for early detection and timely treatment for young ACG patients.

Differential Effects of TGF-ß2 on the Low-Density Lipoprotein Receptor Expression in Three Types of Human Subconjunctival Fibroblasts.

Purpose: To investigate whether TGF-ß2 had a different effect on the expression levels of low-density lipoprotein receptor (LDLr) in the subconjunctival fibroblasts from glaucoma patients who underwent a reoperation (RGSFs) compared with those from glaucoma patients who underwent first filtering surgery (GSFs) and control patients with cataracts (HSFs). Methods: Human subconjunctival fibroblasts were obtained from the three groups of patients. Different concentrations of TGF-ß2 were added to the fibroblasts for 1, 3, and 5 days. The proliferation of the fibroblasts was determined by CCK-8 assays. Real-time PCR and western blotting were performed to analyze the mRNA and protein levels of LDLr. The uptake of DiI-labeled LDL was determined by confocal microscopy. Results: The results revealed that under TGF-ß2 exposure, fibroblast proliferation was positively correlated with LDLr expression (all p < .001). The LDLr mRNA and protein levels were affected by TGF-ß2 in a concentration-dependent and time-dependent manner in the RGSFs, GSFs and HSFs. The maximal expression of LDLr after TGF-ß2 stimulation was consistent with the peak uptake of DiI-LDL, which was obviously highest in the RGSFs, followed by the GSFs, and then the HSFs (all p < .05). All 3 groups took up DiI-LDL in a similar time-dependent manner, with maximal uptake at 6 h following DiI-LDL incubation (all p < .05). In addition, there were significant differences in the LDLr protein levels in the subconjunctival tissues isolated from the glaucoma patients during reoperation, the glaucoma patients during first filtering surgery and the control patients at day 3 (p < .05). The highest protein expression of LDLr was observed in the RG group. Conclusion: These data suggested that the RGSFs had the highest LDLr expression and the highest peak uptake of LDL among three groups. The LDLr-drug-LDL delivery system could potentially be used for targeted delivery of antimetabolite agents in anti-scarring therapy for glaucoma patients after filtering surgery.

miR-587 promotes cervical cancer by repressing interferon regulatory factor 6.

BACKGROUND: Interferon regulatory factor 6 (IRF6) exhibits tumor-suppressive functions in several cancer types. In the present study, the antitumor properties and related pathway mechanism of IRF6 were investigated in cervical cancer. METHODS: Forty-one pairs of cervical cancer specimens and para-carcinoma tissues were collected to evaluate IRF6 expression using immunohistochemical staining and miR-587. The effects of miR-587 and IRF6 on cervical cancer cell growth were explored by MTT assays and in a HeLa tumor xenograft mouse model. The migration and invasion of cervical cancer cells were monitored using transwell assays. RESULTS: IRF6 expression in cervical cancer specimens and cell lines was significantly reduced compared to that in the corresponding control group. In addition, IRF6 expression was negatively correlated with miR-587 in cervical cancer tissues. Bioinformatics algorithms and luciferase assays revealed that IRF6 is a potential target of miR-587, and miR-587 mimic transfection led to a significant repression of IRF6 protein levels in cervical cancer cells. We also discovered that the antineoplastic properties of IRF6 could be reversed by overexpressing miR-587 in cervical cancer cells. The up-regulation of miR-587 was correlated with poor overall survival in cervical cancer. In an in vivo experiment, miR-587 silencing induced HeLa tumor growth inhibition, which was associated with the up-regulation of IRF6 protein in the tumor. CONCLUSIONS: miR-587 post-transcriptionally represses IRF6 protein expression to abrogate the antineoplastic activity of IRF6. The miR-587/IRF6 signaling pathway plays a crucial role in the progression of cervical cancer and serves as a potential therapeutic target for the treatment of cervical cancer.

B-cell lymphoma 2 family genes show a molecular pattern of spatiotemporal heterogeneity in gynaecologic and breast cancer.

OBJECTIVES: BCL2 family proteins have been widely studied over the past decade due to their essential roles in apoptosis, oncogenesis and anti-cancer therapy. However, the similarities and differences in the spatial pattern of the BCL2 gene family within the context of chromatin have not been well characterized. We sought to fill this knowledge gap by assessing correlations between gene alteration, gene expression, chromatin accessibility, and clinical outcomes in gynaecologic and breast cancer. MATERIALS AND METHODS: In this study, the molecular characteristics of the BCL2 gene family in gynaecologic cancer were systematically analysed by integrating multi-omics datasets, including transcriptomics, chromatin accessibility, copy number variation, methylomics and clinical outcome. RESULTS: We evaluated spatiotemporal associations between long-range regulation peaks and tumour heterogeneity. Differential expression of the BCL2 family was coupled with widespread chromatin accessibility changes in gynaecologic cancer, accompanied by highly heterogeneous distal non-coding accessibility surrounding the BCL2L1 gene loci. A relationship was also identified between gene expression, gene amplification, enhancer signatures, DNA methylation and overall patient survival. Prognostic analysis implied clinical correlations with BAD, BIK and BAK1. A shared protein regulatory network was established in which the co-mutation signature of TP53 and PIK3CA was linked to the BCL2L1 gene. CONCLUSIONS: Our results provide the first systematic identification of the molecular features of the BCL2 family under the spatial pattern of chromatin in gynaecologic and breast cancer. These findings broaden the therapeutic scope of the BCL2 family to the non-coding region by including a significantly conserved distal region overlaying an enhancer.

Clinical significance of genotyping for human papillomavirus (HPV) 16 18/45 combined with cytology in cervical exfoliated cells in HPV oncogenic mRNA-positive women.

OBJECTIVES: This study aimed to assess human papilloma virus (HPV) 16 18/45 typing test results combined with cytology for cervical exfoliated cells from women who screened positive in an HPV E6/E7 mRNA assay (Aptima HPV, AHPV). METHODS: In total, 3257 AHPV-positive women aged 25-65 years were underwent AHPV 16 18/45 Genotype assay (AHPV-GT) testing with cytology. Women were referred for colposcopy and further biopsy if indicated. Different triaging strategies were compared. RESULTS: Overall, 624 women (19.2%) tested AHPV-GT positive. When identifying CIN2+, compared with cytology, AHPV-GT achieved a similar AUC (0.72 vs. 0.69, P = 0.158) but a higher specificity (85.1% vs. 79.3%, P < 0.001) and positive predictive value (PPV) (29.6% vs. 23.2%, P < 0.001). When identifying CIN2+, compared with cytology, the cotesting strategy (cytology combined with AHPV-GT) increased the AUC from 0.69 to 0.76 (P < 0.001), with a higher sensitivity (84.6% vs. 59.5%, P < 0.001), higher NPV (97.6% vs. 94.9%, P < 0.001) and similar PPV (21.6% vs. 23.2%, P = 0.054). When identifying CIN2+, the results of combination strategy (AHPV-GT genotyping plus reflex cytology in women positive for the 11 other hrHPV genotypes) were consistent with those of the cotesting strategy. Similar results were achieved when identifying CIN3 + . CONCLUSIONS: The AHPV-GT test may be a promising triage approach with high specificity in women receiving AHPV-positive primary screening tests. Although a combination strategy and cotesting strategy detected the same CIN2+ and CIN3+ cases, the former required significantly fewer screening tests.

Targeting circular RNA-ZRANB1 for therapeutic intervention in retinal neurodegeneration.

Glaucoma is a neurodegenerative disease characterized by retinal ganglion cell (RGC) loss, optic disc excavation, and progressive visual field loss. Direct or indirect ameliorating retinal neurodegeneration is a promising therapeutic therapy for glaucoma. Circular RNAs (circRNAs) are a class of covalently closed circular RNA transcripts and have emerged as potential regulators in several neurodegenerative diseases. In this study, we show that cZRANB1 expression is significantly upregulated in retinal neurodegeneration induced by glaucoma. cZRANB1 knockdown decreases retinal reactive gliosis, glial cell activation, and facilitates RGC survival in vivo. cZRANB1 knockdown directly regulates Müller cell function and indirectly regulates RGC function in vitro. cZRANB1 acts as miRNA sponge to regulate Müller cell function through cZRANB1/miR-217/RUNX2 network. Intervention of cZRANB1 expression would become an effective strategy for treating retinal neurodegeneration.

A proper triage for detecting women with high-risk human papillomavirus genotypes other than HPV16/18.

OBJECTIVE: To evaluate the effect of human papillomavirus (HPV) L1 capsid protein detection in cervical exfoliated cells as a proper triage for women with high-risk human papillomavirus (hrHPV) genotypes other than HPV 16/18. STUDY DESIGN: From January 2013 to June 2015, a total of 513 women aged 30-65 years infected with non-16/18 hrHPV were enrolled into the study. Primary HPV testing, HPV 16/18 genotyping and Papanicolaou (Pap) test were performed in all eligible women. HPV L1 capsid protein was detected by immunocytochemistry in cervical exfoliated cells. All hrHPV positive women underwent colposcopy and further biopsy if indicated. Relationships between HPV L1 capsid protein expression and histologic diagnosis, as well as cytology were analyzed. RESULTS: The positive expression rate of HPV L1 capsid protein in CIN2+ group was significantly lower than that in CIN1 or better group (16.3% vs. 66.7%, P=0.000). Compared with the Pap test, HPV L1 detection achieved higher sensitivity (83.7% vs. 51.2%, P=0.000), higher negative predictive value (NPV) (95.3% vs. 88.6%, P=0.002), and significant lower specificity (66.7% vs.76.1%, P=0.002) while identifying CIN2+. Compared with the Pap test, HPV L1 detection achieved higher sensitivity (89.7% vs. 51.7%, P=0.008), higher NPV (99.0% vs. 96.2%, P=0.043), and significant lower specificity (61.2% vs.73.8%, P=0.000) while identifying CIN3+. CONCLUSION: Because of its higher sensitivity and NPV than that of Pap test, HPV L1 capsid protein detection in cervical exfoliated cells reduces the missed identification of high-grade cervical intraepithelial neoplasia (CIN) in women with hrHPV genotypes other than HPV 16/18. HPV L1 capsid protein detection could be a potential triage for women with non-16/18 hrHPV infection.

Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus.

BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.

Silencing Of Circular RNA-ZNF609 Ameliorates Vascular Endothelial Dysfunction.

Vascular dysfunction is a hallmark of ischemic, cancer, and inflammatory diseases, contributing to disease progression. Circular RNAs (circRNAs) are endogenous non-coding RNAs, which have been reported to be abnormally expressed in many human diseases. In this study, we used retinal vasculature to determine the role of circular RNA in vascular dysfunction. We revealed that cZNF609 was significantly up-regulated upon high glucose and hypoxia stress in vivo and in vitro. cZNF609 silencing decreased retinal vessel loss and suppressed pathological angiogenesis in vivo. cZNF609 silencing increased endothelial cell migration and tube formation, and protected endothelial cell against oxidative stress and hypoxia stress in vitro. By contrast, transgenic overexpression of cZNF609 showed an opposite effects. cZNF609 acted as an endogenous miR-615-5p sponge to sequester and inhibit miR-615-5p activity, which led to increased MEF2A expression. MEF2A overexpression could rescue cZNF609 silencing-mediated effects on endothelial cell migration, tube formation, and apoptosis. Moreover, dysregulated cZNF609 expression was detected in the clinical samples of the patients with diabetes, hypertension, and coronary artery disease. Intervention of cZNF609 expression is promising therapy for vascular dysfunction.

Overexpression of low-density lipoprotein receptors stimulated by vascular endothelial growth factor in fibroblasts from pterygium.

The activation of subconjunctival fibroblasts is believed to be responsible for the pathogenesis of pterygium. Vascular endothelial growth factor (VEGF) appears to be the most potent stimulator of formation and progression of pterygium. Pterygium excision is a common procedure, although the recurrence rates remain high. Various postoperative adjuvant therapies are now attempted to lower the recurrence rate, with severe side effects. To offer a greater therapeutic effect and lower side effects, it's necessary to discover a constant nanoparticle drug delivery targeting to subconjunctival fibroblasts in pterygium (PSFs). This study was designed to investigate the expression of low-density lipoprotein receptor (LDLr) stimulated by VEGF in PSFs. We found that after exposure to VEGF, mRNA and protein levels of LDLr were both increased significantly in PSFs, assessed using relative quantitative real-time RT-PCR and Western blot. Moreover, it's demonstrated that the expression of LDLr were positively correlated with the cells proliferation. Uptake of DiI-LDL via live PSFs was increased with time, estimated by confocal microscopy. The protein expression of LDLr in pterygium subconjunctival tissues was significantly higher than in normal subconjunctival tissues. These results suggest that LDLr in the activated PSFs may become a novel target receptor for controlled drug delivery to lower postsurgical recurrence rate.

Long Noncoding RNA-GAS5: A Novel Regulator of Hypertension-Induced Vascular Remodeling.

Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA-growth arrest-specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through ß-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension.

Anti-angiogenic activity and mechanism of kaurane diterpenoids from Wedelia chinensis.

BACKGROUND: Wedelia chinensis is a traditional medicinal herb used in Asia and it has been reported to possess various bioactivities including anti-inflammatory and anticancer effects. However, its anti-angiogenic activity has never been reported. PURPOSE: To determine the most potent anti-angiogenic component in W. chinensis and its molecular mechanism of action. STUDY DESIGN: Initially, the active fraction of the plant was studied. Then, we determined the active components of the fraction and explored the mechanism of the most active compound. METHODS: The ethanol extract of W. chinensis and its four fractions with different polarities were evaluated for their anti-angiogenic activity in the Zebrafish model using quantitative endogenous alkaline phosphatase (EAP) assay. The molecular mechanism of the most active compound from the active fraction was studied using the real-time polymerase chain reaction (PCR) assay on Zebrafish embryos. The inhibitory effect of the most active compound on the proliferation, invasion and tube formation steps of angiogenesis was evaluated using the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) model, and the influences of the active compound on tyrosine phosphorylation of VEGF receptor (VEGFR-2) and its downstream signal pathway were evaluated by western blotting assay. Moreover, its anti-angiogenic effect was further evaluated by the VEGF-induced sprouts formation on aortic ring assay and the VEGF-induced vessel formation of mice on matrigel plug assay, respectively. RESULTS: Petroleum ether (PE) fraction of the plant displayed potent anti-angiogenic activity. Twelve kaurane diterpenoids (1-12) isolated from this fraction showed quite different effects. Compounds 9-12 could dose-dependently inhibit vessel formation in the Zebrafish embryos while the others showed little inhibitory effect. Among the active diterpenoids, compound 10, 3α-cinnamoyloxy-9ß-hydroxy-ent-kaura-16-en-19-oic acid (CHKA), possessed the strongest effect, and it affected multiple molecular targets related to angiogenesis including VEGF and angiopoietin in Zerbrafish. Moreover, CHKA significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, invasion, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways in HUVECs. CHKA also obviously inhibited sprouts formation of aortic ring, and block vessel formation in mice. CONCLUSION: Our findings demonstrate that kaurane diterpenoids is one of anti-angiogenic components in W. chinensis, and CHKA may become a promising candidate for the development of anti-angiogenic agent.

Diterpenoids from the roots of Croton crassifolius and their anti-angiogenic activity.

Six diterpenoids [crassifolin J, K, L, M, N and O] along with eleven known ones were isolated from the supercritical fluid extract (SFE) of the roots of Croton crassifolius (Euphorbiaceae). Their structures were elucidated using spectroscopic methods (IR, UV, HRESIMS, 1D and 2D NMR). The structure and stereochemistry of crassifolin J was confirmed by single-crystal X-ray diffraction analysis, and the absolute configurations of crassifolin K-M were determined by CD spectra. Twenty-three diterpenoids from this plant were screened for their anti-angiogenic activity using a wild-type zebrafish in vivo model. Four of the known compounds were active, of which penduliflaworosin possessed the best activity relative to the positive control (SU5416). Further study demonstrated that penduliflaworosin could inhibit vessel formation on Tg(fli1a:EGFP)y1-type zebrafish embryos.

Potent anti-angiogenic component in Croton crassifolius and its mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Croton crassifolius Geisel is traditionally used in China for the treatment of snake bites, stomach ache, sternalgia, joint pain, pharyngitis, jaundice and rheumatoid arthritis, while in Thailand, it has been used as an anticancer herbal medicine by the indigenous people. Yet, its pharmacological studies are still limited, especially towards its anticancer property. Anti-angiogenesis is a promising therapeutic strategy in the anti-cancer treatment. Previous studies have shown strong anti-angiogenic activity in the low polar fraction of the herb. Nevertheless, the potent compound which is responsible for the anti-angiogenesis, and its molecular mechanism have never been reported. AIM OF THE STUDY: To determine the potent anti-angiogenic component in C. crassifolius and its molecular mechanism of action. MATERIALS AND METHODS: C. crassifolius was extracted using supercritical fluid extraction and steam distillation. The anti-angiogenic activities of the two extracts were evaluated in the zebrafish model by quantitative endogenous alkaline phosphatase assay. The chemical compounds in the active extract were isolated using chromatographic methods, and their structures were elucidated using different spectroscopic techniques. The content/quantity of the active compounds in this extract was determined with HPLC analysis. The molecular mechanism of the most active compound was further studied using the real-time PCR assay. Besides, its cytotoxicity on various cancer and normal cell lines was evaluated using the cell-counting kit. RESULTS: Supercritical fluid extract (SFE) of C. crassifolius showed better anti-angiogenic activity than that of steam distillation extract (SDE). Three sesquiterpenes, namely, cyperenoic acid, 8-hydroxy-α-guaiene and (+)-guaia-l(10),ll-dien-9-one, were isolated and identified in the SFE. Among them, cyperenoic acid displayed the strongest anti-angiogenic activity by 51.7% of the control at 10µM, while the others showed little effect. HPLC results showed that cyperenoic acid was the major component in the SFE with 9.97% (w/w). Results of the real-time PCR assay suggested that the cyperenoic acid affected multiple molecular targets related to angiogenesis including vascular endothelial growth factor (Vegfa), angpiopoietin (Angpt), and their receptors. Cytotoxicity assay showed cyperenoic acid possessed little toxicity toward cancer and normal cells. CONCLUSIONS: Cyperenoic acid is an important anti-angiogenic component present in C. crassifolius and serve as a potent inhibitor in the angiogenesis in the zebrafish embryo model. The anti-angiogenic property, but not the cytotoxicity, of C. crassifolius provides a scientific basis for its traditional use in cancer treatment.