Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: a HUVECs were treated with normal glucose (5.5 mM, NG) or high glucose (15 or 30 mM, HG) for 0, 24, 48 or 72 h, and cell viability was examined with MTT ( n = 3). HG-treated HUVECs were co-cultured with BMSC-exos or equal volume of PBS. NG and HG-treated cells were used as controls. b HUVEC viability was examined with MTT ( n = 3). c EdU incorporation analysis of HUVECs ( n = 3, scale bar, 100 µm). d Cell apoptosis analysis by TUNEL ( n = 3, scale bar, 100 µm). e Transwell assays for HUVEC migration analysis ( n = 3, scale bar, 100 µm). f Tube formation analysis of HUVECs ( n = 3, scale bar, 100 µm). g The concentration of VEGFA in the supernatants of HUVECs ( n = 3). h The expression of VEGFA, VEGFR2, PDGF and EGF was assessed by western blot ( n = 3). GAPDH was used a normalization control. n = 3 represents biologically independent experiments. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Cell Culture, TUNEL Assay, Migration, Concentration Assay, Expressing, Western Blot, Control

Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: a RT-qPCR analysis of circMYO9B in BMSCs and BMSC-exos ( n = 3). b RT-qPCR analysis of circMYO9B in BMSCs (control), BMSCs transfected with oe-NC, oe-circMYO9B, sh-NC or sh-circMYO9B ( n = 3) and BMSC-Exos. HG-treated HUVECs were co-cultured with Exo-oe-NC, Exo-oe-circMYO9B, Exo-sh-NC or Exo-sh-circMYO9B. HG-treated HUVECs were used as control cells. c EdU incorporation analysis of HUVECs ( n = 3, scale bar, 100 µm). d Cell apoptosis analysis by TUNEL ( n = 3, (scale bar, 100 µm). e Transwell assays for HUVEC migration analysis ( n = 3, scale bar, 100 µm). f Tube formation analysis of HUVECs ( n = 3, scale bar, 100 µm). g The concentration of VEGFA in the supernatants of HUVECs ( n = 3). h The expression of VEGFA, VEGFR2, PDGF and EGF was assessed by western blot ( n = 3). GAPDH was used a normalization control. n = 3 represents biologically independent experiments. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Quantitative RT-PCR, Control, Transfection, Cell Culture, TUNEL Assay, Migration, Concentration Assay, Expressing, Western Blot

Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: a KDM1A and VEGFA were detected in HUVECs transfected with sh-NC or sh-KDM1A via western blot ( n = 3). GAPDH was used a normalization control. b Three potential binding sites (BS1: −1485/−1470, BS2: −289/−268 and BS3: −226/−205) for KDM1A in VEGFA . c ChIP assays for evaluating the interaction between KDM1A and BS1, BS2 or BS3 ( n = 3). d Luciferase activity of VEGFA -WT and VEGFA -MUT reporters in HUVECs co-transfected with sh-NC or sh-KDM1A ( n = 3). HUVECs transfected with sh-NC, sh-KDM1A, vector or KDM1A were co-cultured with Exo-oe-circMYO9B or Exo-sh-circMYO9B. HG-treated HUVECs were used as control cells. The expression of KDM1A was assessed by RT-qPCR ( e , n = 3) and western blot ( f , n = 3). GAPDH was used a normalization control. g EdU incorporation analysis of HUVECs ( n = 3, scale bar, 100 µm). h Cell apoptosis analysis by TUNEL ( n = 3, scale bar, 100 µm). i Transwell assays for HUVEC migration analysis ( n = 3, scale bar, 100 µm). j Tube formation analysis of HUVECs ( n = 3, scale bar, 100 µm). k The concentration of VEGFA in the supernatants of HUVECs ( n = 3). n = 3 represents biologically independent experiments. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Transfection, Western Blot, Control, Binding Assay, Luciferase, Activity Assay, Plasmid Preparation, Cell Culture, Expressing, Quantitative RT-PCR, TUNEL Assay, Migration, Concentration Assay

Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: a The interaction between hnRNPU and VEGFA mRNA was evaluated by RIP assays ( n = 3). b , c RT-qPCR and western blot analysis of KDM1A and ZNF24 in HUVECs transfected with oe-NC or oe-circMYO9B ( n = 3). GAPDH was used a normalization control. d The interaction between hnRNPU and KDM1A was analyzed by IP assays ( n = 3). e Potential E3 ubiquitin ligases which interacted with KDM1A. f , g RT-qPCR and western blot analysis of CBL in HUVECs transfected with oe-NC or oe-circMYO9B ( n = 3). GAPDH was used a normalization control. h Enrichment of circMYO9B, CBL and U6 in the hnRNPU antibody or normal IgG-immunoprecipitated fractions was analyzed by RIP assays ( n = 3). i Western blot analysis of hnRNPU and CBL in HUVECs transfected with oe-NC or oe-hnRNPU ( n = 3). GAPDH was used a normalization control. j The stability of CBL mRNA in HUVECs transfected with oe-NC or oe-circMYO9B after Act D treatment for 0, 2, 4, 6 or 8 h. n = 3 represents biologically independent experiments. Data were presented as mean ± SD. ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Control, Immunoprecipitation

Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: a Western blot analysis of the ubiquitination of immunoprecipitated KDM1A in HUVECs transfected with si-NC or si-CBL ( n = 3). b The stability of KDM1A in HUVECs transfected with si-NC or si-CBL after CHX treatment for 0, 2, 4, 6 or 8 h ( n = 3). c Western blot analysis of the ubiquitination of immunoprecipitated KDM1A in HUVECs transfected with oe-NC or oe-circMYO9B ( n = 3). d Western blot analysis of the expression of CBL, VEGFA and KDM1A in HUVECs transfected with oe-NC, oe-circMYO9B, oe-circMYO9B+oe-NC, oe-circMYO9B+oe-CBL ( n = 3). GAPDH was used as a normalization control. HUVECs were transfected with oe-NC or oe-CBL and treated with HG, and Exo-oe-circMYO9B or Exo-sh-circMYO9B was added for coculture. HG-treated HUVECs were used as control cells. e Western blot analysis of CBL, VEGFA and KDM1A. GAPDH was used a normalization control. f EdU incorporation analysis of HUVECs (n = 3, scale bar, 100 µm). g Cell apoptosis analysis by TUNEL ( n = 3, scale bar, 100 µm). h Transwell assays for HUVEC migration analysis ( n = 3, scale bar, 100 µm). i Tube formation analysis of HUVECs (n = 3, scale bar, 100 µm). n = 3 represents biologically independent experiments. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Control, TUNEL Assay, Migration

Journal: Communications Biology

Article Title: MSC-derived exosomal circMYO9B accelerates diabetic wound healing by promoting angiogenesis through the hnRNPU/CBL/KDM1A/VEGFA axis

doi: 10.1038/s42003-024-07367-z

Figure Lengend Snippet: A mouse model of DFU was established, and PBS (control), MSC-Exo-oe-NC, MSC-Exo-oe-circMYO9B or MSC-Exo-sh-circMYO9B was injected into surrounding areas of wound ( n = 8). a Representative images and calculation of wound healing rates at day 0, 3, 7 and 13. b H&E and Masson’s Trichrome staining of wound tissues (scale bar, 100 µm). c Cell apoptosis analysis in wound tissues by TUNEL (scale bar, 100 µm). d IHC staining of CD31 in wound tissues (scale bar, 100 µm). e Western blot analysis of the expression of CBL, KDM1A and VEGFA in wound tissues ( n = 8). GAPDH was used as a normalization control. n = 8 represents biologically independent animals. Data were presented as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The secretion of VEGFA in the supernatants of HUVECs was examined with the human VEGFA ELISA kit (Sino Biological, Beijing, China).

Techniques: Control, Injection, Staining, TUNEL Assay, Immunohistochemistry, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Ubiquitin C-terminal hydrolase L1 promoted pro-angiogenic capacity of periodontal ligament stem cells via HIF-1α/YAP signaling in periodontitis

doi: 10.1186/s13287-025-04399-y

Figure Lengend Snippet: The elevated expression level of UCHL1 was correlated with the elevated expression levels of VEGFA and ANGPT1 in P-PDLSCs. A Representative immunofluorescent images and the quantification of the immunofluorescence show CD31 expression in the PDL tissues from healthy individuals and the granulation tissues from periodontitis patients. B The heatmap displays gene expressions of upregulated pro-angiogenic factors in P-PDLSCs analyzed in GSE78074 . C The mRNA expression levels of VEGFA , ANGPT1 , VEGFB , VEGFC , and FGF1 in H-PDLSCs and P-PDLSCs were measured using RT-qPCR. D The mRNA expression levels of UCHL1 in H-PDLSCs and P-PDLSCs were measured using RT-qPCR. E The correlations of UCHL1 mRNA expression with VEGFA or ANGPT1 were analyzed using Spearman’s correlation analysis. F – K . The protein expression of VEGF, ANGPT1, and UCHL1 in the PDL tissues from healthy individuals and granulation tissues from periodontitis patients was evaluated using immunofluorescence. F Representative immunofluorescent images show the expressions of VEGFA. G Mean fluorescence intensity of VEGFA protein expression was calculated from immunofluorescence images. H Representative immunofluorescent images show the expressions of ANGPT1. I Mean fluorescence intensity of ANGPT1 protein expression was calculated from immunofluorescence images. J Representative immunofluorescent images show the expressions of UCHL1. K Mean fluorescence intensity of UCHL1 protein expression was calculated from immunofluorescence images. H represents the healthy group; P represents the periodontitis group. Statistical comparisons were analyzed using Mann–Whitney U test; Correlations were analyzed using Spearman’s correlation analysis; data are presented as mean ± S.E.M; ns indicates not significant, * indicates P < 0.05, and ** indicates P < 0.01

Article Snippet: To monitor the secretion of VEGFA and ANGPT1 in PDLSCs, the concentrations of VEGFA and ANGPT1 in the collected serum-free media were measured using a human VEGFA ELISA kit (Solarbio, Beijing, China) and a human ANGPT1 ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instruction.

Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Fluorescence, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Ubiquitin C-terminal hydrolase L1 promoted pro-angiogenic capacity of periodontal ligament stem cells via HIF-1α/YAP signaling in periodontitis

doi: 10.1186/s13287-025-04399-y

Figure Lengend Snippet: Knockdown of UCHL1 abrogated the increased levels of VEGFA and ANGPT1 in PDLSCs. PDLSCs were transfected with lentivirus controls (shScramble) or lentivirus shRNA targeted UCHL1 (shUCHL1). A – C shScramble and shUCHL1 P-PDLSCs were cultured for 72 h. A The concentration of VEGFA and ANGPT1 in the culture media of shScramble and shUCHL1 P-PDLSCs was measured using ELISA assay at 72 h post-culture with serum-free media. B The mRNA expression levels of VEGFA and ANGPT1 in shScramble and shUCHL1 P-PDLSCs were measured using RT-qPCR. C The protein expression levels of VEGFA, ANGPT1, UCHL1, and GAPDH in shScramble and shUCHL1 P-PDLSCs were measured using western blotting. Full-length blots are presented in Supplementary Fig. . D – G shScramble and shUCHL1 H-PDLSCs were treated with or without TNF-α (2 ng/ml) and IL-1β (5 ng/ml) for 72 h. D The concentration of VEGFA and ANGPT1 in the culture media of shScramble and shUCHL1 H-PDLSCs was measured using ELISA assay. E The mRNA expression levels of VEGFA and ANGPT1 in shScramble and shUCHL1 H-PDLSCs were measured using RT-qPCR. F The protein expression levels of VEGFA, ANGPT1, UCHL1, and GAPDH in shScramble and shUCHL1 H-PDLSCs were measured using western blotting. Full-length blots are presented in Supplementary Fig. . G The protein expression levels of VEGFA and ANGPT1 in shScramble and shUCHL1 H-PDLSCs were measured using immunofluorescence. H Mean fluorescence intensity of VEGFA and ANGPT1 protein expression was calculated from immunofluorescence images. T represents TNF-α; I represents IL-1β. Statistical comparisons were analyzed using Mann–Whitney U test or two-way ANOVA; data are presented as mean ± S.E.M; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001

Article Snippet: To monitor the secretion of VEGFA and ANGPT1 in PDLSCs, the concentrations of VEGFA and ANGPT1 in the collected serum-free media were measured using a human VEGFA ELISA kit (Solarbio, Beijing, China) and a human ANGPT1 ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instruction.

Techniques: Knockdown, Transfection, shRNA, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Ubiquitin C-terminal hydrolase L1 promoted pro-angiogenic capacity of periodontal ligament stem cells via HIF-1α/YAP signaling in periodontitis

doi: 10.1186/s13287-025-04399-y

Figure Lengend Snippet: Knockdown of UCHL1 abrogated the increased YAP activity of PDLSCs under inflammatory conditions. A-C. Gene expressions in H-PDLSCs and P-PDLSCs were evaluated by analyzing GSE78074 . A KEGG analysis shows that in upregulated genes of P-PDLSCs, the top 10 significantly enriched signaling pathways in the map of environmental information processing, cellular processes, metabolism, genetic information processing, and organismal systems. B Line plots show the gene expressions of UCHL1 , YAP , and TAZ in H-PDLSCs and P-PDLSCs analyzed in GSE78074 . C The correlations of the gene expression level of UCHL1 with YAP and TAZ were analyzed using Spearman’s correlation analysis. D YAP expression in the PDL tissues from healthy individuals and granulation tissues from periodontitis patients was evaluated using immunofluorescence. E YAP nucleus/cytoplasm ratios in the PDL tissues from healthy individuals and granulation tissues from periodontitis patients were calculated from immunofluorescence images. F – I H-PDLSCs were transfected with lentivirus controls (shScramble) or lentivirus shRNA targeted UCHL1 (shUCHL1). shScramble and shUCHL1 H-PDLSCs were treated with or without TNF-α (2 ng/ml) and IL-1β (5 ng/ml) for 72 h. F. The protein expression levels of YAP, p-YAP, TAZ, p-TAZ, UCHL1, and GAPDH in shScramble and shUCHL1 H-PDLSCs were measured using western blotting. Full-length blots are presented in Supplementary Figs. – . G The expression and the location of YAP in shScramble and shUCHL1 H-PDLSCs were measured using immunofluorescence. H YAP nucleus/cytoplasm ratios immunofluorescence were calculated from immunofluorescence images. I The mRNA expression level of YAP in shScramble and shUCHL1 H-PDLSCs was measured using RT-qPCR. J H-PDLSCs were pretreated with or without Verteporfin (0.1 μM) for 24 h and then treated with or without TNF-α (2 ng/ml) and IL-1β (5 ng/ml) for 72 h. The protein expression levels of VEGFA, ANGPT1, and GAPDH were measured using western blotting. Full-length blots are presented in Supplementary Fig. . H represents the healthy group; P represents the periodontitis group; T represents TNF-α; I represents IL-1β. Statistical comparisons were analyzed using two-way ANOVA; Correlations were analyzed using Spearman's correlation analysis; data are presented as mean ± S.E.M; ns indicates not significant, *indicates P < 0.05, and *** indicates P < 0.001

Article Snippet: To monitor the secretion of VEGFA and ANGPT1 in PDLSCs, the concentrations of VEGFA and ANGPT1 in the collected serum-free media were measured using a human VEGFA ELISA kit (Solarbio, Beijing, China) and a human ANGPT1 ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instruction.

Techniques: Knockdown, Activity Assay, Protein-Protein interactions, Gene Expression, Expressing, Immunofluorescence, Transfection, shRNA, Western Blot, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: Ubiquitin C-terminal hydrolase L1 promoted pro-angiogenic capacity of periodontal ligament stem cells via HIF-1α/YAP signaling in periodontitis

doi: 10.1186/s13287-025-04399-y

Figure Lengend Snippet: UCHL1 inhibition reduced aberrant angiogenesis, inflammation, and alveolar bone resorption in murine experimental periodontitis. A Schematic illustration shows that UCHL1 inhibitor LDN57444 was administered to mice for 10 days after the ligation. B Representative images of H&E staining show the destruction of the gingival tissues triggered by ligature (red arrows) and the blood vessels surrounding the ligature place (black arrows). C Representative images of immunohistochemical staining show the expression of CD31 and indicate blood vessels (black arrows) in the gingival and PDL tissues. The area occupied by blood vessels in the gingival and PDL tissues was statistically quantified. D Representative images of immunohistochemical staining show the expression of VEGFA and ANGPT1. The expression of VEGFA and ANGPT1 was statistically quantified. E The mRNA expression level of Yap in gingival tissues was measured using RT-qPCR. F Representative images of immunohistochemical staining show the expression of YAP. The expression of YAP was statistically quantified. G The mRNA expression level of Hif1a in gingival tissues was measured using RT-qPCR. H Representative images of immunohistochemical staining show the expression of HIF-1α. The expression of HIF-1α was statistically quantified. I Representative immunofluorescent images and the quantification of the immunofluorescence show CD3-positive T cells (red) and CD20-positive B cells (green) in the gingival and PDL tissues in mice. J The mRNA expression levels of Tnfa , Il1b , Il6 , and Ifng in gingival tissues were measured using RT-qPCR. K Representative images of 3D reconstructions and 2D slices were performed using micro-CT. In 3D reconstructions, the red area between CEJ and ABC denotes bone resorption area. Gray and pseudocolored 2D images show alveolar bone resorption (red arrows) between the maxillary M1 and M2 and at M1 furcation sites in a sagittal and horizontal plane (color map: CT values were outlined using an RGB pseudocolor scale). L The bone resorption area between CEJ and ABC, bone mineral density, and bone histomorphometry parameters, BV/TV and Tb.Th, were quantitatively analyzed. H represents the healthy group; P represents the periodontitis group; P + LDN represents the LDN57444-treated periodontitis group. Statistical comparisons were analyzed using Mann–Whitney U test or one-way ANOVA; data are presented as mean ± S.E.M; ns indicates not significant, and * indicates P < 0.05

Article Snippet: To monitor the secretion of VEGFA and ANGPT1 in PDLSCs, the concentrations of VEGFA and ANGPT1 in the collected serum-free media were measured using a human VEGFA ELISA kit (Solarbio, Beijing, China) and a human ANGPT1 ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instruction.

Techniques: Inhibition, Ligation, Staining, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Immunofluorescence, Micro-CT, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Ubiquitin C-terminal hydrolase L1 promoted pro-angiogenic capacity of periodontal ligament stem cells via HIF-1α/YAP signaling in periodontitis

doi: 10.1186/s13287-025-04399-y

Figure Lengend Snippet: Schematic diagram of the mechanism through which UCHL1 confers the pro-angiogenic capacity of PDLSCs in periodontitis. Under inflammatory conditions, the upregulated UCHL1 could stabilize and activate HIF-1α, thereby promoting YAP transcription, and resulting in the secretion of pro-angiogenic factors, VEGFA and ANGPT1 in PDLSCs. The increased pro-angiogenic capacity of PDLSCs subsequently promoted the angiogenesis of endothelial cells and thus played a role in periodontitis

Article Snippet: To monitor the secretion of VEGFA and ANGPT1 in PDLSCs, the concentrations of VEGFA and ANGPT1 in the collected serum-free media were measured using a human VEGFA ELISA kit (Solarbio, Beijing, China) and a human ANGPT1 ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instruction.

Techniques:

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: The antibodies used in the article.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques:

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: Primers used for PCR.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques:

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: The anti‐angiogenic factor AIP1 protects against VECs dysfunction induced by the OIR and hypoxia in vitro. (A and B) Protein and mRNA levels of VEGF‐associated signaling components in HUVECs transfected with control siRNA or AIP1 siRNA (n = 3 cultures/group). (C and D) Protein and mRNA levels of AIP1, VEGFA, VEGFB, and VEGFR2 in control HUVECs and HUVECs transfected with AIP1 overexpression plasmid (n = 3 cultures/group). (E) Representative images of the retinal flat mounts stained with IB4 from WT and AIP1‐deficient mice (n = 6 mice/group). Vaso‐obliteration and neovascularization are indicated by the white area and white dots, respectively. Scale bar: 1 mm for 4× images and 100 µm for 40× images. Avascular area (F), neovascular area (G), and fluid permeability measured by Evans blue dye (H) were quantified. (I) Representative images and quantitative measurements of tube formation assays using HUVECs transfected with empty vector or AIP1 overexpression plasmid before hypoxia exposure. (J) Representative images and quantitative measurements from in vitro tube formation assays using HUVECs transfected with control siRNA or AIP1 siRNA before hypoxia exposure. Scale bar: 200 µm. NV: neovascularization. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Statistical analyses were performed using GraphPad Prism software, with one‐way ANOVA followed by Dunnett's post hoc tests and independent‐sample t‐test.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques: In Vitro, Transfection, Control, Over Expression, Plasmid Preparation, Staining, Permeability, Software

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: The NLRP12–CASP8 inflammasome enhances VEGF signaling. (A and B) NLRP12 knockdown reduced the protein and mRNA levels of VEGFA, VEGFB, and VEGFR2 in HUVECs exposed to hypoxia (n = 3 cultures/group). (C and D) CASP8 knockdown reduced the protein and mRNA levels of VEGFA, VEGFB, and VEGFR2 in HUVECs exposed to hypoxia (n = 3 cultures/group). (E and F) Representative images and quantitative measurements of tube formation assays using HUVECs transfected with NC siRNA, NLRP12 siRNA, or CASP8 siRNA before hypoxia exposure. Scale bar: 200 µm. (G to N) Representative images and quantitative measurements of the retinal flat mounts stained with IB4 from WT, NLRP12 KO (G), and CASP8 +/− (K) mice (n = 6 mice/group). Vaso‐obliteration and neovascularization are indicated by the white area and white dots, respectively. Scale bar: 1 mm for 4× images and 100 µm for 40× images. Analyses of whole‐mount retinal immunofluorescence images, including nonperfused areas (H,L), new vessel tuft formation (I,M), and Evan blue dye permeability (J,N). Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Statistical analyses were performed using GraphPad Prism software, with one‐way ANOVA followed by Dunnett's post hoc tests and the independent‐sample t‐test.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques: Knockdown, Transfection, Staining, Immunofluorescence, Permeability, Software

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: GSDMD‐mediated pyroptosis and VEGF signaling of VECs drive OIR. (A) Retinal vasculature in WT and GSDMD KO mice was visualized using IB4 immunofluorescence staining (n = 6 mice/group). Scale bar: 1 mm for 4× images and 100 µm for 40× images. (B–D) Analysis of whole‐mount retinal immunofluorescence images, including avascular areas (B), neovascular tuft areas (C), and Evans blue dye permeability (D). (E) Flow cytometry analysis of VEGF‐PE expression in CD31‐positive cells (vascular endothelial cell marker) in the retina of WT and GSDMD KO mice (n = 6 mice/group). (F) GSDMD knockdown reduced VEGFA, VEGFB, and VEGFR2 protein levels in HUVECs exposed to hypoxia (n = 3 cultures/group). (G) VEGFA levels in the supernatant of HUVECs exposed to hypoxia were measured using ELISA (n = 3 cultures/group). (H) Representative images and quantitative measurements of tube formation assays using HUVECs transfected with NC siRNA and GSDMD siRNA before hypoxia exposure. Scale bar: 200 µm. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. Statistical analyses were performed using GraphPad Prism software, with one‐way ANOVA followed by Dunnett's post hoc tests and the independent‐sample t ‐test.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques: Immunofluorescence, Staining, Permeability, Flow Cytometry, Expressing, Marker, Knockdown, Enzyme-linked Immunosorbent Assay, Transfection, Software

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: The NLRP12–CASP8 inflammasome promotes GSDMD‐mediated pyroptosis and VEGF expression of VECs. (A and B) Cleavage of GSDMD was detected by Western blot in HUVECs with AIP1 overexpression or AIP1 knockdown, respectively (n = 3 cultures/group). (C and D) Components of the pyroptotic‐ and VEGFA‐associated pathways were assayed by Western blot following CASP1 inhibition (n = 3 cultures/group). (E and F) Western blot analysis of pyroptotic hallmarks, including CASP1, GSDMD cleavage and IL‐1β maturation, after NLRP12 or CASP8 knockdown (n = 3 cultures/group). Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. Statistical analyses were performed using GraphPad Prism software, with one‐way ANOVA followed by Dunnett's post hoc tests.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Inhibition, Software

Journal: Advanced Science

Article Title: AIP1 Regulates Ocular Angiogenesis Via NLRP12‐ASC‐Caspase‐8 Inflammasome‐Mediated Endothelial Pyroptosis

doi: 10.1002/advs.202405834

Figure Lengend Snippet: A new model of ROP pathogenesis involving AIP1 downregulation, NLRP12–CASP8inflammasome activation, GSDMD‐dependent VECs pyroptosis, excessive VEGFA production, and neovascularization. All images were generated by the authors.

Article Snippet: Concentrations of VEGFA in HUVEC supernatants were measured using a human VEGFA ELISA kit (ABclonal Biotechnology, #RK00023) according to the manufacturer's instructions.

Techniques: Activation Assay, Generated

Journal: Investigative Ophthalmology & Visual Science

Article Title: The Effects of STRA6 Regulation of the Circadian Rhythm on Choroidal Neovascularization

doi: 10.1167/iovs.65.11.21

Figure Lengend Snippet: Correlation analysis of STRA6 and the influence of circadian rhythm and CNV on it. ( A ) Schematic diagram of complete transcriptome sequencing in mouse tissue. ( B ) Venn diagram of mRNA upregulation and downregulation in sequencing results. ( C ) Analysis of STRA6 gene expression differences in public dataset GSE29801 ( n = 293). ( D ) Heat map of GSVA analysis in patients with AMD with high and low STRA6 expression. ( E ) STRA6 protein-protein interaction. ( F ) A possible binding site to the CLOCK gene. ( G ) Luciferase assay showed the regulatory effects of BMAL1 and CLOCK on STRA6. ( H ) The expression of STRA6 mRNA in each group was detected by qRT-PCR ( n = 3 independent experiment). ( I and J ) STRA6 protein expression levels in each group ( n = 3 independent experiments). ( K to M ) The expression of STRA6 and VEGFA protein in RPE-choroid-sclera complex in CNV model mice induced by laser at different time points ( n = 3 independent experiments). ZT, zeitgeber time; error bars represent mean ± SEM; ns was not statistically significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The Human VEGFA ELISA kit (Bosterbio, China) was used to detect the VEGFA content in the cell supernatant.

Techniques: Sequencing, Gene Expression, Expressing, Binding Assay, Luciferase, Quantitative RT-PCR

Journal: Investigative Ophthalmology & Visual Science

Article Title: The Effects of STRA6 Regulation of the Circadian Rhythm on Choroidal Neovascularization

doi: 10.1167/iovs.65.11.21

Figure Lengend Snippet: Construction of circadian rhythm cell model and effect of STRA6 knockdown on RPE cell function in vitro. ( A ) Immunofluorescence staining of ARPE-19 cells ( n = 3 independent experiments; red = STRA6, blue = DAPI; scale = 200 µm). ( B ) The effects of different concentrations of COCL2 on the proliferation of ARPE-19 cells were detected by CCK8 (µmol/L; n = 3 independent experiments). ( C ) The effects of different concentrations of DEX on the proliferation of ARPE-19 cells was detected by CCK8 (nmol/L; n = 3 independent experiments). ( D ) Schematic diagram of cell synchronization circadian rhythm model construction. ( E to H ) Expression of clock gene BMAL1 and CLOCK protein in ARPE-19 cells in each group ( n = 3 independent experiments). ( I and J ) STRA6 protein expression levels in each group ( n = 3 independent experiments). ( K ) STRA6 knockdown verification ( n = 3 independent experiments). ( L to N ) Transwell showed the migration of ARPE-19 cells ( n = 3 independent experiments; scale = 6–12 hours 200 µm, 8 hours 50 µm). ( O to Q ) The wound healing assay showed the migration of ARPE-19 cells in each group at 24 hours and 48 hours ( n = 3 independent experiments; scale = 50 µm). ( R ) The proliferation of ARPE-19 cells in each group was detected by CCK8 ( n = 3 independent experiments). ( S ) VEGF in cell supernatant was detected by ELISA ( n = 3 independent experiments). ZT, zeitgeber time; error bars represent mean ± SEM; ns was not statistically significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The Human VEGFA ELISA kit (Bosterbio, China) was used to detect the VEGFA content in the cell supernatant.

Techniques: Knockdown, Cell Function Assay, In Vitro, Immunofluorescence, Staining, Expressing, Migration, Wound Healing Assay, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: The Effects of STRA6 Regulation of the Circadian Rhythm on Choroidal Neovascularization

doi: 10.1167/iovs.65.11.21

Figure Lengend Snippet: Relationship between STRA6 and inflammation and VEGF. ( A ) Laser induced immunofluorescence staining of paraffin sections of CNV model mice ( n = 3/group; red = STRA6; green = F4/80, IB4; blue = DAPI; scale = 100 µm). ( B and C ) Expression of VEGFA protein in ARPE-19 cells at different time points in the non-combined hypoxia model ( n = 3 independent experiments). ( D and E ) Expression of VEGFA protein in ARPE-19 cells at different time points in the non-combined hypoxia model ( n = 3 independent experiments). ( F and G ) STRA6 protein expression in ARPE-19 cells at different time points after circadian rhythm synchronization in the combined hypoxia model ( n = 3 independent experiments). ZT, zeitgeber time; error bars represent mean ± SEM; ns was not statistically significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The Human VEGFA ELISA kit (Bosterbio, China) was used to detect the VEGFA content in the cell supernatant.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: The Effects of STRA6 Regulation of the Circadian Rhythm on Choroidal Neovascularization

doi: 10.1167/iovs.65.11.21

Figure Lengend Snippet: Effect of STRA6 on CNV formation by activating JAK2/STAT3/VEGFA signaling in RPE cells. ( A and B ) The expression of JAK2 protein in circadian rhythm ( n = 3 independent experiments). ( C and D ) The expression of STAT3 protein in circadian rhythm ( n = 3 independent experiments). ( E and F ) Expression of P-JAK2, JAK2, P-STAT3, STAT3, STRA6, and VEGFA in ARPE-19 cells of each group ( n = 3 independent experiments). ( G and H ) Expression of P-JAK2, JAK2, P-STAT3, STAT3, and VEGFA proteins in ARPE-19 cells that did not synchronize circadian rhythm after addition of JAK2 pathway inhibitors ( n = 3 independent experiments). ZT, zeitgeber time; error bars represent mean ± SEM; ns was not statistically significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The Human VEGFA ELISA kit (Bosterbio, China) was used to detect the VEGFA content in the cell supernatant.

Techniques: Expressing