Journal: Frontiers in medicine

Article Title: Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

doi: 10.3389/fmed.2022.1012827

Figure Lengend Snippet: FIGURE 1 Biomarker plasma levels in sarcoidosis and healthy controls. Plasma levels medians are plotted. Compared to healthy controls, each biomarker exhibited higher values in plasma of sarcoidosis patients. Except for IL-8 all of plasma biomarkers were significantly higher in sarcoidosis. eNAMPT (2.1 vs. 1.2 ng/ml), IL-6 (1.2 vs. 0.6 pg/ml), ANG-2 (2.7 vs. 1.0 pg/ml), HBEGF (5.9 vs. 1 pg/ml), and IL-1 RA (227 vs. 82.3 pg/ml). Mann–Whitney test was calculated for each comparison; ****p-values < 0.0001 and *p < 0.05.

Article Snippet: 150 μl of plasma was used to quantify HBEGF levels using Quantikine R© ELISA Human HBEGF immunoassay (R&D Systems R©), quantitative sandwich enzyme immunoassay technique.

Techniques: Biomarker Discovery, Clinical Proteomics, MANN-WHITNEY, Comparison

Journal: Frontiers in medicine

Article Title: Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

doi: 10.3389/fmed.2022.1012827

Figure Lengend Snippet: FIGURE 2 Circulating biomarkers in complicated sarcoidosis and pulmonary fibrosis. (A) The plasma levels of HBEGF were significantly elevated (**p < 0.003) in a group of 62 complicated sarcoidosis (parenchymal lung disease by CT scan, radiographic Scadding stages III and IV; Forced vital capacity (FVC) < 50%; cardiac or neurological involvement in addition to lung involvement) compared to non-complicated sarcoidosis. (B) ANG-2 plasma levels were significantly (p < 0.017) elevated in 19 subjects with sarcoidosis and pulmonary fibrosis (confirmed fibrotic changes on high-resolution chest CT scan or radiographic stage IV) compared to those with non-fibrosis.

Article Snippet: 150 μl of plasma was used to quantify HBEGF levels using Quantikine R© ELISA Human HBEGF immunoassay (R&D Systems R©), quantitative sandwich enzyme immunoassay technique.

Techniques: Clinical Proteomics, Computed Tomography

Journal: Frontiers in medicine

Article Title: Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

doi: 10.3389/fmed.2022.1012827

Figure Lengend Snippet: FIGURE 3 The receiver-operator characteristics curves (ROC) assessing the diagnostic accuracy power. (A–E) Sarcoidosis vs. controls. eNAMPT (A), IL-6 (B), IL-1RA (C), ANG-2 (D), and HBEGF (E). IL-1RA showed the highest discriminatory accuracy between sarcoidosis and healthy controls (AUC of 0.93, 95% CI of 0.90–0.95), followed by ANG-2 (AUC 0.85, 95% CI of 0.79–0.90). (F,G) Progressive sarcoidosis. ANG-2 diagnostic accuracy performed well to distinguish sarcoidosis pulmonary fibrosis vs. sarcoidosis without pulmonary involvement (Stages I and II) (F) and HBEGF showed a good discriminatory power to differentiate complicated sarcoidosis.

Article Snippet: 150 μl of plasma was used to quantify HBEGF levels using Quantikine R© ELISA Human HBEGF immunoassay (R&D Systems R©), quantitative sandwich enzyme immunoassay technique.

Techniques: Diagnostic Assay

Journal: Frontiers in medicine

Article Title: Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

doi: 10.3389/fmed.2022.1012827

Figure Lengend Snippet: FIGURE 4 (A–F) Representative immunohistochemical staining of lymph nodes (A,C,E) and lung tissues (B,D,F) from sarcoidosis compared healthy controls 40 × magnification. ANG-2 (A,B,G), HBEGF (C,D,H), NAMPT (E,F,I). (G–I) Bar graphs (G) illustrate quantification of mean proportions of ANG-2, HBEGF, and NAMPT *p < 0.05. Sarcoidosis in dark gray and normal tissue in light gray. ANG-2 expression in lung tissue and lymph node in sarcoidosis was 32.2 and 33.7%, respectively, in contrast to the expression observed in normal lung and lymph nodes was 9.8 and 11.3%, respectively (p = 1.2 e-04). HBEGF expression in sarcoidosis lymph nodes was 44.8% vs. 4.8% in normal lymph nodes (p = 3.8 e-05); in normal lungs was 18.3% contrasting with 60.4% in lung of sarcoidosis (p = 4.0 e-05). NAMPT mean proportions in lymph nodes were 12.7% in normal tissue and 52% in sarcoidosis (p = 4.9 e-04); while in normal lungs was 23.3% vs. 53% in sarcoidosis (p = 9.2 e-040. in lymph nodes).

Article Snippet: 150 μl of plasma was used to quantify HBEGF levels using Quantikine R© ELISA Human HBEGF immunoassay (R&D Systems R©), quantitative sandwich enzyme immunoassay technique.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in medicine

Article Title: Biochemical and genomic identification of novel biomarkers in progressive sarcoidosis: HBEGF, eNAMPT, and ANG-2.

doi: 10.3389/fmed.2022.1012827

Figure Lengend Snippet: FIGURE 5 Enrichment analysis of DEGs in lung tissues from subjects with sarcoidosis compared to healthy lung. Functional protein network interaction showing eNAMPT and HBEGF relationship with regulators of lung fibrosis (MMP9), inflammation, and lung remodeling and NOTCH4, a T-cell activity and angiogenesis regulator. Each node represents a coded protein. The edges represent protein–protein associations (line color indicates type of interaction: green—gene neighborhood, red—gene fusion, blue—co-occurrence, and black—co-expression). Pathway analysis and first shell interactors members included are indicated by the color code. A complete list of dysregulated pathways is presented in Supplementary Table 2.

Article Snippet: 150 μl of plasma was used to quantify HBEGF levels using Quantikine R© ELISA Human HBEGF immunoassay (R&D Systems R©), quantitative sandwich enzyme immunoassay technique.

Techniques: Functional Assay, Activity Assay, Expressing

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Sequencing

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: Endothelial Notch signaling affects RPE barrier function by regulating the HBEGF paracrine of EC. ( A ) Analysis for previously published transcriptome sequencing data of normal HUVECs and DAPT treated HUVECs: the GSEA results for ECM ( left ) and heatmap of the expression of ECM-related genes ( right ). ( B ) mRNA and protein levels of HBEGF in normal HUVECs and DAPT-treated HUVECs. ( C ) mRNA and protein levels of HBEGF in normal HUVECs and NICD adenovirus-infected HUVECs. ( D ) The secretion levels of HBEGF protein in normal HUVECs and DAPT-treated HUVECs or NICD adenovirus-infected HUVECs were tested using an ELISA kit. ( E ) Evaluating the RPE barrier function in RPE cells cultured in normal medium, RPE cultured in the EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF) using ZO-1 staining (top), fibronectin staining ( middle ), and collagen IV staining (bottom). The number of RPEs with intact tight junctions were quantitatively compared. Fibronectin- and collagen IV-positive areas were quantitatively compared. Scale bars: 50 µm. ( F ) Evaluating the RPE barrier function in the RPE, RPE with HUVECs, RPE with siHBEGF-treated HUVECs, and RPE with siNC-treated HUVECs using ZO-1 staining (top), fibronectin staining ( middle ), and collagen IV staining (bottom). The number of RPEs with intact tight junctions were quantitatively compared. Fibronectin- and collagen IV-positive areas were quantitatively compared. Scale bars: 50 µm. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Sequencing, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Staining

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: r-HBEGF improved the RPE barrier dysfunction of Notch signaling deletion mice. ( A ) In the SI-induced RPE barrier disfunction model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJ f/f intravitreally injected with IgG or r-HBEGF, and control mice (RBPJ f/+ ) at p30, were analyzed using ZO-1 staining. The number of RPE cells with intact tight junctions were quantitatively compared. Red circles indicate the location of the optic disk. Scale bars: 50 µm. ( B ) In the laser-induced CNV model, whole-mount choroidal staining for EC-specific Notch signaling deletion mice CDH5-CRE-RBPJ f/f intravitreally injected with IgG or r-HBEGF and control mice (RBPJ f/+ ) were analyzed using IB-4 staining. IB4 positive areas were quantitatively compared. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). Scale bars: 150 µm. * p < 0.05, ** p < 0.01.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Staining, Injection, Control

Journal: Antioxidants

Article Title: Endothelial Notch Signaling Regulates the Function of the Retinal Pigment Epithelial Barrier via EC Angiocrine Signaling

doi: 10.3390/antiox12111979

Figure Lengend Snippet: EC regulates MMP-9 expression in RPE cells via Endothelial Notch signaling-driven HBEGF secretion. ( A ) Protein interaction prediction analysis results of HBEGF and MMP-9, using the String database. ( B ) mRNA level, protein level, and enzyme activity level of MMP-9 in the RPE, RPE with HUVEC, and RPE with Notch signaling-inhibited HUVECs were tested via qRT-PCR, Western blot, and gelatin assay kit. ( C ) mRNA levels of MMP-9 in normal HUVECs and DAPT-treated HUVECs were tested via qRT-PCR. ( D , E ) mRNA and protein levels of MMP-9 in the RPE, RPE with HUVECs, RPE with siNC-treated HUVECs, and RPE with siHBEGF-treated HUVECs were tested via qRT-PCR and Western blot. ( F ) Protein levels of MMP-9 in RPE cells cultured in normal medium, RPE cultured in EC-conditioned medium, RPE cultured in Notch signaling-inhibited EC-conditioned medium, and RPE cultured in Notch signaling-inhibited EC-conditioned medium supplemented with recombinant HBEGF (r-HBEGF) were tested using Western blot. Three biological replicates were performed and values are presented as the mean ± SEM ( n = 3). NS, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the concentration of secreted HBEGF in the media was measured using the human HBEGF ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Cell Culture, Recombinant