Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: CircNPHP1 Expression in the Human LV and Cultured ECs Cardiac CircNPHP1 levels are higher in patients with IHD with/without associated T2DM and in cultured endothelial cells (human cardiac microvascular endothelial cells and human umbilical vein endothelial cells) exposed to disease-mimicking conditions, such as hypoxia and hypoxia, combined with increased D-glucose level (High glu). (A) Preliminary screening of circRNAs (identified from predicted networks) in endothelial cells: human cardiac microvascular endothelial cells were cultured in normal conditions, hypoxia (1% Oxygen), and hypoxia-high glucose (High Glu) (25 mmol/L D-glucose) conditions, respectively. After 48 hours, cells were harvested for quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis and validation of indicated circRNAs. RNA expression is relative to normoxia. 18S is used as housekeeping gene; n = 3. Data are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. CircSTX17 was also tested but the levels were undetectable; hence, it is not included in the plot. CircNPHP1 emerges as an interesting candidate for further exploration and validation. (B) Comparison of expression levels of circNPHP1 and its linear counterpart in different patient groups—RNAseq (LV biopsies, cohort 1). Log-transformed normalized (FPM) counts of circNPHP1 and linear NPHP1 are plotted as a boxplot showing the median and IQR. Comparison between patient groups and calculation of P values was done using the R package Limma. CircNPHP1 is significantly up-regulated in IHD+T2DM compared with IHD and non-IHD ( P < 0.05). Linear NPHP1 is expressed at very low levels and does not change between the different patient groups (IHD [n = 12], IHD+T2DM [n = 11], non-IHD [n = 12]). (C) qRT-PCR of circNPHP1 (left) and linear NPHP1 (right) of LV biopsies (cohort 1) from non-IHD (n = 12), IHD (n = 12), and IHD+T2DM (n = 11), and (D) of LV biopsies (cohort 2) from non-IHD (n = 10), IHD (n = 10), IHD+T2DM (n = 10) of patients. RNA expression is relative to non-IHD; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by Kruskal-Wallis test followed by Dunn's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: Human cardiac microvascular endothelial cells (HCMECs) (Promocell: C-12285 [mono-doner], Lot numbers: 440Z021.5 [male], 440Z021.4 [male], 446Z001.1 [female], 492Z009.4 [female]) and human umbilical vein endothelial cells (HUVECs) (Promocell: C-12208 [poly-donor], Lot numbers: 447Z004, 450Z015, 466Z022, 503Z026) were cultured on 0.2% gelatin (Sigma-Aldrich) in endothelial cell growth medium MV and endothelial cell growth medium 2 (EGM2) (PromoCell).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, RNA Expression, Comparison, Transformation Assay

Journal: JACC: Basic to Translational Science

Article Title: Human Left Ventricle circRNA-miRNA-mRNA Network Analyses Reveal a Novel Proangiogenic Role for circNPHP1 Under Ischemic Conditions

doi: 10.1016/j.jacbts.2025.101468

Figure Lengend Snippet: CircNPHP1 and Linear NPHP1 Expression in Different Cell Types (A) Single-cell and single-nuclei RNAseq data from healthy donor hearts (human heart atlas ) of NPHP1 gene as log2 transformed counts per million (CPM). Each dot indicates the data point as 1 donor (source: human heart atlas ). (B) RNAseq: A preliminary screen was carried out in different cells (sample size = 2 each) to determine absolute levels of circNPHP1 and linear NPHP1 in human umbilical vein endothelial cells (HUVECs or HU), human cardiac microvascular endothelial cells (HCMECs or HC), human AC16 cardiomyocytes (ACs), and cardiac fibroblasts (CFs). (C) HCMEC (left) and HUVEC cells (right) were cultured in either normal conditions, hypoxia (1% oxygen), or hypoxia combined with high glucose (25 Mm D-glucose). After 48 hours, cells were harvested for quantitative real-time polymerase chain reaction for the analysis of circNPHP1 (n = 3). Fold change in RNA expression is relative to normoxia; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by 1-way analysis of variance with Dunnett's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. (D) 2 μg of total RNA from HUVEC cells were digested with RNase R enzyme followed by quantitative real-time polymerase chain reaction analysis of circNPHP1 and linear NPHP1 (n = 3). Fold change in RNA expression is relative to undigested RNA; 18S is used as housekeeping gene. Data are expressed as mean ± SEM and were assessed by unpaired Student's t -test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns = not significant. Abbreviations as in .

Article Snippet: Human cardiac microvascular endothelial cells (HCMECs) (Promocell: C-12285 [mono-doner], Lot numbers: 440Z021.5 [male], 440Z021.4 [male], 446Z001.1 [female], 492Z009.4 [female]) and human umbilical vein endothelial cells (HUVECs) (Promocell: C-12208 [poly-donor], Lot numbers: 447Z004, 450Z015, 466Z022, 503Z026) were cultured on 0.2% gelatin (Sigma-Aldrich) in endothelial cell growth medium MV and endothelial cell growth medium 2 (EGM2) (PromoCell).

Techniques: Expressing, Transformation Assay, Cell Culture, Real-time Polymerase Chain Reaction, RNA Expression

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells treated with Stx1a or Stx2a exhibited prominent morphological alterations when observed under a fluorescence microscope at 20X magnification, whereas cells treated with Stx1a mut or Stx2a mut did not show such changes. Images were collected from cells incubated with or without Stxs for 24, 48, and 72 h; ( B ) hCMEC/D3 cells were seeded in 6-well plates (5.0 ×10 5 cells/well) and incubated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) or Stx2a mut (10 ng/ml) for 24, 48, 72 h. Cell viability was determined using WST-8 assays, expressed as percentage viability and fold change relative to untreated controls ( left panel ). hCMEC/D3 cells (1.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and caspase-3/7 activity was measured using the caspase Glo-3/7 assay. Under the same conditions, hCMEC/D3 cells (5.0 × 10 5 cells/well) were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml) and Stx2a mut (10 ng/ml) for 24 h, and then protein samples were subjected to Western blotting using an anti-cleaved caspase-3 antibody ( right panel ). β-Actin was used as a control for equal protein loading. The results are a representative experiment obtained from three independent experiments; ( C ) Gb3 expression in hCMEC/D3 cells was detected by staining with Alexa Fluor 647-conjugated anti-CD77/Gb3 antibody or isotype control (mouse IgM-Alexa Fluor 647) for 1 h at 4oC. Representative results from three independent experiments are shown. Statistical significance. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panel B). * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Fluorescence, Microscopy, Incubation, Activity Assay, Caspase-Glo Assay, Western Blot, Control, Expressing, Staining

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates (5.0 × 10 5 cells/well). After washing with culture medium, cells were fixed and nuclei were stained with DAPI reagent. Representative DAPI-positive cells were visualized by fluorescence microscopy. To detect Stx translocation to the ER, cells were stimulated with complete growth medium containing 50 nM ER-tracker (red) live cell staining dye 2 h after treatment with Alexa Fluor 488-conjugated Stx1a (100 ng/ml). After washing, cells were captured using a fluorescence microscope EVOS M5000. Yellow fluorescence indicates co-localization of Stx1a with the ER marker. The scale bars represent 150 μm. The bar graph represents the mean ± SEM of the Pearson's correlation coefficient for the co-localization of Stx1-Alexa Fluor 488/ER tracker. At least 30 cells per condition were analyzed. Asterisks indicate statistically significant differences between the control and Stx1-treated groups. *** = p < 0.001; ( B ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 3, 6, 9, 12 and 24 h. After washing, cells were lysed at the indicated time points, and the presence of activated ER stress sensors and downstream targets in the cell lysates was determined by Western blotting. Untreated cells served as controls, and β-actin was used as a loading control; ( C ) hCMEC/D3 cells were treated as described above, and CHOP and DR5, spliced XBP1/unspliced XBP1 mRNA expression was measured by RT-qPCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); ( D ) hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 0, 24, 48, and 72 h. At the indicated time points, cells were lysed, and the expression of ROS-related markers (Nrf-2, HO-1, SOD-2, NO2) in cell lysates was measured by RT-qPCR and normalized using GAPDH. Asterisks indicate significant differences between control cell values and Stxs-treated cells (Panels C and D) at each time point. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Staining, Fluorescence, Microscopy, Translocation Assay, Marker, Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx1B (100 ng/ml), Stx2a (10 ng/ml), Stx2a mut (10 ng/ml), or Stx2B (10 ng/ml) for 3 h. After 3 h, cells were washed and cell lysates were collected. Phosphorylation of p38, JNK, and ERK were assessed by Western blotting. β-Actin was used as a loading control. The graphs show the mean ± SEM of band densities normalized by the division of β-Actin band densities and compared to untreated control cell values ( right panel ). Statistical analyses of densitometric scans from at least three independent experiments are shown; ( B ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 24, 48, and 72 h. At each time point, cells were lysed and mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2) were measured by RT-qPCR and normalized to GAPDH; ( C ) Under the same conditions, supernatants were collected and protein levels of IL-1β, IL-6, IL-8, TNF-α and CCL2 were quantified by ELISA using kit standards. Data are presented as mean ± SEM from three independent experiments. Asterisks indicate significant differences between control and Stx-treated cells (Panels B, C) at the indicated time points. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Phospho-proteomics, Western Blot, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of approximately 5.0 × 10 5 cells/well and treated with Stxs for 0, 3, 6, 9, 12, and 24 h. At each time point, cells were lysed, and mRNA expression of ZO-1, CLDN3, Occludin and JAM2 was measured by RT-qPCR. Expression levels were normalized to GAPDH; ( B ) hCMEC/D3 cells were seeded as above and treated with Stx2a for 0, 6, 12, 24, and 48 h. At each time point, cells were lysed, and the tight junction proteins CLDN1, ZO-1, and Ecadherin were analyzed by Western blotting. β-Actin was used as a loading control; ( C ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well. Cells were treated with Stx1a (100 ng/ml), Stx1a mut (100 ng/ml), Stx2a (10 ng/ml), or Stx2a mut (10 ng/ml) for 24 h. Fluorescein-conjugated 45kDa ovalbumin was added to the toxin-treated insert wells, and supernatants were collected from the lower chamber after 1 h. Cell permeability was evaluated by measuring the fluorescence of ovalbumin translocated from the insert wells to the lower chamber using a microplate reader. Asterisks indicate significant differences between the control cell values and the Stxs-treated cells (Panels A, C) at each time point. * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Permeability, Fluorescence

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/ D cells were seeded in 6-well plates. Cells were pretreated with z-VAD (20 μM) for 1 h before Stx2a exposure and divided into the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. After 24 h of Stx2a treatment, cell viability was assessed using the WST-8 assay ( left panel ). Subsequently, Western blot analysis showed that the total levels of caspase-3/7 in the cell lysates were decreased only in the Stx2a-treated group. Protein samples were prepared using anti-caspase-3, anti-cleaved caspase-3, and anti-β-Actin antibodies ( right panel ). β-Actin was used as a control for equal protein loading; ( B ) hCMEC/D3 cells were seeded as described above and treated with z-VAD 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: untreated control, Stx2a-treated, z-VAD only, and Stx2a + z-VAD pre-treated. Culture supernatants were collected, and the levels of secreted IL-1β, IL-6, IL-8, TNF-α and CCL2 were measured using ELISA ( left panel ). Total RNA was isolated from cell lysates, and mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1, and CCL2 were quantified using RT-qPCR ( right panel ). RNA expression levels were normalized using GAPDH; ( C ) hCMEC/D3 cells were seeded in 6-well plates at a total cell density of 5.0 × 10 5 cells/well and treated with NAC (5μM) 1 h before Stx2a exposure. The experimental groups included control, Stx2a-treated, NAC only, and Stx2a + NAC pre-treated. After 24 h of Stx2a treatment, the cytotoxicity level was measured by LDH assay using the cell culture supernatant. mRNA expression levels of Nrf-2, HO-1, and SOD-2 were measured by RT-qPCR and normalized to GAPDH, and the degree of NO expression was measured by NO assay; ( D ) hCMEC/D3 cells were seeded in 6-well plates at a density of 5.0 × 10 5 cells/well and treated with z-VAD and NAC 1 h before Stx2a treatment. Cell lysates were harvested from the following groups: control, Stx2a-treated, z-VAD + Stx2a, and NAC + Stx2a groups. After 24 h of Stx2 treatment, washed cell lysates were harvested, and the mRNA expression of ZO-1, CLDN3, Occludin and JAM2 were measured using RT-qPCR. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences compared to the control group, Stx-treated group, or between monotherapy vs. combination therapy (e.g., z-VAD alone vs. z-VAD+Stx2a) (Panels A, B) and compared to the control group, Stx-treated group, or between monotherapy vs. pre-treatment therapy ( e.g. , NAC alone vs. NAC+Stx2a) (Panel C). Significant differences are indicated between the control and cells treated with Stx, Stx+z-VAD, or Stx+NAC (Panel D).* = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Quantitative RT-PCR, RNA Expression, Lactate Dehydrogenase Assay, Cell Culture

Journal: Journal of Microbiology and Biotechnology

Article Title: Shiga Toxin Induces Apoptosis via ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

doi: 10.4014/jmb.2512.12006

Figure Lengend Snippet: ( A ) hCMEC/D3 cells were seeded in the insert wells of Trans-well plates at 3.0 × 10 5 cells/well, and A172 cells were seeded in the bottom wells of Trans-well plates at 5.0 × 10 5 cells/well. The insert wells containing hCMEC/D3 cells were stimulated with Stx2a (10 ng/ml) for 24 h. After 24 h, the supernatants were collected and cytotoxicity in A172 cells was assessed by LDH assay ( left panel ). A representative flow-cytometry readout of Gb3 in A172 astrocytes ( right panel ). ( B ) Subsequently, the A172 cells in the bottom wells were washed, and the cell lysates were harvested and the protein levels of caspase-3 and cleaved caspase-3 were determined by Western blotting. Anti-caspase-3, anticleaved caspase-3, and anti-β-Actin antibodies were used for protein samples. β-Actin served as a loading control ( C-D ) Culture supernatants of A172 cells seeded in the same manner as above were harvested, and the secretion levels of IL-1β, IL-6, IL-8, TNF-α, and CCL2 were determined using ELISA. Subsequently, the mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, CXCL1 and CCL2 were assessed by using RT-qPCR in cell lysates. RNA expression levels were normalized using GAPDH. Asterisks indicate significant differences between the control group and cells treated with Stx, Stx+z-VAD, and Stx+NAC (Panels A, C, D). * = p < 0.05; ** = p < 0.01; ***= p < 0.001.

Article Snippet: Collect the supernatant from hCMEC/D3 cells treated with Stx2a (10 ng/ml) and NAC (MedChemExpress, USA).

Techniques: Lactate Dehydrogenase Assay, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, RNA Expression

Journal: bioRxiv

Article Title: Endothelial RRM2B-dependent mitochondrial DNA signalling drives doxorubicin-cardiotoxicity

doi: 10.64898/2026.01.19.700350

Figure Lengend Snippet: Consequences of Doxorubicin exposure in cardiac endothelial cells in vitro : senescence, mtDNA instability and inflammation. A. Experimental model of endothelial cells transiently exposed to Doxorubicin . B. Representative immunoblot image in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). C. SA-β-galactosidase assay of HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). **** p<0.0001 in a two-sample t-test with Welch’s post hoc analysis for mean comparison. D. Quantification of mtDNA copy per cell determined by qPCR in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). Hydroxyurea (HU) was added one day after Dox withdrawal. N=3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. E. Expression level of dGUOK and TK2 by qRT-PCR in HCMECs, during Dox exposure (1d) and after drug withdrawal (8d). TK2, thymidine kinase 2. dGUOK, deoxyguanosine kinase. N=3 independent experiments. * p<0.05 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. F. Representative immunoblot image and G. quantification of the canonical RRM2 and alternative p53 inducible RRM2B small RNR subunit in HCMECs, not treated (NT), during Dox exposure (1d) and after drug withdrawal (8d). N=3 independent experiments. **** p<0.0001 in in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison. H-J. Immunostaining of HCMECs not treated (NT) and after Dox withdrawal (8d). H. Representative super-resolution Airyscan confocal images of dsDNA (green) and HSP60 (red) Scale bar 10 µm. At bottom, the magnified images show mtDNA (green) and mitochondria (red), showing that most DNA foci are located within mitochondria with some foci in the cytoplasm of Dox treated HCMECs. Arrows indicate cytosolic dsDNA signals. I. Quantification of the colocalization coefficient between mtDNA and mitochondria. N=3 independent experiments. ** p<0.01 in a two-sample t-test with Welch’s post hoc analysis for mean comparison. J. Quantification of mitochondrial length. NT (n=317), 8d (n=379). N=3 independent experiments. **** p<0.0001 in a Mann-Whitney test. K. Expression levels of indicated genes measured by qRT-PCR in HCMECs, not treated (NT) and after drug withdrawal (8d). Hydroxyurea (HU) or Sting inhibitor H151 (H151) was added one day after Dox withdrawal. N=3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001 in a one-way ANOVA test with Tukey’s post hoc analysis for mean comparison.

Article Snippet: Human endothelial cell lines HUVECs (pooled donors, C2519A, Lonza) and HCMECs (Caucasian male, C-12285 PromoCell) were used between passage 3 and 6.

Techniques: In Vitro, Western Blot, Comparison, Expressing, Quantitative RT-PCR, Immunostaining, MANN-WHITNEY