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: 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