human microvascular ecs  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Small molecule intervention of actin-binding protein profilin1 reduces tumor angiogenesis in renal cell carcinoma

doi: 10.1016/j.jbc.2025.111033

Figure Lengend Snippet: Discovery of a novel analog of C74 with superior antiangiogenic activity in vitro and in vivo . A and B , docking models of C74 and UP-6 interaction with Pfn1—the binding modes of C74 ( panel A ) and UP-6 ( panel B ) as predicted by GNINA. Blue arrow indicates the altered structure on each compound. The unmodified hydroxypyrazoles are predicted to make an identical network of hydrogen bonds in both compounds while the modified aryl ring can potentially interact with R74 and H119. C and D , representative images ( panel C ; 4x magnification) and quantification ( panel D ; based on 4x field images) of cord formation by HmVECs on Matrigel treated with either 20 μM compound UP6 or equivalent DMSO control. Cord length is determined by the distance between each cord junction, i.e. cord intersections. Each data point represents quantification of cord formation in a single field of observation (two-tailed Student’s t test; ∗∗∗∗, p < 0.0001; n = 3 experiments; the scale bar represents 200 μm). E - F , representative fluorescence images ( panel E ) and quantification ( panel F ) of cord formation by HmVECs (labeled with cell tracker dye) subjected to 10 μM of either C74 or UP6 versus DMSO (control) (One-way ANOVA with Tukey multiple comparison post hoc test, ∗ - p < 0.05; ns – not significant; the scale bar represents 200 μm). G - H , representative images of CD31 immunohistochemistry with hematoxylin counterstaining ( panel G ; 4 × magnification) and quantification ( panel H ; based on 20X field images) of CD31+ cell infiltration in the subcutaneously implanted Matrigel plugs in Balb/C mice coinjected with a single 200 μM dosage of either C74 or UP-6 or DMSO control. Data are summarized from quantification of 10 bilaterally injected plugs (n = 5 mice) per treatment group (One-way ANOVA with Tukey post hoc test, ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; the scale bar represents 200 μm). Pfn1, profilin1; HmVEC, human microvascular EC; DMSO, dimethyl sulfoxide.

Article Snippet: Human microvascular ECs (HmVECs, ATCC, CRL-3243) were cultured in MCDB-131 media with growth supplements as described previously ( ).

Techniques: Activity Assay, In Vitro, In Vivo, Binding Assay, Modification, Control, Two Tailed Test, Fluorescence, Labeling, Comparison, Immunohistochemistry, Injection

Journal: The Journal of Biological Chemistry

Article Title: Small molecule intervention of actin-binding protein profilin1 reduces tumor angiogenesis in renal cell carcinoma

doi: 10.1016/j.jbc.2025.111033

Figure Lengend Snippet: . In vitro proof-of-concept of angiogenesis inhibition by C74 upon release by UTMD of lipid microbubbles. A and B , representative cord-formation images of HmVECs for various treatment settings ( panel A ): NT: DMSO (vehicle control); C74: treatment with unencapsulated C74 (this group shows effect of C74 alone); MB US: empty MB with UTMD (this control group tests if bubble cavitation alone causes reduction in cord formation), C74 MB: C74-encapsulated MB with no UTMD (this group demonstrates that uncavitated bubbles do not cause an effect even though they are dosed with C74); C74 MB US: C74-encapsulated MB with UTMD (this is the true test group showing that when bubbles are destroyed and C74 is released, we see reduction in cord formation), and C74 US: C74-encapsulated MB with UTMD followed by immediate washout and replacement with C74-devoid culture media (this group tests whether C74 immediately enters cells post bubble destruction). The bar graph in panel B depicts the relative cord lengths between the different treatment groups—cord length is defined as the distance between the junctional i.e. cord-intersectional points. (One-way ANOVA with Tukey post hoc test, ∗∗∗∗ p < 0.0001 from n = 3 experiments). HmVEC, human microvascular EC; UTMD, ultrasound-targeted microbubble destruction; DMSO, dimethyl sulfoxide.

Article Snippet: Human microvascular ECs (HmVECs, ATCC, CRL-3243) were cultured in MCDB-131 media with growth supplements as described previously ( ).

Techniques: In Vitro, Inhibition, Control

Journal: QJM: An International Journal of Medicine

Article Title: Acute endothelial stresses identify microRNA let-7b-5p and non-coding SLC11A2 (NRAMP2/DMT1) exon as biomarkers that overlap with those detected in malignant and non-malignant diseases

doi: 10.1093/qjmed/hcae235

Figure Lengend Snippet: Pre-miRNA and mature miR responses to ferric citrate treatment of normal primary ECs. ( A ) RNA-seq data for all pre-miRNAs detected in 1 and 6 h rRNA-depleted libraries from EC treated with media or 10 μmol/l ferric citrate, presented as fold change of respective media-treated EC represented at 0 h. One hour data are from normal HDMEC compared to paired 1 h media-treated HDMEC. Six hour data are from normal HPMEC compared to paired 6 h media-treated HPMEC. By Dunn’s test post Kruskal–Wallis, there was a fall (* P < 0.05) after 1 h treatment, and no significant change after 6 h. ( B ) RNA-seq data for let-7 pre-miRNAs as in (A) (let-7a-1, let-7a-2, let-7a-3, let-7b, let-7d, let-7f-1, let-7g, let-7i), with P values (* P < 0.05) calculated by Dunn’s test post Friedman. ( C ) RNA-seq data for mRNA changes in HPMEC after 6 h 10μmol/l ferric citrate, as fold change of respective values in paired media-treated HPMEC, categorized by whether mRNAs were identified by TargetScan 5.2[28] as a let-7 target based on mature miRNAs from miRbase: 570 of 10 851 mRNAs had 1-6 (mean 1.11) let-7 family 8mer, 7mer-m8 or 7mer-1A binding sites . ( D ) Comparisons of let-7b by qRT-PCR and RNA-seq. (i) Mature miRNA let-7b-5p, assayed at stated durations of 10μmol/l ferric citrate as quantified in HPMEC by qRT-PCR using Applied Biosystems miRNA assay 002619. Ct values were converted to concentrations following spiking of cell extracts with known concentrations of cel-miR-39 as described in the . (ii) RNA-seq comparisons (as also represented in A and B). ( E ) Dose–response curves for mature let-7b-5p in four cultures of primary HDMEC or primary HUVEC, after 1 h treatment with media (‘0’), 4 or 10 μmol/l ferric citrate treatments, quantified by qRT-PCR using miRNA assay 002619 (Applied Biosystems). Threshold cycles (Ct) values were converted to concentrations based on known concentrations of spiked cel-miR-39 (described further in ), before fold-changes from the media-treated EC were calculated for graphical presentation.

Article Snippet: For treatment duration, no morphological changes were observed in primary human pulmonary microvascular ECs (HPMEC) or primary human dermal microvascular ECs (HDMEC, PromoCell GmbH, Heidelberg) cultured for 24 h with 10 μmol/l ferric citrate: 1 h and 6 h responses were evaluated.

Techniques: RNA Sequencing, Binding Assay, Quantitative RT-PCR