Journal: Molecular Cancer

Article Title: Combination therapy targeting integrins reduces glioblastoma tumor growth through antiangiogenic and direct antitumor activity and leads to activation of the pro-proliferative prolactin pathway

doi: 10.1186/1476-4598-12-144

Figure Lengend Snippet: Effects of conditioned medium from encapsulated PAE cells overexpressing ES and Tum. (A) Secretion of ES and Tum from stably transfected PAE cells was determined using Western blot analysis of culture supernatants after protein concentration with heparin sepharose for ES and Nickel Cam for Tum. (B) Transfected PAE cells were encapsulated in alginate/PLL as described. Phase contrast photomicrograph shows transfected cells in microbeads at a magnification of x10. (C) HUVECs cultivated in CM containing ES, Tum or ES + Tum showed reduced proliferation rates when compared with cells cultivated in CM from PAE WT cells. Bars represent mean values ± SE ( n = 3); * on bars indicates significant differences vs. WT (p < 0.05); */** on connecting lines indicates significant differences between respective groups (p < 0.05 / p < 0.005). (D) Wound healing assay. CM containing ES, Tum or ES + Tum reduced migration of HDMECs. Wound closure data are normalized to results obtained with CM from PAE WT cells.

Article Snippet: Commercially available HUVECs and HDMECs were cultured in EGM-2 medium (Lonza, Basel, Switzerland) containing 2% fetal calf serum (FCS).

Techniques: Stable Transfection, Transfection, Western Blot, Protein Concentration, Wound Healing Assay, Migration

Journal: The Journal of Cell Biology

Article Title: Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

doi: 10.1083/jcb.201706041

Figure Lengend Snippet: High levels of barrier resistance correlate with continuous intercellular junctions and cortical arrangement of the actin cytoskeleton. (A) Confocal images of confluent endothelial monolayers displayed in order of low (HAEC, HBMVEC, and HUVEC) and high levels of barrier resistance (HLMVEC and HDMEC) left to right (bar, 20 µm; representative of n = 3). HLMVEC and HDMEC exhibit a linear and organized junctional staining of VE-cadherin, claudin-5, and ZO-1 compared with an irregular junctional pattern in cell types with lower levels of resistance. Phalloidin staining reveals a strict cortical arrangement of the actin cytoskeleton in HLMVEC and HDMEC, whereas cells with low resistance exhibit more stress fibers. Localization of cortactin is more peripheral and less cytoplasmic in HLMVECs and HDMECs. F-actin fibers aligned along the cell periphery, minimizing radial tension forces at cell junctions, indicated by linear junctional pattern of VE-cadherin/phalloidin overlay images (arrows) and lower presence of pMLC2 in HLMVECs and HDMECs. ( B–F) Graphs presenting mean values of fluorescence intensity across multiple cell–cell junctions per cell type (Fig. S2 B) for each barrier protein shown in A. Junctional components as well as phalloidin and cortactin are concentrated along the cell borders in HLMVECs and HDMECs compared with cell types with low barrier resistance.

Article Snippet: Specifically, we obtained HUVECs (C2519A; Lonza; C-12203; PromoCell), HSaVECs (HSVEC/A; VEC-Technologies; cAP-0019; Angio-Proteomie), HAECs (PCS-100-011; ATCC; 6100; ScienCell), HIAECs (CC-2545; Lonza; cAP-0020; Angio-Proteomie), HBMVECs (ACBRI 376 V; Cell-Systems; cAP-0002; Angio-Proteomie), HUMVECs (C-12295; PromoCell; 7000; ScienCell), HLMVECs (3000; ScienCell; C-12281; PromoCell), HAMVECs (7200; ScienCell), and HDMECs (C-12212; PromoCell; 2000; ScienCell).

Techniques: Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

doi: 10.1083/jcb.201706041

Figure Lengend Snippet: Knockdown of VAV3 reduces barrier strength and alters cytoskeletal arrangement in HDMECs and HLMVECs. (A) siRNA knockdown of three candidate genes with a strong correlation of expression to barrier resistance ( BAIAP2, VAV3, and SORBS2 ) validates the effect of VAV3 as an important regulator. (B) Efficiency of siRNA knockdown for BAIAP2, VAV3, and SORBS2 . (C and E) Effect of VAV3 silencing on barrier resistance compared with combined siRNA knockdown of VAV2 and VAV3 or CLDN5 (positive control) in HDMECs and HLMVECs. (D and F) Efficiency of siRNA knockdown for VAV2 , VAV3 , and CLDN5 (ECIS dataset C and E) in HDMECs and HLMVECs versus control siRNA. (G and H) Bar graphs of barrier resistance levels at 48 h for HDMECs and HLMVECs (ECIS dataset C and E). Error bars show mean ± SEM; *, P < 0.05; n = 3. (I) Table presenting ECIS data modeling values of R b , α , and C m at 48 h for HDMECs and HLMVECs with siRNA knockdown for VAV3 , VAV2/3 , and CLDN5 versus control siRNA, respectively (mean ± SEM; n = 3). (J) Immunofluorescence staining of VE-cadherin, ZO-1, F-actin (phalloidin), and cortactin in HDMEC monolayers subjected to either siRNA knockdown of VAV3 and VAV2/3 or siRNA control (bars, 20 µm). Magnification of phalloidin staining highlights loss of cortical actin and gain of stress fibers upon VAV3 and VAV2/3 knockdown. Translocation of cortactin to the cell periphery (filled arrows) is reduced in monolayers with siRNA knockdown of either VAV3 alone or VAV2/3 (open arrows). (K) Fluorescence intensity across cell–cell junctions (mean of n = 6) in cells exposed to siRNA control, siRNA VAV3 , and siRNA VAV2/3 (for VE-cadherin, ZO-1, phalloidin, and cortactin; as shown in J).

Article Snippet: Specifically, we obtained HUVECs (C2519A; Lonza; C-12203; PromoCell), HSaVECs (HSVEC/A; VEC-Technologies; cAP-0019; Angio-Proteomie), HAECs (PCS-100-011; ATCC; 6100; ScienCell), HIAECs (CC-2545; Lonza; cAP-0020; Angio-Proteomie), HBMVECs (ACBRI 376 V; Cell-Systems; cAP-0002; Angio-Proteomie), HUMVECs (C-12295; PromoCell; 7000; ScienCell), HLMVECs (3000; ScienCell; C-12281; PromoCell), HAMVECs (7200; ScienCell), and HDMECs (C-12212; PromoCell; 2000; ScienCell).

Techniques: Knockdown, Expressing, Positive Control, Control, Immunofluorescence, Staining, Translocation Assay, Fluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5

doi: 10.1016/j.omtn.2022.07.002

Figure Lengend Snippet: miR-200 and miR-466 contribute to the increased endothelial cell (EC) permeability in hyperglycemia ECs from healthy and type 2 diabetic (T2D) donors were cultured to passage 4–7 before high glucose treatment or gene manipulation. (A) FITC-dextran Transwell assay showing T2D ECs have higher permeability than healthy ECs. n = 5 per group. (B) FITC-dextran Transwell assay showing healthy ECs exposed to 25 mM glucose have higher permeability than control group exposed to 20 mM mannitol and 5 mM glucose. n = 5 per group. (C) Stem-loop structure of miR-466 and miR-200 family, including miR-200a/b/c, miR-141 and miR-429, and miR-466. (D) Real-time quantitative polymerase chain reaction (qPCR) confirmed higher expression levels of miR-200a/b/c, miR-141, miR-429, and miR-466 in T2D ECs than healthy ECs. n = 5 per group. (E) qPCR showing higher expression levels of miR-200a/b/c, miR141, miR-429, and miR-466 in healthy ECs exposed to 25 mM glucose for 72 h compared to the control group exposed to 20 mM mannitol and 5 mM glucose. n = 5 per group. (F) qPCR confirmed the upregulation of miR-200 family members and miR-466 in healthy ECs transfected with miR-200 or miR-466 precursors compared to scrambled controls. n = 5 per group. (G) FITC-dextran Transwell assay indicating increased permeability of healthy ECs transfected with miR-200 or miR-466 precursors is compared to scrambled controls. Data are presented as means ± SDs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Mann-Whitney U test for statistical analysis (A, B, D, and E); Kruskal-Wallis test, and Mann-Whitney U test with Benjamini, Krieger, and Yekutieli’s adjustment for multiple comparisons if significant (F and G).

Article Snippet: HDMECs from healthy donors and patients with T2D were purchased from Lonza and maintained in microvascular EGM-2 (EGM-2 MV, Lonza) in 37°C, 5% CO 2 .

Techniques: Permeability, Cell Culture, Transwell Assay, Real-time Polymerase Chain Reaction, Expressing, Transfection, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5

doi: 10.1016/j.omtn.2022.07.002

Figure Lengend Snippet: miR-466 and miR-200 decrease expression level of Claudin-5 ECs were cultured to passage 4–7 before collection or transfection with miR precursors. (A) Protein expression of Claudin-5, VE-cadherin, and Connexin43 in healthy and T2D human aortic ECs (HAECs). n = 5 per group. Representative bands of western blot are shown below the quantification analysis. (B) Protein expression of Claudin-5, VE-cadherin, and Connexin43 in healthy and T2D human dermal microvascular ECs (HDMECs). n = 3–5 per group. Representative bands of western blot are shown below the quantification analysis. (C) The binding details of miR-200 family, including miR-200a/b/c, miR-141, and miR-466 on CLDN5 mRNA. (D) Luciferase activity assay illustration of rationale. (E) 3′ UTR luciferase activity assay confirmed reduced luciferase activity in healthy ECs transfected with miR-200 inhibitor or miR-466 inhibitor. Data are presented as means ± SDs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. M, marker. Mann-Whitney U test for statistical analysis (A and B); Kruskal-Wallis test and Mann-Whitney U test with Benjamini, Krieger, and Yekutieli’s adjustment for multiple comparisons if significant (E).

Article Snippet: HDMECs from healthy donors and patients with T2D were purchased from Lonza and maintained in microvascular EGM-2 (EGM-2 MV, Lonza) in 37°C, 5% CO 2 .

Techniques: Expressing, Cell Culture, Transfection, Western Blot, Binding Assay, Luciferase, Activity Assay, Marker, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5

doi: 10.1016/j.omtn.2022.07.002

Figure Lengend Snippet: miR-466 and/or miR-200 inhibitors accelerated wound healing in T2D mice A mouse model of leptin receptor-deficient mice (BKS.Cg-m +/+ Lepr db /J, db/db) and their healthy littermates (BKS.Cg-m −/− Lep db/− lean, db/+) was used. A full-thickness standard-size excisional wound was created on the dorsal skin. (A) Wound closure curves in db/db mice that received topical delivery of 100 nM miR-466 inhibitor or scramble controls onto the excisional wound. n = 4–5 per group. (B) Representative images of cutaneous wounds taken on days 0, 2, 4, 6, 12, 20, and 26 after wounding. (C) The dosage and time of the administration of miR-466 and/or miR-200 inhibitor, or scramble control on db/db or db/+ mice. (D and E) Real-time-PCR of miR-200 and miR-466 levels in wound tissues isolated from the mice received administration of miR-466 or miR-200 inhibitors, or scramble control onto the wounds. (F) Wound closure curves in db/db or db/+ mice which received topical delivery of miR-466 and/or miR-200 inhibitor, or scramble control onto the excisional wound. (G) Representative images of cutaneous wounds taken on days 0, 2, 4, 6, 12, 14, 16, 18, and 20 after wounding. (H) Representative images of CD31 staining of the wound bed isolated from db/db or db/+ mice that received miR-200 and/or miR-466 inhibitor, or scramble controls. Scale bar, 200 μm. (I) Capillary density on wound samples from db/db or db/+ mice that received miR-200 and/or miR-466 inhibitor, or scramble controls. (J) Immunohistochemistry staining of Claudin-5 protein on wound samples from db/db or db/+ mice that received miR-200 and/or miR-466 inhibitor or scramble controls. Scale bar, 200 μm. (K) Claudin-5 + area on wound samples from db/db or db/+ mice that received miR-200 and/or miR-466 inhibitor, or scramble controls. Data are presented as means ± SDs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. The statistical analysis used for each bar graph: Mann-Whitney U test for (A); Kruskal-Wallis test and Mann-Whitney U test with Benjamini, Krieger, and Yekutieli’s adjustment for multiple comparisons if significant for (D)–(F), (I), and (K).

Article Snippet: HDMECs from healthy donors and patients with T2D were purchased from Lonza and maintained in microvascular EGM-2 (EGM-2 MV, Lonza) in 37°C, 5% CO 2 .

Techniques: Real-time Polymerase Chain Reaction, Isolation, Staining, Immunohistochemistry, MANN-WHITNEY