hdmecs from healthy donors and patients with t2d  (Lonza)

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