es007 rnd systems  (R&D Systems)

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Discovery, validation, and prodrug design of an ACE2 activator for treating bacterial infection-induced lung inflammation.

doi: 10.1016/j.jconrel.2023.10.025

Figure Lengend Snippet: Fig. 1. Identification of ACE2 activator H4 and its ability to boost ACE2 activity in lung epithelial cells. (A) ACE2 activity in the conditional medium of cultured Calu- 3 cells treated with different drugs. The lead drug is a 288Da molecule with the formula C18H24O3, herein named “H4” with its chemical structure illustrated to the right. (B) A dose-response curve of H4 for ACE2 activity in Calu-3 cells. (C) The ACE2 activity in the conditional medium of cultured Calu-3 cells treated with 25 μg/ mL H4 and harvested at different time points. ****p < 0.0001, pvalues obtained either from two-sided t-tests. Each dot in the graphs represents data from an in dividual well of Calu-3 cells.

Article Snippet: Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15].

Techniques: Activity Assay, Cell Culture

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Discovery, validation, and prodrug design of an ACE2 activator for treating bacterial infection-induced lung inflammation.

doi: 10.1016/j.jconrel.2023.10.025

Figure Lengend Snippet: Fig. 2. Administration of H4 reduces lung injury and impacts neutrophil infiltration in bacterial pneumonia model. (A) Experimental procedure of H4 administration following bacterial injection. (B–D) The Ace2 mRNA expression (B), ACE2 protein expression (C) and ACE2 activity (D) in the lung tissues of control mice (Ctrl, n = 5), mice infected with P. aeruginosa (P.a, n = 5), and mice infected with P. aeruginosa and then treated with H4 (P.a H4, n = 5). (E) Body weight changes at the time of sacrifice for indicated experimental groups. (F) H&E-stained representative images of indicated experimental groups. Scale bars, 100 μm. (G-H) The expression of proinflammatory cytokines TNFγ (G) and IFNγ (H) in the lung tissues of indicated experimental groups. (I) Neutrophil numbers in BAL as determined by flow cytometry, gated as CD45+CD11b+Ly6G+ in live cells. (J) Representative immunofluorescence im ages for neutrophil marker MPO in the lung tissue of indicated experimental groups. Scale bars, 100 μm. (K-M), The expression of neutrophil recruitment chemokines KC (K) CXCL5 (L) and MIP-2 (M). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p values obtained from one-way ANOVA followed by multiple comparisons. Each dot in the graphs represents data from an indi vidual mouse.

Article Snippet: Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15].

Techniques: Injection, Expressing, Activity Assay, Control, Infection, Staining, Flow Cytometry, Immunofluorescence, Marker

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Discovery, validation, and prodrug design of an ACE2 activator for treating bacterial infection-induced lung inflammation.

doi: 10.1016/j.jconrel.2023.10.025

Figure Lengend Snippet: Fig. 3. H4 reduces lung injury in a bacterial pneumonia model via ACE2. (A-C) The Ace2 mRNA expression (A), ACE2 protein expression (B) and ACE2 activity (C) in the lung tissue of wildtype mice (WT, n = 7) and Ace2−/−mice (Ace2−/−, n = 7). (D) Body weight changes at the time of sacrifice of Ace2−/−mice infected with P. aeruginosa (P.a, n = 3) and Ace2−/−mice infected with P. aeruginosa and treated with H4 (P.a H4, n = 3). (E) H&E-stained representative images of indicated experimental groups. Scale bars, 100 μm. (F) The expression of proinflammatory cytokine IFNγ in the lung tissue of indicated experimental groups. (G) Neutrophil numbers in BAL as determined by flow cytometry, gated as CD45+CD11b+Ly6G+ in live cells. (H) Representative immunofluorescence images for neutrophil marker MPO in the lung tissue of indicated experimental groups. Scale bars, 100 μm. (I) The expression of neutrophil recruitment chemokine MIP-2. ****p < 0.0001, p values obtained from two-sided t-tests. Each dot in graphs represents data from an individual mouse.

Article Snippet: Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15].

Techniques: Expressing, Activity Assay, Infection, Staining, Flow Cytometry, Immunofluorescence, Marker

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Discovery, validation, and prodrug design of an ACE2 activator for treating bacterial infection-induced lung inflammation.

doi: 10.1016/j.jconrel.2023.10.025

Figure Lengend Snippet: Fig. 6. Zn depletion reversed the increases ACE2 activity by H4. (A) The expression of ACE2 mRNA in cultured Calu-3 cells treated with 25 mg/mL H4 and harvested at different times. (B-C) The expression of ACE2 mRNA in the lungs and ACE2 levels in BALF from mice inoculated with P.a for 48 h with or without H4 administration (10mg/kg) 18 h post bacterial inoculation. (D) The ACE2 activity of the recombinant ACE2 treated with 200 ng/mL H4 or/and 25 mM DTPA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, p values obtained from one-way ANOVA followed by multiple comparisons. Each dot in graphs represents data from an individual well of treatment. n≥4 in all in vivo studies.

Article Snippet: Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15].

Techniques: Activity Assay, Expressing, Cell Culture, Recombinant, In Vivo

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Discovery, validation, and prodrug design of an ACE2 activator for treating bacterial infection-induced lung inflammation.

doi: 10.1016/j.jconrel.2023.10.025

Figure Lengend Snippet: Fig. 5. H4 prodrug shows similar beneficial effects in bacterial pneumonia model to H4. (A) Cell viability of cultured NIH/3 T3 cells treated with different doses of H4 and prodrug. (B–D) The Ace2 mRNA expression (B), ACE2 protein expression (C) and ACE2 activity (D) in the lung tissue of mice infected with P. aeruginosa and treated with H4 (P.a H4, n = 5) and mice infected with P. aeruginosa and treated with prodrug (P.a prodrug, n = 5). (E) Body weight changes at the time of sacrifice of indicated experimental groups. (F) H&E-stained representative images of indicated experimental groups. Scale bars, 100 μm. (G-H) The expression of proin flammatory cytokines TNFγ (G) and IFNγ (H) in the lung tissue of indicated experimental groups. (I) Neutrophil numbers in BAL as determined by flow cytometry, gated as CD45+CD11b+Ly6G+ in live cells. (J) Representative immunofluorescence images for neutrophil marker MPO in the lung tissue of indicated experimental groups. Scale bars, 100 μm. (K-M) The expression of neutrophil recruitment chemokines KC (K), CXCL5 (L) and MIP-2 (M). p values obtained from two-sided t-tests. Each dot in graphs represents data from an individual mouse.

Article Snippet: Calu-3 cells were treated serially with individual compounds contained within the Johns Hopkins Drug Library (JHDL), which includes a series of FDA-approved drugs [46] (kindly provided by Dr. Jun O. Liu, Johns Hopkins University) at 10μM for 18h, and the ACE2 activity in cell culture medium was determined by measuring the fluorescence intensity of ACE2 substrate Mca-YVADAPK(Dnp)-OH (Catalog no. ES007; R&D Systems) [15].

Techniques: Cell Culture, Expressing, Activity Assay, Infection, Staining, Flow Cytometry, Immunofluorescence, Marker