Journal: American Journal of Physiology - Cell Physiology

Article Title: Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction

doi: 10.1152/ajpcell.00391.2023

Figure Lengend Snippet: Pentastatin (PS) promotes β1-integrin subunit activation on human pulmonary arterial endothelial cells (hPAECs). A : ITGB1 gene expression in healthy-hPAECs ( n = 5) compared with IPAH-hPAECs ( n = 4 derived from n = 3 different IPAH-hPAECs. * P < 0.05; determined by unpaired t test. IPAH, idiopathic pulmonary arterial hypertension; ITGB1 , integrin subunit beta 1. B : pull-down of NC1 with β1-integrin subunit along with schematic representation of immunoprecipitation workflow. hPAECs were exposed to either vehicle (veh) or NC1 for 10 min. Consequently, hPAECs/NC1 were cross-linked and protein complexes were immunoprecipitated. IP, immunoprecipitated material; Iso, isotype-matched control. Created with BioRender and published with permission. C : direct-binding of β1-integrin subunit to pentastatin (PS) and to NC1 was revealed using a solid-phase binding assay. β1-integrin subunit was allowed to bind surface coated with equal concentration of veh, NC1, PS, random peptide (RP), and bovine serum albumin (BSA). Binding was measured in absorbance, normalized to BSA. Y -axis set to log2 scale. * P < 0.05, *** P < 0.001; determined by one-way ANOVA with Tukey’s post hoc test. D and E : hPAECs were exposed to veh or PS (50 µg/mL), and β1-integrin subunit levels on the cell surface were analyzed by flow cytometry. Representative dot blot plot from a single experiment and quantification of total β1-integrin subunit (clone MB1.2; D ) and active β1-integrin subunit (clone 12G10; E ) on cell surface upon PS stimulation. veh vs. PS: * P < 0.05 or ** P < 0.01; determined by one-way ANOVA for repeated measured followed by Tukey’s post hoc; n = 5 independent experiments from n = 3 different hPAECs. F : barrier integrity of hPAECs pretreated with β1-integrin neutralizing antibody (2 µg/mL, clone P5D2) for 3 h prior to veh or PS (50 µg/mL) treatment at 240 min after veh/PS the stimulation. Barrier integrity was calculated by percentage (%) of the normalized endothelial resistance at given time point compared with baseline. * P < 0.05; determined by paired t test; n = 6 independent experiments from n = 5 different hPAECs. G : barrier integrity of hPAECs pretreated with ROCK inhibitor Y-27632 (20 µM) for about 70 min before veh or PS (50 µg/mL) at 30 and 60 min after the veh/PS stimulation presented as %. PS effect alone vs. PS effect with Y-27632 on hPAEC monolayer: * P < 0.05; determined by two-way ANOVA followed by Tukey’s post hoc test; n = 6 independent experiments from n = 3 different hPAECs donors. Error bars represent the standard deviation (SD).

Article Snippet: To pharmacologically block Rho/ROCK pathway and β1-integrin, hPAECs were pretreated with the ROCK inhibitor Y-27632 (#10005583, Cayman Chemical Company, Michigan) and an anti-β1-integrin subunit antibody (#MAB17781, clone P5D2, R&D Systems, Minnesota).

Techniques: Activation Assay, Expressing, Derivative Assay, Immunoprecipitation, Control, Binding Assay, Concentration Assay, Flow Cytometry, Dot Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Extra-Cellular Matrix Proteins Induce Matrix Metalloproteinase-1 (MMP-1) Activity and Increase Airway Smooth Muscle Contraction in Asthma

doi: 10.1371/journal.pone.0090565

Figure Lengend Snippet: (a) ASM cells processed for flow cytometry were incubated with antibodies against the integrin subunits β1, β3 and the αvβ5 receptor prior to incubation with a fluorescently conjugated secondary antibody. Histogram depicts fluorescence intensity detected by flow cytometry (one representative experiment of three shown) for antigens (shaded) and their respective isotype control (broken line). (b) ASM cells were incubated with the same antibodies as listed above prior to stimulation with tenascin-C (10 µg/ml). Supernatant was harvested after 24 hours and assayed for MMP-1 protein. Expression levels analysed using a 2-way ANOVA with post-hoc Dunnett’s Multiple comparison test (*P≤0.05. ****P≤0.0001). Graph depicts results normalised to the isotype control (stimulated with tenascin-C (10 µg/ml)) at equivalent concentration, representative experiment of three separate donors is shown.

Article Snippet: Cells were incubated on ice for 45 minutes with antibodies against integrin subunits β1 (R&D, MAB17781) and β3 (Millipore, MAB2023Z), the αvβ5 integrin (VWR, AB24694) and toll like receptor 4 (TLR4) (R&D, AF1478).

Techniques: Flow Cytometry, Incubation, Fluorescence, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Extra-Cellular Matrix Proteins Induce Matrix Metalloproteinase-1 (MMP-1) Activity and Increase Airway Smooth Muscle Contraction in Asthma

doi: 10.1371/journal.pone.0090565

Figure Lengend Snippet: (a) Six asthma derived and six control ASM cultures were incubated for 24 hours with and without the addition of soluble tenascin-C (10 µg/ml). The supernatant was harvested and levels of total MMP-1 assessed by ELISA. Expression levels were analysed using an unpaired one-tailed t-test (*P<0.05. **P<0.01). (b) ASM cells from four asthma and three control donors were incubated with a β3 integrin subunit function blocking antibody prior to stimulation with tenascin-C (10 µg/ml). Supernatant was harvested after 24 hours and assayed for MMP-1 expression. Expression levels for control and asthma ASM were separately normalised to an equivalent concentration of an isotype control, stimulated with tenascin-C (10 µg/ml). (c) Three control ASM cell cultures were seeded on ECM deposited by ASM cells from six asthma and six control donors. After 24 hours total MMP-1 was assessed by ELISA. Expression was analysed using a 1 way ANOVA with post-hoc Dunnett’s Multiple comparison test (**P<0.01. ***P≤0.001). (c) Serum starved ASM cells from four asthma and three control donors were seeded onto tissue culture plastic for six hours and TNC or COL1A1 mRNA assessed by qRT-PCR. Levels are expressed as mean fold change in cycle threshold number relative to GAPDH and compared by paired t-test (*P<0.05).

Article Snippet: Cells were incubated on ice for 45 minutes with antibodies against integrin subunits β1 (R&D, MAB17781) and β3 (Millipore, MAB2023Z), the αvβ5 integrin (VWR, AB24694) and toll like receptor 4 (TLR4) (R&D, AF1478).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, One-tailed Test, Blocking Assay, Concentration Assay, Quantitative RT-PCR