Figure 2.
Intracellular MKK3/P38 pathway affected by heme. (A-F) HLMVECs were treated with heme (50 µM) for different time points, and cell lysate was prepared in RIPA buffer with protease and phosphatase inhibitors. The MKK3/p38/HSP27 axis proteins were found to be activated after heme treatment. Representative blots of proteins and corresponding proteins phosphorylation levels for P-p38, p38, P-MK2 (T334), P-MK2 (T222), P-HSP27, HSP27, P-MKK3 (S189), and MKK3 (A), quantified using BioRad software (B-F). (H-L) MKK3−/− mice had attenuated barrier disruption and diminished p38 pathway activation. Wild-type (WT) and MKK3−/− mice were treated with heme (250 µM IV) or with vehicle for 6 hours, their were lungs excised, and lysates were prepared in RIPA buffer with protease and phosphatase inhibitors. Our data indicate activation of MKK3/P38 on heme treatment that was blunted in MKK3−/− lungs. TJ proteins showed a decrease only in WT mice treated with heme. Representative blots of proteins ZO-1, CLDN-5, P-p38, p38, P-MK2 (T334), MK2, P-MKK3(S189), and MKK3 (K), quantified using BioRad software (H-L). (M-P) Heme mildly activated adhesion molecules, specifically ICAM-1. Representative blots of E-selectin, ICAM-1, and VCAM-1, and quantifications are shown. All protein levels were normalized to β-actin. (Q) Assessment of heme-induced barrier dysfunction in mouse lungs. Mice were injected with heme or vehicle (6 hours) and, in the end, injected with fluorescein isothiocyanate (FITC)–dextran to visualize FITC-dependent fluorescence in the lungs. Our data indicated a high level of extravasation of FITC-dextran into the lungs in WT rats treated with heme, whereas MKK3−/− rats showed protection against heme-mediated lung barrier disruption. (R) Quantification of FITC-dextran extravasation into the lungs showed that MKK3−/− mice were significantly protected from heme-induced barrier damage. Experiments were repeated at least 3 times. Values are mean ± standard error of the mean. (S) Heme injected into mice activates the MKK3/p38MAPK/HSP27 pathway, leading to disruption of TJs, causing cytoskeletal derangements and ultimately causing barrier disruption (red). Compromised endothelial barrier is reflected by flow of FITC-dextran from bloodstream and into lung tissue. MKK3−/− mice are highly protected (green) from heme-induced endothelial barrier disruption. *P < .05 compared with WT, **P < .05 compared with MKK3 vehicle, ***P < .05 compared with WT heme group.

Intracellular MKK3/P38 pathway affected by heme. (A-F) HLMVECs were treated with heme (50 µM) for different time points, and cell lysate was prepared in RIPA buffer with protease and phosphatase inhibitors. The MKK3/p38/HSP27 axis proteins were found to be activated after heme treatment. Representative blots of proteins and corresponding proteins phosphorylation levels for P-p38, p38, P-MK2 (T334), P-MK2 (T222), P-HSP27, HSP27, P-MKK3 (S189), and MKK3 (A), quantified using BioRad software (B-F). (H-L) MKK3−/− mice had attenuated barrier disruption and diminished p38 pathway activation. Wild-type (WT) and MKK3−/− mice were treated with heme (250 µM IV) or with vehicle for 6 hours, their were lungs excised, and lysates were prepared in RIPA buffer with protease and phosphatase inhibitors. Our data indicate activation of MKK3/P38 on heme treatment that was blunted in MKK3−/− lungs. TJ proteins showed a decrease only in WT mice treated with heme. Representative blots of proteins ZO-1, CLDN-5, P-p38, p38, P-MK2 (T334), MK2, P-MKK3(S189), and MKK3 (K), quantified using BioRad software (H-L). (M-P) Heme mildly activated adhesion molecules, specifically ICAM-1. Representative blots of E-selectin, ICAM-1, and VCAM-1, and quantifications are shown. All protein levels were normalized to β-actin. (Q) Assessment of heme-induced barrier dysfunction in mouse lungs. Mice were injected with heme or vehicle (6 hours) and, in the end, injected with fluorescein isothiocyanate (FITC)–dextran to visualize FITC-dependent fluorescence in the lungs. Our data indicated a high level of extravasation of FITC-dextran into the lungs in WT rats treated with heme, whereas MKK3−/− rats showed protection against heme-mediated lung barrier disruption. (R) Quantification of FITC-dextran extravasation into the lungs showed that MKK3−/− mice were significantly protected from heme-induced barrier damage. Experiments were repeated at least 3 times. Values are mean ± standard error of the mean. (S) Heme injected into mice activates the MKK3/p38MAPK/HSP27 pathway, leading to disruption of TJs, causing cytoskeletal derangements and ultimately causing barrier disruption (red). Compromised endothelial barrier is reflected by flow of FITC-dextran from bloodstream and into lung tissue. MKK3−/− mice are highly protected (green) from heme-induced endothelial barrier disruption. *P < .05 compared with WT, **P < .05 compared with MKK3 vehicle, ***P < .05 compared with WT heme group.

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