Fig 2: (a), (d) Immunohistochemistry staining images showing expression of eNOS in cardiac muscle ×100 (inset: ×400) and thoracic aorta ×400 respectively of control and tramadol treated mice. Arrows show the specific staining for the antibody. (b), (e) Relative quantification of immunohistochemistry staining for eNOS in cardiac muscle and thoracic aorta, respectively of both groups, n = 10. Data are expressed as means ± SEM, *p < 0.05 for control versus tramadol. (c), (f) Relative quantitative expression of mRNA levels of eNOS in cardiac muscle and thoracic aorta respectively of control and tramadol treated mice. All gene expression levels were normalized to GAPDH, n = 10. Data are expressed as means ± SEM, *p < 0.05 for control versus tramadol. (g) Immunohistochemistry staining images ×100 (inset: ×400) showing VEGF expression in cardiac muscle of control and tramadol treated mice. Arrows show the specific staining for the antibody. (h) Relative quantification of immunohistochemistry staining for VEGF in cardiac muscle of control and tramadol treated mice, n = 10. Data are expressed as means ± SEM, *p < 0.05 for control versus tramadol.

Fig 3: The over-expression of a constitutively active PKCa mutant mimics the effects of TRPV4 activation in pulmonary arterial endothelial cells. PAEC were transiently transfected with a constitutively active PKCa mutant (Myr-PKCa) for 48 h. Myr-PKCa over-expression (A) increases pT495eNOS levels (B) and NOS-derived superoxide (C). Myr-PKCa over-expression also increases mitochondrial ROS (D) and reduces the mitochondrial membrane potential in PAEC (E). Mitochondrial bioenergetics are also disrupted (F) with significant reductions in basal mitochondrial respiration, O2 consumed for ATP synthesis as well as the spare-, and maximal-respiratory capacity (G). Immunofluorescence analysis shows that Myr-PKCa over-expression induces the mitochondrial redistribution of eNOS (H). Values are mean ± SEM; n = 3–10. *P < 0.05 vs. untreated.

Fig 4: PKC activation disrupts mitochondrial bioenergetics in pulmonary arterial endothelial cells. Exposing PAEC to the PKC activator, PMA (100 nM, 0–60min) induces a transient increase in pT495eNOS levels (A) and eNOS uncoupling (B). PMA also affects mitochondrial bioenergetics (C) with significant reductions in the spare- and maximal-respiratory capacities (D). Immunofluorescence analysis shows that PMA induces the mitochondrial redistribution of eNOS (E&F). Values are mean ± SEM; n = 3–10. *P < 0.05 vs. untreated. †P < 0.05 vs prior time point.

Fig 5: Blocking eNOS phosphorylation at T495 attenuates endothelial barrier disruption in vitro and in vivo. The T495 eNOS decoy peptide (d-peptide) is able to bind efficiently to purified PKCa, but not eNOS (A). Treating PAEC with the d-peptide (1 µg/ml) attenuates the PMA (100 nm, 2 h)-mediated increase in pT495eNOS (B) and prevents the PMA-mediated increase in eNOS-derived superoxide (C). Immunofluorescence analysis shows that d-peptide attenuates the PMA-mediated mitochondrial redistribution of eNOS (D&E) and the 4aPDD (10 µM) dependent disruption of the EC barrier and endothelial junctional integrity as determined by ECIS (F) and VE-cadherin immunofluorescence staining (G&H). In the mouse model of VILI, the d-peptide decreases the stretch dependent increase in pT495eNOS (I) and attenuates the increase in capillary permeability as determined by reductions in the cell number (J) and protein concentration (K) in the BALF. H&E stained mouse lung sections (L) show that VILI causes atelectasis and thickened alveolar walls (*), alveolar edema and fibrin (red arrow), as well as vascular injury and evidence of perivascular edema (black arrow). The presence of the d-peptide prevents the development of these injurious events (L). Representative images are shown. Values are mean ± SEM; n = 3–10. *P < 0.05 vs. Control. †P < 0.05 vs PMA or VILI alone. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)