Figure 1.
Chronic thrombin treatment increases Cx43 protein expression.
HPAECs were left untreated or treated hourly with thrombin (0.5 U/mL) over 6 hr. and Cx43 protein and mRNA were measured. A.) Representative immunoblot shows Cx43 protein levels in thrombin (thr) or control (C) HPAECs. Panel images for control and thrombin bands were recombined from images generated from a single membrane of a Western blot. B.) Graph shows densitometric analysis of Cx43 protein expression. Cx43 protein levels were normalized to β-actin levels and divided by the average control value. *, p<0.05, n = 9. C.) Cx43 mRNA levels were measured by real-time RT-PCR and normalized to GAPDH mRNA and divided by the average control value. *p<0.05, n = 14.
Figure 2.
LPS increases Cx43 expression.
HPAECs were treated with LPS (200 ng/mL) for 6 hr or 24 hr and collected for Western Blot or qRT-PCR analyses. A.) A representative Western blot is shown. B.) Cx43 protein expression was normalized to β-actin and expressed relative to control. **, p<0.001. n = 6 for control, n = 3 for LPS (6 hr), n = 7 for LPS (24 hr). C.) Real-time RT-PCR analysis was performed on cells treated with LPS (24 hr) or control cultures. Cx43 mRNA levels were normalized to GAPDH mRNA. *, p<0.05, n = 14. C = control.
Figure 3.
Cx43 distribution between appositional membranes and cytoplasm is unaffected by thrombin or LPS treatment.
HPAECs were grown to confluence on glass cover slips and left untreated (A, Control) or treated with LPS (B, 200 ng/mL for 24 hr) or chronic thrombin (C, 0.25 U/mL hourly for 6 hrs). Then, cells were subjected to indirect immunofluorescent staining using a Cx43 antibody.
Figure 4.
LPS and chronic thrombin treatments increase intercellular communication.
HPAECs were grown to confluence on glass cover slips and treated with LPS (200 ng/mL for 24 hr) or chronic thrombin (0.25 U/mL; once every hr for 6 hr.) Single cells were injected with Lucifer yellow for 1 min., and cells were photographed after an additional 5 min. A.) Photomicrographs show representative fluorescence images of an injected cell and its neighbors after injection of Lucifer yellow in a control culture or a culture treated with LPS. The injected cell is marked with a black arrow; neighboring cells which show dye transfer are marked with a white asterisk. B.) Graph shows the composite data for Lucifer yellow dye transfer in control or LPS-treated HPAECs. Values shown are normalized by dividing the number of dye-containing neighbors by the average value obtained by injections in control cultures. **, p<0.001. n = 4. C.) Graph shows the composite normalized data for Lucifer yellow dye transfer in control or thrombin-treated HPAECs. *, p<0.05, n = 3.
Figure 5.
Cyclic stretch increases Cx43 expression.
HPAECs were grown to confluence on flexible Bioflex plates; subjected to pathological cyclic stretch (18% elongation), physiological cyclic stretch (5% elongation), or static stretch (0% elongation) for 24 hr.; and Cx43 protein or mRNA was measured. A.) 18% cyclic stretch (18% C.S.) is compared to physiological (5%) stretch control (5% C.S.) and static stretch control. A representative Western blot is shown. Cx43 is normalized to β-actin housekeeping protein levels and expressed relative to static stretch. *, p<0.05. n = 7 for 18% stretch and static stretch; n = 5 for 5% stretch. B.) To measure Cx43 mRNA, cells were subjected to 18% cyclic stretch or static stretch control as before and samples were analyzed by qRT-PCR. Cx43 mRNA was normalized to GAPDH housekeeping mRNA. n = 6. *p<0.05.
Figure 6.
Gap junction inhibitor carbenoxolone attenuates permeability response to thrombin.
Confluent HPAECs were treated with carbenoxolone (carb) for >5 min (red triangle; 100 µM) followed by treatment with thrombin (arrow; 0.5 U/mL) and transendothelial resistance (TER) was measured. TER was recorded prior to any treatments and continued an additional 1.5 hr after treatments. A.) Representative TER traces are shown. B.) Peak permeability response is given as [1–(minimum normalized TER)], where normalization is to TER at the time point just prior to the first treatment. **, p<0.001; *, p<0.05; n = 13 for control, n = 11 for carb alone, and n = 15 for thr alone and thr+carb. C.) Permeability response after recovery is given as (1–normalized TER), where TER is taken at the time point in recovery where the thrombin alone group recovers to reach a permeability of <0.25; this is roughly 1 hr after thrombin treatment. As before, TER is normalized to the time point just prior to the first treatment. *p<0.001, n = 13 for each group. Carb = carbenoxolone, Thr = thrombin.
Figure 7.
Gap junction inhibitor carbenoxolone attenuates thrombin-induced MLC phosphorylation.
HPAECs were treated with carbenoxolone for >5 min followed by thrombin treatment (0.5 U/mL; 30 min) and collected for Western Blot. A.) Representative Western Blot is shown. B.) Phospho-MLC protein was normalized to β-actin relative to control. **, p<0.001. *, p<0.05. n = 5 for control (C), thrombin alone (thr), and thrombin+carbenoxolone (thr+carb) groups. n = 1 for carbenoxolone (carb).
Figure 8.
Transfection with siRNA effectively reduces expression and function of Cx43 in HPAECs.
Cultures of HPAECs were transfected with Cx43 or control (non-targeting) siRNA 48 hrs prior to various analyses. A.) Western Blots show levels of Cx43 in untreated HPAECs (C) or in cells transfected with different concentrations of non-targeting (NT) or Cx43 (si) siRNA. B.) Fluorescence microscope images show HPAECs micro-injected with Lucifer yellow after transfection with non-targeting (NT) or Cx43 (siRNA-Cx43) siRNA. C.) Graph shows the degree of dye transfer in cultures of HPAECs microinjected with Lucifer yellow after transfection with non-targeting (NT) or Cx43 (siRNA-Cx43) siRNA. Dye transfer was normalized by dividing the number of dye containing neighbors by the average value obtained in NT transfected controls. **, p<0.001, n = 6 for each group.
Figure 9.
Cx43 knockdown attenuates permeability induced by thrombin or LPS.
(A.) Graphs show the normalized permeability responses in HPAECs that were transfected with non-targeting (NT) or Cx43 (si) siRNAs for 48 hr and then left untreated or treated with 0.5 U/mL thrombin for 3 hr. (NT+thr and si+thr). Permeability response is given as (1– minimum normalized TER), where normalization is to TER at time point just prior to the first treatment. **, p<0.001. *, p<0.05. n = 8 for NT or siRNA alone; n = 11 for NT+thr group; n = 13 for si+thr group. (B.) Graphs show the normalized permeability responses in Pulmonary Microvascular Endothelial Cells (PMVECs) that underwent identical siRNA transfection and thrombin treatment. **, p<0.001. n = 3 for NT+thr and si+thr groups; n = 4 for NT group; n = 3 for si groups. (C.) HPAECs were transfected with Cx43 or control siRNA for 48 hr, treated with thrombin (0.5 U/mL; 30 min) and collected for Western Blot. Phospho-MLC protein was normalized to β-actin relative to control. *, p<0.05. n = 6 for each condition. (D.) Graphs show the normalized permeability responses in siRNA-transfected HPAECs that had been treated with 200 ng/mL lipopolysaccharide (LPS) for 24 hr. **, p<0.001. n = 7 for each group. NT = non-targeting siRNA, si = siRNA-Cx43. Thr = thrombin.