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Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

ABSTRACT: Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

SUBMITTER: Wang Z

PROVIDER: S-EPMC2898399 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

Wang Zhen Z Ginnan Roman R Abdullaev Iskandar F IF Trebak Mohamed M Vincent Peter A PA Singer Harold A HA

The Journal of biological chemistry 20100504 28

Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) mo ...[more]

PMID: 20442409

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Project description:ImportanceAfter anterior ST-segment elevation myocardial infarction (STEMI), left ventricular (LV) remodeling results in heart failure and death. Calcium/calmodulin-dependent protein kinase II delta (CaMKIId) is a key molecular mediator of adverse LV remodeling.ObjectiveTo determine whether NP202, an orally active inhibitor of CaMKIId, prevents LV remodeling in patients after anterior STEMI with early residual LV dysfunction.Design, setting, and participantsA randomized, double-blind, placebo-controlled multicenter clinical trial of NP202 vs placebo in patients after primary percutaneous coronary intervention (PCI) for anterior STEMI was performed from November 19, 2015, to August 1, 2018. The study was performed at 32 sites across the US, Australia, and New Zealand. Patients presenting with anterior STEMI who underwent PCI within 12 hours of symptom onset and left ventricular ejection fraction (LVEF) less than 45% on screening echocardiogram 48 hours after primary PCI were included in the study. Baseline cardiovascular magnetic resonance (CMR) imaging was performed within 5 days of the STEMI and before administration of the study drug. Follow-up CMR was performed after 3 months. Data were analyzed from November 19, 2015, to August 1, 2018.InterventionsPatients were randomly assigned to NP202, 1000 mg, daily for 3 months vs corresponding placebo.Main outcomes and measuresThe primary end point was change in LV end-systolic volume index (LVESVi) on CMR. Secondary end points were change in LV end-diastolic volume index, change in LVEF, change in infarct size, and change in diastolic function. Safety and tolerability were also assessed.ResultsA total of 147 patients (mean [SD] age, 58 [11] years; 129 men [88%]; 130 White patients [88%]) who experienced anterior STEMI treated with primary PCI were randomized to receive NP202 (73 [49.7%]) or placebo (74 [50.3%]). Baseline LVEF was similar between groups. At baseline, patients randomized to NP202 had greater LVESVi (48.2 mL/m2) than that in the placebo group (41.3 mL/m2; P = .03). However, the groups were otherwise well matched. For the primary end point of change in LVESVi from baseline to 3 months, there was no significant difference between the placebo (median [interquartile range] change, -0.60 [-9.28 to 5.99] mL/m2) and NP202 groups (-3.53 [-9.24 to 4.81] mL/m2) (P = .78). There was also no difference in the secondary efficacy end points assessed by CMR. NP202 was well tolerated and demonstrated an acceptable safety profile. Major adverse cardiac and cerebrovascular event rates were similar between groups. Two deaths occurred in each group during the follow-up period.Conclusions and relevanceThree months of treatment with NP202 after primary PCI for anterior STEMI with residual LV dysfunction did not improve LV remodeling. The drug was safe and well tolerated.Trial registrationClinicalTrials.gov Identifier: NCT02557217.

Project description:BackgroundVascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively.ResultsWe found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α.ConclusionsThe present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane. Video abstract.

Dataset Information

Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

Publications

Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction.

Similar Datasets

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