Abstract
Rationale: Antibiotic treatment of patients infected with G− or G+ bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion.Methods: Barrier function measurements were done in HL-MVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALF) was measured by multiplex analysis. PKA and PKC-α activity were assessed by Western blotting.Results: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-α activity in PLY-treated HL-MVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HL-MVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HL-MVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers.Conclusions: GHRH signaling in HL-MVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter.
Highlights
Of the approximately 9 million children aged less than 5 years that die each year worldwide, 1.6 million succumb from pneumonia (Rudan et al, 2008)
Growth Hormone-releasing Hormone (GHRH) signaling in human lung microvascular endothelial cells (HL-MVEC) protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective protein kinase A (PKA)-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter
We investigated whether GHRH agonists are able to activate both PKA-mediated barrier-protective and PKC-mediated barrier-disruptive pathways in human lung microvascular endothelial cells (HL-MVEC) and how these pathways interact
Summary
Of the approximately 9 million children aged less than 5 years that die each year worldwide, 1.6 million succumb from pneumonia (Rudan et al, 2008). GHRH protects pulmonary barrier function of antibiotics, mortality during the first several days of pneumonia has not decreased appreciably over the past 75 years (Evans and Gaisford, 1938; Waterer et al, 2011). Even though the lungs are essentially free of bacteria following antibiotic treatment, many patients still die from complications, the most important one of which is pulmonary permeability edema. One of the main causes of pneumonia-associated permeability edema in patients treated with antibiotics is the massive release of toxins, including pneumolysin (PLY), a virulence factor from G+ pneumococci and lipopolysaccharide (LPS), an endotoxin from G− bacteria (Anderson et al, 2007), following bacterial lysis. Inducing very different pathways leading to endothelial dysfunction, both toxins are important mediators of pneumonia-associated capillary leak, which is an important problem contributing to permeability edema
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