Chronic severe asthma is characterized by airways inflammation and airway hyperresponsiveness (AHR). Cytokines and growth factors promote smooth muscle cell proliferation, deposition of extracellular matrix and microvascular remodeling that are prominent features of airway remodeling. New clinical evidence supports an important role for vascular endothelial growth factor (VEGF), a growth factor that regulates vascular permeability and angiogenesis, as a modulator of airway inflammation and AHR in asthma. We present exciting new data demonstrating that VEGF enhances agonist-induced tracheal ring force generation. VEGF also activates RhoA and Rac1 and induces formation of reactive oxygen species (ROS) in ASM cells. In light of these findings, we hypothesize that VEGF activates the Rho family GTPases in ASM cells, leading to enhanced agonist-induced force generation through disruption of microtubules and formation of ROS. To test these hypotheses, we propose the following specific aims:
Aim 1 will define the in vivo effects of VEGF on Rho activation and agonist-induced force generation, using a murine model of allergen-induced airway inflammation or transgenic overexpression of VEGF. The relative efficacy of VEGF receptor-specific ligands to activate Rho, phosphorylate myosin light chain (MLC) and enhance agonist-induced force will be characterized. We will also determine whether VEGF induces calcium sensitization in ASM cells.
Aim 2 will determine whether microtubule (MT) disruption mediates VEGF-induced augmentation of force generation induced by agonists. We will determine whether MT disruption or stabilization alters agonist-induced tracheal ring force generation and characterize the effect of VEGF on phosphorylation of the MT-associated protein Tau. Finally, we will determine whether Rho activation is necessary and/or sufficient for mediating VEGF effects on MT.
Aim 3 will determine whether reactive oxygen species are necessary and/or sufficient to mediate VEGF-induced augmentation of agonist-induced force generation. We will determine the receptor specificity of VEGF-induced ROS generation and whether inhibition of ROS formation abrogates the effects of VEGF on agonist-induced force. Agonist-induced force generation in tracheal rings derived from gp91phox-deficient or superoxide dismutase transgenic mice will be compared to the effects of antioxidants or enzyme inhibitors. Finally, we will determine whether expression of VEGF modulates Rac activation in vivo and if Rac is necessary and/or sufficient for VEGF-induced ROS and MLC phosphorylation in vitro. Elucidating mechanisms of VEGF-induced alterations in smooth muscle cell force generation fits into the broader program of understanding the contribution of ASM cells to airway remodeling in asthma, and may provide a basis for the development of anti-VEGF therapies aimed at modulating the pathophysiological changes associated with asthma. ? ?
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