4th year graduate student in the laboratory of Dr. David A. Dean.
"Role of MRCKa and Na+, K+-ATPase Signaling in Alveolar Barrier Function in the Mouse Lung"
Project Summary: Acute Respiratory Distress Syndrome (ARDS) is a severe medical condition, which is characterized by significant alveolar fluid accumulation and insufficient gas exchange. Cardiac surgery, ECMO, and use of cardiac medications are all known risk factors for ARDS which also complicates management of these and other cardiovascular diseases. Effective alveolar fluid clearance and repair of a functional alveolar-capillary barrier are considered the primary mechanisms for edema resolution in ARDS. Apart from enhancing fluid clearance, the Na+,K+-ATPase has been shown important for alveolar barrier function. Our lab showed that overexpression of the Na+,K+-ATPase b1 subunit into lungs enhances alveolar barrier integrity in previously injured lungs in mice and pigs. Previous in vitro data indicated that MRCKa mediates the upregulation of tight junction (TJ) proteins and epithelial barrier integrity by b1 overexpression. I hypothesize that the b1-Na+,K+-ATPase regulates alveolar barrier function through MRCKa in vivo. I will determine 1) whether MRCKa is required for the upregulation of TJ proteins and barrier function by b1 gene delivery in vivo, and 2) whether overexpression of MRCKa alone is sufficient to protect and/or treat lipopolysaccharides (LPS) induced lung injury in mice. LPS will be delivered by oropharyngeal aspiration to induce lung injury. Various plasmids will be delivered to mouse lungs by electroporation. For aim 1, plasmid to knockdown MRCKa or an MRCKa inhibitor will be delivered to lungs 24 hours before gene transfer of plasmid to overexpress b1. If required for signaling, MRCKa knockdown or inhibition will abolish b1's induction of TJ proteins and barrier upregulation. For aim 2, mice will be challenged with LPS 24 hours after (protection study) or before (treatment study) gene transfer. At end points, various assays will be performed to assess lung injury. It is expected that overexpression of MRCKa alone will be sufficient to protect and treat LPS induced acute lung injury, decreasing lung injury and increasing TJ expression; overexpression of both MRCKa and b1 subunit will augment the protection and treatment of LPS injured lungs to give the greatest reduction in lung injury and improvement in barrier function. These studies will increase our understanding of the pathogenesis and treatment of ARDS, improve lung health, and ultimately decrease cardiovascular complications.
5th year graduate student in the laboratory of Dr. Chen Yan.
"The Role of PDE1C in Vascular Smooth Muscle Cell Lysosomal Dysfunction and Atherosclerosis"
Project Summary: The objective of this project is to investigate the function and underlying mechanism of the cyclic nucleotide phosphodiesterase 1C (PDE1C) in pathological vascular remodeling during atherogenesis. Cyclic AMP and cyclic GMP regulate vascular functions. PDEs by hydrolyzing cyclic nucleotides, regulate cyclic nucleotide signaling. Vascular smooth muscle cells (SMCs), upon endothelium damage, transit from contractile phenotype to synthetic phenotype. In vasculature, PDE1C expression is selectively induced in synthetic SMCs, but not in contractile SMCs or endothelial cells. Synthetic SMCs can accumulate oxidized low-density lipoprotein (oxLDL) in lysosomes, referred to as SMC-derived foam cells, that have been suggested to contribute significantly in atherosclerotic lesions. oxLDL accumulation in lysosome causes lysosome membrane permeabilization and lysosome dysfunction, which accelerates atherosclerosis progression. Our preliminary data demonstrate PDE1C deficiency significantly decreases atherosclerotic lesions in vivo. In synthetic SMCs in vitro, we found that PDE1C inhibition reduces lysosomal oxLDL accumulation and ameliorates lysosomal permeabilization. Therefore, we propose two specific aims. Aim1: Determine the roles and underlying mechanisms of PDE1C in the regulation of oxLDL accumulation and lysosomal permeabilization in synthetic SMCs in vitro. In SMCs culture, PDE1C deficiency will be examined by PDE1 activity inhibitor, PDE1C wildtype vs PDE1C knockout mouse SMCs, PDE1C shRNA, and PDE1C reconstitution by adenovirus. SMCs oxLDL accumulation and lysosomal permeabilization will be assessed by Acridine Orange staining, cathepsin B/D staining, and lysosomal galectin puncta assay. Underlying mechanistical studies will use pharmacological inhibitors, siRNA or shRNA. Aim2: Evaluate the effect of PDE1C deficiency on SMC lipid accumulation and lysosomal dysfunction in atherosclerotic lesions in vivo. We will use spontaneous atherosclerotic model induced by 4 months high fat diet in mouse for biochemical assessments in the lesion areas. We will also examine the treatment potential of PDE1 inhibition on pre-trapped lipid deposition, using an accelerated atherosclerosis mouse model induced by carotid artery partial ligation. We hypothesize that PDE1C plays an essential role in atherosclerotic vascular modeling by promoting oxLDL induced-lysosomal dysfunction in synthetic SMC. This study may have significant therapeutic impact on atherosclerosis treatment.