My laboratory has been investigating for over 25 years the role of reactive oxygen species (ROS) and bioactive lipids in vascular endothelial signaling, injury and barrier integrity. ROS have been implicated in the pathophysiology of several respiratory diseases including ARDS, COPD, pulmonary hypertension and bronchopulmonary dysplasia and our current primary focus is on the role and regulation of NADPH Oxidase and NOX proteins in hyperoxia- and sepsis-induced lung injury. We were the first to demonstrate that sphingosine-1-phosphate (S1P) is an agonist in endothelial cell signal transduction and S1P is the most potent angiogenic naturally occurring bioactive lipid that is present in plasma and tissues. My laboratory has been studying mechanisms of generation of intracellular S1P mediated by sphingosine kinases and degradation catalyzed by lipid phosphate phosphatases and S1P lyase in the endothelium and S1P lyase as a novel target of sepsis-mediated lung injury. Our investigations suggest a role of intracellular S1P in lung inflammation, injury, cell motility and NADPH Oxidase dependent ROS production. More recently, we have been investigating the role of HATS and HDACs in Mesothelioma and potential regulation of HATs/HDACs by sphingosine kinases and S1P lyase. These ongoing projects involve basic and translational research to develop novel therapeutic strategies and targets to limit the adverse effects of inflammatory lung injury.
Ongoing Projects in Natarajan’s Laboratory
1. Regulation of NADPH Oxidase by Phospholipase D and the EC Cytoskeleton
The NADPH Oxidase is a major source of reactive oxygen species (ROS) in the vasculature with increased generation of ROS linked to inflammation and vascular leak. Acute hyperoxia activates endothelial NADPH Oxidase and we have demonstrated that activation of NADPH Oxidase was partly regulated by Src dependent tyrosine phosphorylation of cortactin and p47phox and interactions between cortactin, p47phox and Src in cytoskeletal regulation of assembly of the Oxidase components in lipid rafts of the endothelium. Currently, the hypothesis that phospholipase D (PLD) activation and EC cytoskeleton are essential regulatory components of NADPH Oxidase mediated ROS production by hyperoxia is being tested in a murine model of lung injury and lung microvascular endothelial cells in culture. These studies include: 1) Role of phospholipase D in MLCK activation and MLC phosphorylation in endothelial NADPH Oxidase activation and barrier function; 2) Potential interactions between specific actin binding proteins and p47phox in hyperoxia-induced NADPH Oxidase activation and barrier function; 3) Role of Rac1 and IQGAP1 in hyperoxia- and VEGF-induced NADPH Oxidase activation and barrier function; and 4) Involvement of lipid rafts and caveolin-1 in hyperoxia- and VEGF-induced assembly and activation of endothelial NADPH Oxidase and barrier function.
Investigators: Peter Usatyuk, PhD; Srikanth Pendyala, MD; Donghong He
Funding: NIH/NHLBI PPG 1PO1 HL05864 – Project 4
Schema outlining ROS generation by NADPH oxidase activation
2. Endothelial Nox Proteins in Hyperoxia-Induced ROS Production, Signaling and Cell Motility
NADPH Oxidase (NOX) family enzymes catalyze the reduction of oxygen to superoxide (O2.-) as the primary product. Depending on the microenvironment or cellular compartment in which it is generated, spontaneous or superoxide dismutase (SOD)-catalyzed reduction of O2·- to hydrogen peroxide (H2O2) may occur, in association with a number of other reactive oxygen species (ROS). ROS function as signaling molecules and regulators of cell function when they are generated in a compartmentalized and regulated manner. NOX enzymes emerged during the evolutionary transition from unicellular to multi-cellular life and number of NOX/DUOX family enzymes increased to seven (NOX1-5; DUOX1-2) in mammals. This project investigates the role and regulation of Nox proteins, specifically Nox4 and Nox2 (gp91phox) endothelial ROS production, signal transduction and cell motility in primary human lung ECs and murine model of hyperoxic lung injury. Investigations include: 1) Characterize expression of Nox4 in HPAECs and mouse lung ECs and determine its role in ROS production; 2) Investigate molecular mechanisms of increased expression and activation of Nox4 in HPAECs; 3) Characterize signaling pathways that regulate Nox4- and Nox2-dependent endothelial cell migration and capillary tube formation via intracellular sphingosine-1-phosphate (S1P) generated by sphingosine kinases 1 and 2 and 4) Investigate the role of Nox2 and Nox4 in ROS generation and vascular leakiness in an in vivo murine model of lung injury using genetically modified Nox4 or Nox2 mice.
Investigators: Srikanth Pendyala, MD; Panfeng Fu, PhD.
Funding: NIH/NHLBI RO1 HL085553
3. Protective Role of Intracellular S1P in Sepsis-Induced Lung Injury
We have identified S1P as a major barrier-protective agent responsible for maintenance of vascular barrier integrity against lipopolysaccharide (LPS)-induced endothelial/lung injury in vitro and in vivo. S1P is a naturally occurring bioactive lipid that acts extracellularly through its G-protein coupled S1PR1 and other S1PRs with subsequent down-stream activation of Rho-GTPases, cytoskeletal reorganization, adherens and tight junction assembly, and focal adhesion formation. Also, there is evidence that supports an intracellular role of S1P in calcium release and proliferation of mouse embryonic fibroblasts. S1P-mediated cellular responses are regulated by its synthesis, catalyzed by sphingosine kinases (SphKs), and degradation mediated by S1P phosphatases (SPPs) and S1P lyase (S1PL) (Fig. X). In this project the hypothesis that modulation of intracellular S1P by sphingosine kinases and S1PL regulates LPS-induced lung inflammation and EC barrier dysfunction is being evaluated. Specific studies include:
1) Define S1PL and SphKs in regulation of intracellular S1P levels during LPS-induced inflammation and lung injury; 2) Define molecular mechanisms by which intracellular S1P, S1PL and SphKs regulate LPS-induced inflammatory responses; 3) Characterize the influence of ALI-associated single nucleotide polymorphisms (SNPs) on SphKs and S1PL expression and activities, and 4) Evaluate S1PL and SphKs as potential therapeutic strategies to limit LPS-induced lung injury in vivo.
Investigators: Wenli Ma, PhD; Panfeng Fu, PhD and Peter Usatyuk, PhD
Funding: NIH/NHLBI RO1 HL079396
4. Sphingosine Kinase 1 is a novel target of Mesothelioma
Malignant mesothelioma is a rapidly growing neoplasm that arises from the serosal surfaces of pleural, peritoneal or pericardial surfaces. The majority of mesothelioma cases diagnosed every year arises from the pleura, but ~10% of cases are mesothelioma of the peritoneum, with rare occurrence in the pericardium and tunica vaginales of the testes. Approximately 2000 new cases are diagnosed each year in the US with an even higher number worldwide. Although the disease is prevalently seen in men, it has been described in women and early childhood. Malignant mesothelioma (MM) is divided into three major categories: epithelioid (50% to 60%), sarcomatoid (7% to 20%), and the mixed/biphasic (20% to 35%). Prolonged exposure to asbestos is a well-known risk factor for MM, and the cooperation of other carcinogens with asbestos in the onset of this neoplasm seems possible (22). Therapy for mesothelioma can involve surgery, radiation therapy, and/or chemotherapy. Recent, initial phase I studies with oral formulation of histone deacetylase (HDAC) inhibitor, Zolinza (vorinostat; suberoylanilide hydroxamic acid, or SAHA) have demonstrated objective response inn patients with MPM (21). Our preliminary results suggest SphK1 and HATs/HDACs to be involved in proliferation of mesothelioma cells, a major focus of this investigation. Specific studies include: 1) Determine the role of SphKs and Histone acetylation modulated by HATs/HDACs in regulation of mesothelioma cell migration and proliferation, and 2) Examine therapeutic efficacy of SphK and histone deacetylase (HDAC) inhibitor(s) in a mouse model of mesothelioma.
Investigators: Hong Li, MD; Ravi Salgia, MD; Yutong Zhao MD, PhD.
Funding: R21 CA152849-01; MPI Pending with a score of 17 percentile