Activation of EGFR and stimulation of arginase in pulmonary hypertension

Pulmonary hypertension is a life-threatening disease characterized by progressive vascular dysfunction and remodeling. Pulmonary hypertension can be a primary disorder, or it can be secondary to an underlying condition. There is currently no cure for pulmonary hypertension and available therapies remain essentially supportive (52). The hallmark of pulmonary hypertension is vascular remodeling, which has recently been postulated to represent a type of malignant transformation (54). Epidermal growth factor receptor tyrosine kinase (EGFR) is critical for promoting growth, survival, and differentiation of many types of cells. EGFR is activated by phosphorylation, which initiates signal transduction cascades leading to a variety of cellular responses including proliferation. EGFR tyrosine kinase has been recognized as a potential therapeutic target to prevent tumor cell proliferation in various types of cancer associated with local hypoxia. Pulmonary hypertension is seen in lung diseases characterized by hypoxia such as chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). We hypothesized that hypoxia causes EGFR activation leading to greater arginase expression and thereby to greater cellular proliferation in human pulmonary microvascular endothelial cells (hPMVEC).;Arginase metabolizes L-arginine to urea and L-ornithine (L-orn). The L-orn produced by arginase can be further metabolized by ornithine decarboxylase and/or ornithine aminotransferase to ultimately produce polyamines and/or proline, respectively. Polyamines and proline are vital to the cellular proliferation necessary for pulmonary vascular remodeling. Our laboratory has previously reported that cytokine-induced arginase up-regulation in bovine pulmonary arterial endothelial cells was dependent on EGFR activation. Mitogen-activated protein kinases (MAPK) are a group of evolutionarily conserved proteins linking cell surface receptors to critical intracellular targets. Activity of the MAPK is regulated through successive activation of a three-tiered cascade of kinases. The extracellular signal-regulated kinases (ERKs), a family member of the MAPK, are activated by the sequential activation of Raf and MEK kinases. A part of the intracellular pool of activated ERK translocates to the nucleus where it phosphorylates and activates transcription factors involved in mitogenic signaling. We hypothesized that EGFR tyrosine kinase would be increased in hypoxia and that arginase would be downstream of this. We further hypothesized that ERK would be involved in hypoxia-induced EGFR activation and subsequent induction of arginase in human pulmonary microvascular endothelial cells.;To test our hypotheses we studied human pulmonary microvascular endothelial cells (hPMVEC). For specific aim 1, the hPMVEC were incubated in either normoxia or hypoxia. The levels of EGFR, proliferating cell nuclear antigen (PCNA), arginase I and arginase II were determined by immunoblotting. EGFR activity assays were performed. Increases in cell proliferation due to EGFR activity were assessed using pharmacological inhibitors of EGFR and arginase. For specific aim 2, pharmacological inhibitors of EGFR or the MAPK (ERK and JNK) were employed in the various experiments. For specific aim 3, we utilized both male and female adult mice and exposed them to hypoxia for a period of 2 weeks while being treated with vehicle or the EGFR inhibitor AG1478. We harvested tissue from the animals to assay mRNA and protein levels of specific genes, as well as performed physiological measurements on the animals.