Our preliminary data show that epoxyeicosatrienoic acids (EETs) are pro-angiogenic in two animal models for angiogenesis, matrigel plugs embedded subcutaneously in rats and chick chorioallantoic membranes.
The aims of this proposal are to measure regioisomer specific EET-mediated growth and capillary morphogenesis of human endothelial cells derived from the lung and heart and to define cellular signaling mechanisms that carry out these functions. This will address our long term goal to induce angiogenesis during pathological injury to the human heart and lung. We present evidence that growth and tubular differentiation of primary cultures of human coronary artery endothelial cells (HCAECs) and human lung microvascular endothelial cells (HLMECs) are stimulated by EETs. Levels of the signal regulator, mitogen-activated protein kinase phosphatase-1 (MKP-1) are also induced by over-expresssion of epoxygenase enzymes that catalyze formation of EETs in HLMECs. Using a novel, sensitive fluorescent assay developed at the Medical College of Wisconsin we have measured 4 EET regiosiomers in human endothelial cells and demonstrated increase in EETs in HCAECs after stimulation with bradykinin. In addition, we have cloned eDNA for 2 functional epoxygenase enzymes, the human endothelial epoxygenase 2C9 and its antisense, as well as rat 2C11. The epoxygenases have been recombined in adenoviral vectors and can be delivered to primary HCAECs and HLMECs with >90% efficiency. Using these data and tools the aims of this proposal are: (1) to determine growth of HCAEC and HLMECs by specific EET-regiosiomers and by over-expression of recombinant epoxygenases in the presence and absence of specific inhibitors 2) measure tube formation in the same cells in vitro after treatment with different EET regioisomers, epoxygenases and their inhibitors 3) determine potency of each regioisomer to mediate angiogenesis in vivo and 4) test the role of MKP-1 in inactivating the p38 mitogen-activated protein kinase pathway to promote tubular differentiation of human endothelial cells and angiogenesis in vivo.
These aims combine analytical, molecular, pharmacological and whole animal protocols to coordinate a timely, detailed, and focused research effort that will evaluate the role of EETs in growth and differentiation of human endothelial cells as well as angiogenesis in animals. It will impact on the potential for development of EETs as therapeutic agents during and after cardiac ischemia and acute lung injury.
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