Endothelial cells are the vascular cells that line the lumen of blood vessels; they are in contact with smooth muscle cells on one side and circulating blood cells on the other. Endothelium modulates vascular tone and provides a nonthrombogenic surface for blood vessels. These actions are mediated through the release of soluble mediators and some of these mediators are metabolites of arachidonic acid. Arachidonic acid undergoes a variety of oxidation reactions to yield several families of biologically active lipids called eicosanoids. The principal enzymatic pathways of arachidonic acid metabolism include cyclooxygenase, lipoxygenases and cytochrome P450 epoxygenase. The products of these pathways include the prostaglandins, thromboxane, leukotrienes and others. Eicosanoids act as local hormones to regulate cell function and to communicate among cells. Endothelial cells, for example, synthesize prostacyclin from arachidonic acid. It causes relaxation of vascular smooth muscle and inhibits platelet aggregation. Research in our laboratory involves the isolation and identification of new metabolites of arachidonic acid and other endothelial mediators of vascular function. Once identified, the biological activities of the mediators are determined, their mechanism of action examined and the regulation of their synthesis investigated. These studies are important to our understanding of the causes of hypertension and ischemic heart disease and provide insights into new therapies.
We are studying the regulation of vascular tone and adrenal steroidogenesis by endothelial cell factors:
Smooth Muscle Cells: Like prostacyclin, epoxyeicosatrienoic acids (EETs) are also synthesized from arachidonic acid by the coronary vascular endothelium. While prostacyclin is made by cyclooxygenase, the EETs are synthesized by cytochrome P450 epoxygenase. The EETs also cause coronary vasodilation. The hormone acetylcholine stimulates the release of prostacyclin and EETs from endothelial cells, and these eicosanoids mediate a portion of the endothelium-dependent relaxations to this hormone. EETs cause vasodilation by opening calcium-activated potassium channels in smooth muscle cells and thereby decreasing the membrane potential of these cells. Thus, the EETs represent endothelium-derived hyperpolarizing factors (EDHFs). Using cultured cells, patch clamp and biochemical assays, we find that EETs activate these potassium channels by activating a guanine nucleotide binding protein. In isolated membranes, 14,15-EET radioligands exhibit specific, saturable, reversible binding that is inhibited by GTP suggesting a receptor-mediated pathway is involved. Future studies will (1) investigate the mechanism of action of the EETs by characterizing the cellular binding sites, receptors, (2) characterize specific EET agonist analogs and (3) identify specific inhibitors of EET synthesis or action.
We are also investigating the hypothesis that endothelial cells produce other metabolites of arachidonic acid that regulate vascular tone. Endothelial cells from aortic and mesenteric arteries release two lipoxygenase metabolites of arachidonic acid that causes vasodilation of smooth muscle by activations of small conductance, calcium-activated potassium channels and membrane hyperpolarization. Thus, they also function as EDHFs. We have identified these new vasodilators as 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites are produced by the sequential action of 15-lipoxygenase and a hydroperoxide isomerase on arachidonic acid. The expression of 15-lipoxygenase and the activity of this vasodilator pathway is enhanced by hypoxia, estrogen, hypercholesterolemia, interleukin-13 and other hormones. Thus, the 15-lipoxygenase pathway represents the inducible EDHF. Studies are in progress to further characterize the regulation of 15-lipoxygenase expression in cardiovascular disease models and determine the mechanism of action of these eicosanoids.
Adrenal Glomerulosa Cells: Adrenal glomerulosa cells synthesize and release aldosterone. This steroid is the major mineralocorticoid of the body. It regulates the excretion of sodium and potassium and is involved in the long-term control of blood pressure. The synthesis of aldosterone is regulated principally by angiotensin II, potassium and adrenocorticotropic hormone. However, evidence from our laboratory indicates that the synthesis of aldosterone is modulated by nitric oxide released by adrenal capillary endothelial cells. This is not surprising since the adrenal is a highly vascular gland and the aldosterone producing cells are in close proximity to the capillary endothelial cells. For the adrenal gland to function, adrenal blood flow must increase with steroid synthesis to deliver oxygen, cholesterol and cofactors and to carry the steroids to target tissues. We have shown that steroidogenic stimuli such as angiotensin II and adrenocorticotropic hormone release EETs from steroidogenic cells that dilate adrenal arterioles increasing adrenal blood flow. Future studies will define the mechanism of action of EETs on adrenal arteriolar smooth muscle cells, define the influence of adrenal steroids, identify other vasoactive factors made by steroidogenic cells and determine the pathways of EET synthesis and degradation by steroiodgenic cells.
Campbell WB and Fleming I: Invited Review - Epoxyeicosatrienoic acids and endothelium-dependent responses. Pflügers Archive Euro J Physiol 2010 459:881-895. PMCID: PMC3373596
Gauthier KM, Goldman DH, Aggarwal NT, Chawengsub Y, Falck JR and Campbell WB: Role of arachidonic acid lipoxygenase metabolites in acetylcholine relaxations of mouse arteries. Am J Physiol Heart Circ Physiol 2011 300:H725-H 735. PMCID: PMC3064304
Kopf PG, Gauthier KM, Zhang DX, Falck JR and Campbell WB: Angiotensin II regulates adrenal vascular tone through zona glomerulosa cell-derived EETs and DHETs. Hypertension 2011 57:323-329. PMCID: PMC3034648
Chen Y, Falck JR, Manthati VL, Jat ML and Campbell WB: 20-Iodo-14,15-epoxyeicosa-8(Z)-enoyl-3-azidophenylsulfonamide: Photoaffinity labeling of a 14,15-epoxyeicosatrienoic acid receptor. Biochemistry 2011 50:3840-3848. PMCID: PMC3100183
Bukhari IA, Shah AJ, Gauthier KM, Walsh KA, Koduru SR, Imig JD, Falck JR and Campbell WB. 11,12,20-Trihydroxy-eicosa-8(Z)-enoic Acid: A selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries. Am J Physiol Heart Circ Physiol 2012 302:H1574-H1583. PMCID: PMC3330801
Kriska T, Cepura C, Magier D, Siangjong L, Gauthier K and Campbell WB. Mice lacking macrophage 12/15-lipoxygenase are resistant to experimental hypertension. Am J Physiol Heart Circ Physiol 2012 302:H2428-2438. PMCID: PMC3378305
Campbell WB and Gauthier KM: Inducible endothelium-derived hyperpolarizing factor (iEDHF): Role of the 15-Lipoxygenase-EDHF pathway. J Cardiovascular Pharmacol 2013 61:176-187. PMCID: PMC3594564