Prostaglandin H Synthases1 and 2 in ECs

Enzymology of PGHS-1 and -2

Prostaglandins (PGs) are predominantly generated through the action of PGHS, of which there are both consti tutive (PGHS-1: stomach, gut, kidney, platelets) and inducible (PGHS-2: fibroblasts, macrophages) isoforms. Synthesis involves a two-step conversion of arachidonic acid. First, the enzyme oxidizes arachidonic acid to a cyclic endoperoxide, prostaglandin-G2 (PGG2), by a cyclooxygenase activity; then a peroxidase reduces the peroxide to a hydroxide, yielding the endoperoxide, prostaglandin-H2 (PGH2).

Biochemically, PGHS-1 and -2 are very similar, with 60 percent sequence homology, identical reaction mechanisms, superimposable x-ray crystal structures, and the same sub-cellular localization at the endoplasmic reticulum and nuclear membrane. However, PGHS isoforms function as two independent prostaglandin synthesis systems utilizing different cellular arachidonate pools in the same cell type, and with very different patterns of expression control.

PGHS in Vascular Disease

In the vasculature, PGHS isoforms regulate vascular homeostasis through generation of PGH2, the precursor for prostacyclin (PGI2, endothelial) or thromboxane (TXA2, platelets). PGHS is transiently activated in platelets or endothelial cells by agonists, such as thrombin, collagen (platelets), bradykinin, or acetylcholine (endothelium). Following this, the PGH2 is rapidly converted into PGI2 or TXA2 by the CYP enzymes, prostacyclin synthase or thromboxane synthase, respectively. Platelet PGHS-1 is the primary source of plasma TXA2 in both healthy humans and patients with vascular disease, whereas endothelial PGHS-2 is the major source of PGI2. These eicosanoids have opposing effects, with PGI2 being vasodilatory and an inhibitor of platelet activation via elevating cAMP, and TXA2 causing vasoconstriction and platelet activation (Figure 2).

Figure 1 Generation of bioactive eicosanoids by healthy and inflammatory-activated endothelium. Following its hydrolysis from the membrane by phospholipase A2 (PLA2), arachidonate is oxidized to prostacy-clin (PGI2) or epoxyeicosatetraenoic acid (EET) by prostaglandin H synthase (PGHS). (see color insert)

Figure 1 Generation of bioactive eicosanoids by healthy and inflammatory-activated endothelium. Following its hydrolysis from the membrane by phospholipase A2 (PLA2), arachidonate is oxidized to prostacy-clin (PGI2) or epoxyeicosatetraenoic acid (EET) by prostaglandin H synthase (PGHS). (see color insert)

Flgure 2 Localization of PGHS isoforms in vascular cells. Platelets contain PGHS-1, which forms prostaglandin H2 (PGH2) and is subsequently metabolized by thromboxane synthase (TXS) to thromboxane A2 (TXA2). Endothelial cells contain PGHS-1 and -2, both of which are responsible for providing PGH2 for PGI2 synthesis by PGI synthase. (see color insert)

Flgure 2 Localization of PGHS isoforms in vascular cells. Platelets contain PGHS-1, which forms prostaglandin H2 (PGH2) and is subsequently metabolized by thromboxane synthase (TXS) to thromboxane A2 (TXA2). Endothelial cells contain PGHS-1 and -2, both of which are responsible for providing PGH2 for PGI2 synthesis by PGI synthase. (see color insert)

The formation of PGHS-derived prostaglandins, including TXA2, PGI2, and isoprostanes, is markedly elevated in vascular disease. For example, urinary 8-epi-prostaglandin F2a is increased 130 percent in hypercholesterolemia. Also, isoprostanes are present in human atherosclerotic lesions along with PGHS-1 and -2.

Endothelial Expression of PGHS Isoforms

It has long been considered that PGI2 is the main prostanoid synthesized by ECs, and TXA2 the main prostanoid from platelets. However, cultured human umbilical vein endothelial cells (HUVECs) and lung microvascu-lar and cerebral ECs express PGHS-1 constitutively, with this enzyme being the major source of EC-derived PGH2 precursor for low-level TXA2 synthesis in HUVECs. Basal expression of PGHS-2 is low or absent in most ECs, but following stimulation with a number of mediators [including laminar flow, HIV-infected monocytes, platelet-derived TXA2, hypoxia, interleukin (IL)-1b, tumor necrosis factor-a (TNFa), fibroblast growth factor, phorbol ester, lipopolysaccharide (LPS) or vascular endothelial growth factor (VEGF)], its upregulation through an immediate early gene leads to generation of PGI2 and PGE2 in a number of microvascular EC types (including human pulmonary, cerebral, and atherosclerotic). Interestingly, IL-1b induces PGI synthase and PGE synthase in tandem with PGHS-2, but not TX synthase. It is therefore likely that the PGHS-2-dependent generation of PGI2 in vivo in both healthy people and patients with vascular disease requires continuous stimulation of gene expression, for example by laminar flow or proinflammatory cytokines. In contrast to HUVECs, PGHS-2 is a significant source of TXA2 generated by human microvascular endothelial cells, which can inhibit migration and angiogenesis in vitro. The in vivo importance of this is unclear, however, since platelet PGHS-1 is the major source of TXA2 in healthy people. PGHS-2 is also negatively regulated at the transcriptional level in ECs. For example, aspirin, sodium salicylate, or nitric oxide inhibits IL-1P-, phorbol-, or LPS-induced PGHS-2 expression in HUVECs and bovine pulmonary artery endothelial cells.

Although PGHS-1 is expressed constitutively by a number of EC types, its expression is also controlled by transcriptional regulation. For example, upregulation of PGI2 synthesis in intrapulmonary vessels rises markedly during late fetal life, because of a developmental increase in PGHS-1 expression that occurs via estrogen stimulation of the estrogen receptor. This may also have implications for PGHS-1 expression in pre- and postmenopausal women where risk of vascular disease increases with decreased estrogen levels, and estrogen replacement is associated with decreased cardiovascular risk.

Regulation of EC Function by PGHS Products

Endothelial cell function is regulated in several ways through PGHS signaling (Figure 3). In particular, recent data have implicated the prostaglandin 15-deoxy-S(12,14)-prostaglandin J2 (15S-PGJ2) in mediating multiple responses through activating peroxisome proliferator-acti-vated receptors (PPARs). These are members of the nuclear receptor superfamily of transcription factors that are important mediators of the inflammatory response. Through this pathway, 15S-PGJ2 activation of endothelial PPARs inhibits leukocyte-endothelial interactions, IFNg-induced expression of CXC chemokines, and TNF-induced oxidized LDL receptor (LOX-1) and induces stress proteins including heme oxygenase and plasminogen activator inhibitor type-1 (PAI-1) in a number of ECs (including brain microvascular). 15S-PGJ2 also signals in a PPAR-independent manner in ECs, inducing apoptosis and synthesis of GSH and IL-8.

In addition to 15S-PGJ2, additional prostaglandins that signal in ECs include PGE2, which induces expression of P-selectin, VEGF, and endothelial nitric oxide synthase (eNOS) through activation of ERK/JNK2 signaling pathways, and PGD2, which can relax vessels through stimulation of eNOS activity in bovine coronary arteries (Figure 3).

In summary, PGHS isoforms expressed in ECs regulate normal vascular function and participate in the pathophysi-ology of vascular disease. In addition, PGHS products generated by adjacent cells are important in regulating numerous microvascular EC functions, including apoptosis, integrin expression, and eNOS activity.

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