Protease-activated receptors , cyclo-oxygenases and pro-angiogenic signalling in endothelial cells

COX (cyclo-oxygenase)-2 and members of the PAR (protease-activated receptor) family (PARs 1–4) are highly overexpressed in a number of angiogenesis-dependent pathologies, including advanced atherosclerosis and cancer. An appreciation of the potential role(s) of PARs and COX enzymes in physiological angiogenesis is, however, currently lacking. Exposure of human endothelial cells to serine proteases (e.g. thrombin) or to PAR-selective agonist peptides leads to a wide range of cellular responses, including enhanced expression of COX-2, and we have shown that this induction depends on activation of classic pro-inflammatory signalling elements [e.g. MAPKs (mitogen-activated protein kinases) and NF-κB (nuclear factor κB)]. Our current studies suggest that COX-2-derived mediators are important autocrine regulators of PAR-stimulated angiogenesis. This mechanism could help us to explain how this novel family of receptors couple vascular inflammation with repair and angiogenesis in health and disease. COXs (cyclo-oxygenases) and endothelial cell function The initial step in the formation of prostanoids by endothelial cells is the stimulus-induced liberation of AA (arachidonic acid) from the sn-2 position of membrane glycerophospholipids by PLA2 (phospholipase A2) enzymes (Figure 1) [1]. The released AA is metabolized by COX enzymes (COX-1 and/or COX-2) to produce the unstable intermediate PGH2 (where PG is prostaglandin), which is then rapidly converted into several bioactive prostanoids by the actions of terminal PG synthases including PGIS (prostacyclin synthase), PGE2 synthases, PGD synthase and thromboxane synthase, all of which are expressed by endothelial cells. The overall profile of prostanoid production is therefore defined by the level of COX(s) activity and additionally influenced by the relative cell-specific expression of the terminal synthases, some of which may be co-induced with COX-2 [6]. We have shown that unperturbed HUVECs (human umbilicalvein endothelial cells) express both COX-1 and COX-2 and that both these enzymes contribute to prostanoid production under basal conditions [2,3], a finding that is consistent with observations that COX-2, as well as COX-1, play physiologically relevant roles in prostanoid production in normal humans [7]. We have also demonstrated that PARs (proteaseactivated receptors; see below) are important regulators of COX-2 induction in endothelial cells [2,3]. Their potential effects on terminal synthase expression are unknown, but it


COXs (cyclo-oxygenases) and endothelial cell function
The initial step in the formation of prostanoids by endothelial cells is the stimulus-induced liberation of AA (arachidonic acid) from the sn-2 position of membrane glycerophospholipids by PLA 2 (phospholipase A 2 ) enzymes (Figure 1) [1].The released AA is metabolized by COX enzymes (COX-1 and/or COX-2) to produce the unstable intermediate PGH 2 (where PG is prostaglandin), which is then rapidly converted into several bioactive prostanoids by the actions of terminal PG synthases including PGIS (prostacyclin synthase), PGE 2 synthases, PGD synthase and thromboxane synthase, all of which are expressed by endothelial cells.The overall profile of prostanoid production is therefore defined by the level of COX(s) activity and additionally influenced by the relative cell-specific expression of the terminal synthases, some of which may be co-induced with COX-2 [6].We have shown that unperturbed HUVECs (human umbilicalvein endothelial cells) express both COX-1 and COX-2 and that both these enzymes contribute to prostanoid production under basal conditions [2,3], a finding that is consistent with observations that COX-2, as well as COX-1, play physiologically relevant roles in prostanoid production in normal humans [7].We have also demonstrated that PARs (proteaseactivated receptors; see below) are important regulators of COX-2 induction in endothelial cells [2,3].Their potential effects on terminal synthase expression are unknown, but it Abbreviations used: AA, arachidonic acid; COX, cyclo-oxygenase; GPCR, G-protein-coupled receptor; HUVEC, human umbilical-vein endothelial cell; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; PAR, protease-activated receptor; PG, prostaglandin; PGIS, prostacyclin synthase; PLA2, phospholipase A2; PI3K, phosphoinositide 3-kinase; TxA2, thromboxane A2; VEGF, vascular endothelial growth factor. 1To whom correspondence should be addressed (email cwheeler@rvc.ac.uk).
is possible that PAR agonists, as well as other stimuli, are capable of differential induction of these enzymes to directly influence COX-mediated prostanoid generation and thus to affect the repertoire of downstream prostanoid-mediated functional effects.A high level of PGIS expression ensures that the endothelium is capable of releasing large quantities of biologically active, vasculoprotective PGI 2 (also known as prostacyclin) after exposure to agonists and to mechanical perturbations or after vessel damage (Figure 2).PGI 2 and other PGs not only have important paracrine actions on a range of cell types (Figure 2) but also have the capacity to interact with a number of prostanoid receptors (Figure 1) expressed on the endothelial cell surface [11], and/or to modify endothelial PPAR (peroxisome-proliferatoractivated receptor) activation [12].These mechanisms provide scope for autocrine regulation of endothelial cell function through the COX-1/-2-terminal synthase pathway.

PARs and endothelial cell prostanoid synthesis
PARs are a novel family of four GPCRs (G-protein-coupled receptors; PAR-1, -2, -3 and -4) with emerging importance in the cardiovascular system [13].A broad range of serine proteases have been identified as activators of these receptors and do so by cleaving the PAR within its extracellular N-terminus.This proteolytic cleavage generates a receptorspecific tethered ligand that initiates intracellular signalling by binding to extracellular receptor domains.Thrombin, a multifunctional serine protease generated during normal haemostasis and at sites of vascular injury, not only influences cellular function principally through PAR-1 but is also capable of cleaving PAR-4.Although thrombin is a recognized physiological activator of endothelial PAR-1, the endogenous proteases involved in PAR cleavage in vivo are not generally Activation of GPCRs including PAR-1 and PAR-2 [2,3], as well as growth factor receptors (e.g.VEGF receptors; [2,4]), leads to increased enzymatic activity of PLA 2 enzymes, generation of AA and subsequent formation of PGs and TxA 2 through the sequential activities of COX-1 and/or COX-2 enzymes and downstream terminal synthases.AA can also be metabolized through the lipoxygenase and cytochrome P450 pathways to generate leukotrienes and other eicosanoids [e.g.EETs (epoxyeicosatrienoic acids)], and endothelial-derived PGH 2 can support transcellular PG formation.The overall profile of prostanoids generated will be critical for determining the functional consequences of changes in COX activity/expression through their paracrine and autocrine actions on PG receptors (DP, EP 1−4 , IP, FP and TP).Top right panel: acute (<15 min) thrombin-induced PGI 2 synthesis depends on MAPK-mediated regulation of cytosolic PLA 2 α (cPLA 2 α) phosphorylation (p-) and activity [5].Lower right panel: prolonged thrombin-stimulated PGI 2 generation (as assessed by measurement of 6-keto PGF 1α formation) is COX-2 dependent and regulated by activation of MAPKs and NF-κB [2,3].Modified from [2,5], used with permission.
known, although extensive studies in vitro have shown that a range of proteases are capable of influencing endothelial cell signalling and function through PAR-1 and PAR-2 [14,15].These studies have made extensive use of PAR-specific activating peptides that mimic the tethered ligand sequences resulting from protease-mediated cleavage and activate the receptors in the absence of proteolysis [2,3,[16][17][18].
The potential for differential regulation of prostanoid release by PAR-1 compared with PAR-2 is highlighted by our ongoing studies, which indicate that human endothelial cells exposed to PAR-1 or PAR-2 activators display differing prostanoid profiles.These results support the recognition that PAR-1 and PAR-2 promote overlapping but mechanistically distinct responses in the vascular endothelium [19,20].Thus activation of PAR-1, but not PAR-2, enhances PGE 2 synthesis; neither receptor stimulates TxA 2 (thromboxane A 2 ) formation, but both PARs are efficiently coupled with PGI 2 generation [3].The time courses of PGI 2 production are also distinct, which probably reflects differences in the utilization of signalling elements upstream of COX activation/expression [3], and these remain to be defined.Together, these results seem to suggest that PAR-1 compared with PAR-2 stimulation of human endothelial cells could promote differential recruitment of enzymes downstream of COX activity resulting in differing prostanoid profiles that are likely to have functional significance.In this respect, our studies using PARselective peptides in vitro have provided clear evidence that activation of PAR-1 and PAR-2 promotes prolonged synthesis of PGI 2 through NF-κB (nuclear factor κB)-dependent up-regulation of COX-2 expression.In marked contrast, PAR-4 stimulation neither activates NF-κB signalling nor induces COX-2 expression, suggesting that endothelial PARs couple selectively to prostanoid-generating pathways [3].

PAR-mediated regulation of angiogenesis: a role for COXs?
Studies in COX-2-deficient mice [20a,20b] and in cells overexpressing COX-2 [20c] provide support for the involvement of COX-2 in both physiological and pathological angiogenesis.The pro-angiogenic responses of endothelial cells to PAR activation have received comparatively little attention.Thrombin, presumably through activation of PAR-1, can promote endothelial cell proliferation and angiogenesis  Endothelial PGI 2 exerts vasculoprotection through paracrine influences on a range of cell types and, in general, these actions oppose those of platelet-derived TxA 2 .Thus PGI 2 increases endothelial permeability, promotes vasodilatation, inhibits platelet activation and limits intimal hyperplasia and vascular wall remodelling by suppressing smooth muscle cell proliferation and pro-fibrotic/proliferative cytokine release from fibroblasts (e.g.[8,9]).Protective effects may also result from PGI 2 -mediated limitation of reactive oxygen species (ROS) production as well as induction of cytoprotection through regulation of anti-apoptotic gene expression (e.g.[10]).Evidence is emerging that PGI 2 regulates pro-angiogenic responses in endothelial cells, raising the possibility that endothelial-derived PGI 2 has autocrine roles to facilitate growth and repair.
[21] and accordingly has been reported to modify the expression of a number of angiogenesis-associated genes, e.g.matrix metalloproteinases and VEGF (vascular endothelial growth factor) [15,10].There is also evidence that PAR-2 activation increases endothelial cell mitogenesis in vitro and potentiates reparative angiogenesis in a model of limb ischaemia [22,23] but, in general, the involvement of PAR-2 in angiogenic processes is less well understood.The relative abilities of active PAR-1 compared with PAR-2 to drive the component responses of angiogenesis and the molecular bases of these functions are, therefore, largely unknown and await clarification.
Functional roles for COX-2 in endothelial cells are not yet defined but in non-vascular cell types (tumour cells in particular) constitutive overexpression of COX-2 protein is associated with increased tumour cell proliferation and angiogenesis [24], providing support for a role for COX-2 activity in cell growth.Indeed, AA-generating pathways are increasingly associated with control of cell growth and there is evidence that several classes of PLA 2 enzymes, as well as COX-2, are involved in regulating the proliferative status of endothelial cells [25].VEGF, as well as PAR agonists, enhances COX-2 expression by endothelial cells [2,26], and pharmacological blockade of COX-2 activity reduces cell proliferation [27].PAR-2 may also recruit TNFα (tumour necrosis factor α)-dependent mechanisms to promote retinal angiogenesis [27a].Thus there is a clear association between the pro-inflammatory and pro-angiogenic actions of PARs and other angiogenic mediators, and increased COX-2 activity may be one factor that couples these events.There is also an emerging role for the downstream products of COX-2 in proliferation and angiogenesis, as well as in the resolution phase of inflammation.For example, co-transfection of PGIS enhances therapeutic angiogenesis [28] and both PGE 2 and PGI 2 promote endothelial cell survival and have pro-angiogenic actions [29][30][31].In support of a potential role for prostanoids in endothelial cell growth, PGE 2 is both an effective mitogen for HUVECs and an efficient inducer of COX-2 expression [26,27].Thus, while the mechanisms underlying the pro-angiogenic actions of PARs are largely unexplored, COX-2 induction is likely to be a key component of this control.Lipid-derived mediators generated through COX activities may therefore play a central role in regulating the pro-angiogenic actions of PARs on endothelial cells.
The intracellular signalling events important for regulating PAR-1-and PAR-2-mediated endothelial cell growth are not defined.Our recent studies have shown that PAR-1 and PAR-2 utilize MAPKs [mitogen-activated protein kinases; principally ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (p38 MAPK)] and NF-κB-dependent mechanisms to regulate COX-2 expression and activity [3,32].An additional pathway with potential relevance for controlling PAR-induced changes in endothelial cell growth and angiogenesis is the PI3K (phosphoinositide 3-kinase)-Akt (also known as protein kinase B) pathway.This mechanism has known importance for cell survival and proliferation and its overexpression in tumours is associated with increased tumour growth and angiogenesis.In accordance with this, studies indicate that the PI3K-Akt pathway is important for PAR-induced COX-2 expression, suggesting that signalling through Akt could contribute to generation of growthpromoting prostanoids by endothelial cells exposed to PAR agonists (E.Garonna, F. Syeda, E. Paleolog and C.P.D. Wheeler-Jones, unpublished work).Furthermore, activation of PI3K-Akt signalling is a recently described effect of PGE 2 acting through PG receptors [33], raising the possibility that autocrine actions of prostanoids facilitate signalling through this pathway.An additional signalling mechanism that has increasing relevance for proliferation induced by GPCRs is transactivation of growth factor receptors [34].Thus tumour cells exposed to PAR activators use the epidermal growth factor receptor as an integrator of proliferative responses [35] and this mechanism may be involved in thrombin-driven effects in VSMCs (vascular smooth muscle cells) [36].The role(s) of growth factor receptors as mediators of PAR-driven angiogenic responses in endothelial cells are unclear but it is conceivable that such receptor cross-talk is a key determinant of PAR-induced COX-2 expression and downstream growth responses.

Summary
There is very limited understanding of the mechanisms used by PARs to regulate pro-angiogenic responses in endothelial cells but current evidence suggests that COX enzymes are potentially important players in this control.The direct vascular actions of COX-derived mediators have relevance for a range of cardiovascular and other disorders characterized by increased inflammation and angiogenesis, including atherosclerosis and tumour growth.In addition to their involvement in pathology, growing evidence suggests that these mediators are likely to play pivotal roles in angiogenesis-dependent processes of physiological relevance.Our present studies support the hypothesis that the PAR-COX-2 axis couples downstream prostanoid production with angiogenesis, suggesting a critical autocrine role for these mediators in repair and remodelling.Continued investigation of the molecular details of this regulatory axis in vitro, together with studies in knockout models, should help us to define the importance of PAR-mediated control of prostanoid synthetic pathways for the pro-angiogenic actions of these receptors.Our results also highlight the potential for COX-2-targeted therapies to limit the reparative processes that accompany inflammation and reinforce the close association between inflammation and angiogenesis.

Figure 1
Figure 1 Synthesis of prostanoids

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The Authors Journal compilation C 2009 Biochemical Society