Subcellular signaling in the endothelium: cyclic nucleotides take their place

Available online at www.sciencedirect.com

Subcellular signaling in the endothelium: cyclic nucleotides take their place Donald H Maurice When lecturing on the topic of cellular signaling I have had occasion to ask the class for examples of cellular processes NOT impacted by cyclic AMP (cAMP) and am struck by how few examples exist. Indeed, studies spanning the past 60 years have detailed how this ubiquitous second messenger impacts virtually all cellular processes, including intermediary metabolism, contractility, motility, proliferation, and gene expression in most mammalian cells. Since the hydrophobic cAMP could in principle diffuse rapidly throughout the cell once formed, the remarkable spatial and temporal specificity of its numerous actions in cells is truly impressive. Herein I introduce the main players involved in coordinating actions of cAMP in vascular endothelial cells (VECs), and focus on the increasing awareness of the dominant role that cyclic nucleotide phosphodiesterases (PDEs), the sole cellular enzymes capable of hydrolytically inactivating cAMP, play in fostering this specificity. Address Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Corresponding author: Maurice, Donald H ([email protected])

Current Opinion in Pharmacology 2011, 11:656–664 This review comes from a themed issue on Endocrine and Metabolic Diseases Edited by Vincent Manganiello Available online 27th October 2011 1471-4892/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2011.10.009

VECs and the vascular endothelium (VE) VECs regulate virtually all aspects of blood vessel function, and the activities of numerous blood-borne cells, either via cell–cell contacts through adherens junctions (AJs) or by releasing modulators of cellular function [1–3]. AJs are formed when vascular endothelial cell cadherins (VECAD) of neighboring VECs interact and recruit b-catenin, a-catenin, vinculin, a-actinin or p120-catenin to the actin cytoskeleton. Far from being static structures, AJs are highly dynamic structures that respond to signals and initiate context-appropriate responses. Indeed, while they keep quiescent VECs growth-inhibited, protected from apoptosis and incapable of migration, their disassembly promotes each of these events [1–3]. Virtually all VEC functions are dependent on these adhesions, and the cellular signaling which they dynamically regulate. Current Opinion in Pharmacology 2011, 11:656–664

In healthy mature blood vessels VECs regulate: coagulation and fibrinolysis, platelet reactivity, vascular myocyte contractility, activation, adhesion and migration of inflammatory cells within vascular and perivascular space, as well as their athero-protective properties, and contractility of vascular myocytes by producing vasoactive agents (e.g. nitric oxide (NO), prostacyclin (PGI2), and endothelin (ET-1)) [4–7]. Adaptively in response to trauma (e.g. wound healing) or mal-adaptively in disease states, VECs, or endothelial progenitor cells, are recruited to initiate angiogenesis, the formation of new blood vessels from existing vascular structures (Figure 1) [7–11]. To initiate angiogenesis, VECs must de-differentiate from ‘quiescent’ cells and adopt an ‘activated’ phenotype. Relative to quiescent VECs, activated VECs are migratory and, due to the loss of AJ-based intercellular adhesions, form a less efficient barrier (Figure 1) [7–11]. A number of factors, including the vascular endothelial growth factor (VEGF), promote VEC activation and act to destabilize AJ-based adhesions [7–11]. Reduced endothelial function (endothelial dysfunction), or frank loss of endothelial coverage, promotes expression of the activated phenotype and precipitates the establishment of hypertensive, procoagulant, pro-thrombotic, and pro-atherogenic states [12–14].

VEC cAMP-signaling Numerous hormones and transmitters, as well as the shear forces exerted on the VEC monolayer, impact VEC functions through actions mediated, at least in part, by changes in cAMP-signaling [15,16,17,18]. Indeed, altered cAMP-signaling impacts the synthesis and release of vasoactive agents as well as the release of factors that regulate coagulation, thrombosis, fibrinolysis or inflammation. Most functions of quiescent VECs are dependent on stable intercellular adhesions as well as integrin-based interactions with elements of the ECM [1–4], and agents that alter VEC cAMP-signaling alter VEC functioning largely by actions at the adhesions [15,16,17,18]. Indeed, cAMP-elevating agents stabilize AJ-based and integrin-based contacts [17]. Early models attempting to describe how cAMP could simultaneously and selectively regulate its myriad effects significantly underestimated the levels of specialization involved [19]. In this context, studies identifying nine families of membranous GPCR-activated adenylyl cyclases (AC-AC9) [20], one soluble HCO3 and Ca2+-stimulated AC (sAC) [21], 11 families of PDEs (Figure 2) [22,23], several cAMP-effectors [24,25], and numerous possible anchors/tethers of the individual components of this system [26,27], have redefined our understanding of this specialization. In terms www.sciencedirect.com

Subcellular signaling in the endothelium Maurice 657

Figure 1

(a)

VEC Activation

(b)

VEC Invasion

(c)

Angiogenic gradient

Angiogenic stimulus

VECs

(d)

Tubule Maturation

Mural Cells

ECM

VECs

ECM

Tubule Formation

VECs

VECs

ECM

Quiescent VECs

Migrating VECs

VEC tubules

VEC tube

Mural Cells 40X Blue: Nuclei Red: VECad Green: Actin

Blue:Nuclei Red: Actin Green: VECad

20X

40X

Red: CellTracker-Red VEC

60X Red: CellTracker-Red VEC Green: CellTracker-Green Mural cells Current Opinion in Pharmacology

From Gatekeepers to Keymakers. Intercellular adhesions between individual quiescent VECs forms a contiguous monolayer that allows these cells to regulate virtually all functions of cells within blood vessels and the activity and reactivity of multiple blood-borne cells. One of these functions, ‘The Gatekeeper Function’, involves the formation of a barrier by the quiescent VECs which limits movement of proteins, or cells, from the blood to the subendothelium. (a) Adaptively in wound healing, or mal-adaptively in disease states, including cancers, an angiogenic stimulus recruits quiescent VECs into angiogenesis, a process that promotes the formation of new blood vessels from pre-existing vascular structures. Since this process promotes access to previously less vascularized structures, I refer to it as the ‘The Keymaker Function’ of VECs. To participate in angiogenesis, VECs must de-differentiate from their quiescent state (gray) and adopt an ‘activated’ (pink) phenotype. (b) Migration/invasion of activated VECs to distant sites down the stimulus gradient, their proliferation at these distant sites, allows these cells to form the tubular structures (c) that form the platform upon which new vessels will form. Investment of VEC tubes by mural cells (vascular smooth muscle cells, or pericytes) allows tubule maturation and stabilization (d).

Figure 2

Targeting

Post-translational

Catalytic

3´,5´-cNMP Alternate splicing/ promoter usage

-Cofactors: Ca2+,cGMP, -Phosphorylation (PKA, PKB, PKC, PKG, ERK) -Inter- & intra-molecular protein-protein interactions (SH2, SH3, PAS,14-3-3, AKAP,c(N)MP-effectors)

Targeting

5´-NMP

Family-selective inhibitors

Alternate splicing

Current Opinion in Pharmacology

Cyclic nucleotide phosphodiesterases are modular proteins. PDEs are modular enzymes and individual domains are involved in targeting, posttranslational regulation or hydrolysis. www.sciencedirect.com

Current Opinion in Pharmacology 2011, 11:656–664

658 Endocrine and Metabolic Diseases

of cAMP-based regulation of the various functions of VECs, evidence supports expression of several ACs (AC3, AC5, AC6, and sAC expression in VECs) and numerous PDEs (PDE2, PDE3, and PDE4) (reviewed in [17]). Although roles for each PKA and EPAC in coordinating the actions of cAMP in human VECs are established, and certain VEC AKAPs have been identified [28], before very recent studies, little was known concerning the importance of compartment-specific signaling in these cells.

Compartmentation of cAMP-signaling allows specific regulation of individual VEC functions Since VEC cAMP synthesis occurs predominantly at the plasma membrane, and diffusion of free cAMP can be very rapid (400 mm2 s1) [29], significant efforts have focused on identifying the mechanisms that allow spatial and temporal resolutions of the many sophisticated cellular actions of this messenger. In this context, a consensus has emerged. Thus, it is generally accepted that protein–protein interactions between individual components of the cAMP-signaling system (ACs, GPCRs, cAMP-effectors, and PDEs) allow formation of distinct macromolecular cAMP-signaling complexes (herein ‘cAMP-signalosomes’) [29,30,31]. Also, it is accepted that selective anchoring, or tethering, of these cAMPsignalosomes to discrete subcellular domains allows their actions to be localized [29,30,31]. Indeed, Drs. Houslay, Baillie and I recently proposed [29] that these complexes likely represent the very ‘tools’ which cells use to simultaneously regulate individual cAMP-dependent cellular events. Our laboratory’s efforts to understand how compartment-specific cAMP-signaling selectively regulates actions of cells in the cardiovascular system [28,32,33,34,35] is built on excellent earlier work in cardiomyocytes, T cells, and spermatozoa, in which tethered cAMP-signalosomes were shown to contain cAMP-effectors (PKA or EPAC1, or both these cAMPeffectors) and individual PDEs, including either PDE3A, PDE3B, PDE4D or PDE7A (reviewed in [22,23]). Previously we reported that human arterial or arteriolar VECs formed numerous distinct cAMP-signalosomes which contained either PKA or EPAC and either PDE3B or PDE4D [28]. More recently, we showed that these cAMP-signalosomes allowed selective regulation of several individual VEC functions independently of one another, or of global cellular levels of cAMP [32,33,34]. These data were consistent with the hypothesis, first proposed in the context of VECs by Stevens and colleagues [35], that cAMP regulates functions in VECs in a manner consistent with the existence of distinct VEC cAMP ‘pools’ or compartments. While the earlier work of Stevens and colleagues was silent on issues related to the number and the complexity of the signaling complexes that operationalized these pools, taken together with our studies, they confirm the existence of Current Opinion in Pharmacology 2011, 11:656–664

multiple distinct non-overlapping cAMP-signalosomes in VECs and provide a mechanistic underpinning for specialized cAMP-signaling in VECs. Below, I describe how compartmentation of cAMP-signaling through two distinct cAMP-signalosomes impact specific human VECs functions. In addition to highlighting how cAMP-signalosomes regulate individual VEC functions, these examples also identify cAMP-signalosomes as novel therapeutic targets. Indeed, prompted by investigations into the functional consequences of compartmented cAMP-signaling, many, including us, have proposed that manipulating the protein–protein interactions which allow formation of these structures, or inhibiting their tethering, could allow greater selectivity than has hitherto been possible using agents acting on these same proteins globally within cells [29,30,31].

A PDE4D/EPAC1/Rap 1 signalosome coordinates local actions of cAMP on VEC permeability As expertly reviewed [17], cAMP-elevating agents reduce VEC permeability to solutes and cells by stabilizing VEC intercellular AJs and promoting formation of cortical actin fibers. Although early reports identified a role for PKA in most of these effects [16,17,18], more recently a critical role for EPAC has been established [36–39]. Since PDEmediated hydrolysis of cAMP controls the magnitude, duration, and subcellular accumulation of cAMP [22,23], recent studies have addressed their role in these actions of cAMP [17,33]. Overall, these studies have identified a dominant role for PDE4, over PDE3 and PDE2 (Figure 4). Four genes (PDE4A–D) yield 20 PDE4 variants and these can be stratified into long or short forms [29]. While long PDE4s contain N-terminal upstream conserved regions (UCR1/2) and unique targeting domains, short PDE4s do not. Individual PDE4s interact with binding partners and this often allows their localization within discrete subcellular domains in cells, including VECs [29,33,40]. Although PDE4A, PDE4B and PDE4D variants are each expressed in VECs, PDE4D is dominant [32,33,34]. PDE4 inhibitors reduce VEC migration, proliferation, and permeability [32,33,34,41–43]. Very recently we uncovered a role for compartmentation of a PDE4D/EPAC1-based cAMP-signalosome in regulating human arterial endothelial cell (HAEC) permeability (Figure 3) [33]. Moreover, we showed that these actions were coordinated through integration of this cAMP-signalosome into VECAD-based adhesive structures. Thus, we observed that PDE4 inhibition with rolipram, but not PDE3 inhibition with cilostamide, reduced HAEC permeability. Consistent with a dominant role for EPAC1 over PKA in mediating this effect, siRNAbased knockdown of EPAC1 markedly increased HAEC permeability while knockdown of PKA-Ca had no such effect. Knockdown of individual PDE4 gene products identified PDE4D, but not PDE4A or PDE4B, as likely www.sciencedirect.com

Subcellular signaling in the endothelium Maurice 659

Figure 3

PDE4D-DP

VECad

EPAC1 activation Reduced Permeability

PDE4D-KD

ns

EPAC1

ni

te

α/β, p120Catenins

PDE4D

ca

PDE4D

Reduced EPAC1/complex Increased Permeability

PDE4D ns

EPAC1

ni

e at

EPAC1

c

Cortical Actin

Actin Stress Fibres Current Opinion in Pharmacology

Scheme depicting how PDE4D/EPAC1 are bound to VECAD in human VECs. Also depicted are the effects associated with incubating VECs with the EPAC1-based PDE4D displacing peptide (PDE4D-DP) or PDE4D knockdown (PDE4D-KD).

dominant in controlling cAMP activation of EPAC1 and its permeability reducing effects. The role of PDE4D as the tether promoting EPAC1 integration into VECADbased structures was revealed when effects of PDE4D knockdown and inhibition were compared. Although PDE4 inhibition with rolipram decreased VEC permeability, PDE4D knockdown INCREASED permeability and caused a loss of EPAC1 from VECAD complexes. Since PDE4 inhibitors had decreased VEC permeability, we had originally anticipated that PDE4D knockdown would similarly reduce HAEC permeability. Knockdown of PDE4A or PDE4B or of the dominant HAEC PDE3 (PDE3B) had no such effect on VEC permeability. Interestingly, although HAEC VECADbased structures also contain PKA, in these cells PDE4D did not locally regulate integration of this cAMP-effector or its ability to impact AJ-based adhesions of permeability. Although our data identified Rap1 as a likely downstream actor in promoting actions of this cAMPsignalosome, involvement of other agents known to impact VEC permeability, including Rac, Cdc42, p21activated kinase (PAK), Krev1 interaction trapped gene (Krit1) (reviewed in [17]), or of other PDE4D-interacting www.sciencedirect.com

proteins, including RACK1, AKAPs and b-arrestins or spectrin (reviewed in [40]), is presently unclear. As discussed briefly above, and depicted in Figure 4, PDE2 and PDE3 have been reported to allow cGMPelevating agents to impact the manner by which cAMPelevating agents can regulate VEC permeability [44]. PDE2 enzymes hydrolyze both cAMP and cGMP, with hydrolysis of either cyclic nucleotide being stimulated when cGMP binds to amino terminal allosteric regulatory sites known as GAF domains [21,22]. In contrast, while PDE3 enzymes also hydrolyze both cAMP and cGMP, due to an intrinsically low Vmax for cGMP hydrolysis by these enzymes, they function as cGMP-inhibited cAMP PDEs in most cells [21,22]. Although PDE2 and PDE3 are expressed in human, bovine, and porcine arterial or venous VECs [17,41,44–48], since neither of these enzymes integrated into the EPAC1/PDE4D-signalosome [33] it is unlikely that this level of control occurs through events coordinated locally at AJs. Of likely translational importance, we identified the region in EPAC1 that promotes the direct interaction Current Opinion in Pharmacology 2011, 11:656–664

660 Endocrine and Metabolic Diseases

Figure 4

Increased Barrier Function

EPAC

PKA

5´AMP

PDE2

cAMP

PDE4 5´AMP

cGMP PDE3

5´AMP Current Opinion in Pharmacology

Multiple PDEs coordinate the VEC barrier stabilizing effects of cAMP and cGMP. In the absence of elevated levels of cGMP, hydrolysis of cAMP by PDE4, and to a lesser degree PDE3, regulates cAMP-mediated activation of PKA and EPAC and coordinates the barrier stabilizing activities of these cAMP-effector proteins. In the presence of cGMPelevating agents, cGMP-mediated activation of cAMP hydrolysis and cGMP-mediated inhibition of cAMP hydrolysis by PDE3 allows further ‘fine tuning’ of cAMP actions on PKA and/or EPAC.

of this enzyme with PDE4D. Moreover, we showed that VEC permeability could be perturbed by antagonizing EPAC1–PDE4D interactions. Thus, a peptide designed to interfere with EPAC1–PDE4D binding reduced the ability of PDE4D to control EPAC1-based effects at the border. These data strongly suggest that small molecules that competed binding between PDE4D–EPAC1 in VECs might represent powerful agents with which to selectively control VEC barrier functions (Figure 3).

A PDE3B/EPAC1/R-Ras signalosome coordinates cAMP-mediated VEC adhesions to ECM proteins, migration and tubule formation Integrin-mediated adhesions: Although cAMP-elevating agents promote integrin-dependent VEC adhesions to ECM proteins and inhibit both random (haptotosis) and directed VEC migration (chemotasis) [28,32,41] the relative roles of PKA or EPAC1 in coordinating these effects remain unclear. Thus, although PKA activation promoted integrin engagement and VEC adhesions, PKA Current Opinion in Pharmacology 2011, 11:656–664

activity itself is reported to be either increased or decreased in response to integrin-ECM engagement, and PKA inhibitors do not antagonize fully these cAMP-stimulated adhesive events [49,50]. On the basis of our work in this area, we have concluded that these somewhat contradictory data are most likely reflective of the fact that while each PKA and EPAC can promote integrin engagement, the activity of these effectors are in turn impacted by events downstream of integrin signaling [28]. Also, the species or vascular structures from which the VECs were derived, the identity of the ECM protein chosen for study and the choice of cAMP-elevating agent, can each impact the relative involvement of PKA or of EPAC in these events. Indeed, in earlier work comparing cAMP-induced adhesions of HAECs (macro-VECs) and HMVECs (micro-VECs) to distinct ECM proteins, we found that the relative involvement of PKA or of EPAC was cell type-dependent and ECM protein-dependent [28]. Briefly, we showed that while cAMP-induced VEC adhesion was more PKA-dependent in HAECs than in HMVECs, the magnitude of the difference was ECM protein-dependent. Thus, although most cAMP-elevating agents tested (isoproterenol, forskolin, rolipram or cilostamide) could promote HAEC adhesion when PKA could be activated, in the presence of PKA inhibitors, these agents only promoted adhesion of HMVECs not HAECs. These data identified a functional link between EPAC and both PDE3-catalyzed or PDE4-catalyzed cAMP hydrolysis that was more dominant in HMVECs than in HAECs and suggest that integrinbased cAMP-signalosomes may underpin these differences [28]. Migration and tube formation: Because initiation of angiogenesis involves VEC adhesion, migration and tubule formation, and because cAMP-elevating agents impact each of these events, the ability of certain cAMP-elevating agents to impact angiogenesis has been investigated [51–54]. Although PKA, or EPAC, likely coordinate these events through numerous separate cAMP-signalosomes, only recently have these events been studied comprehensively. In this context, we identified an EPAC1/ PDE3B/R-Ras-based signalosome that regulates integrin-based adhesions, migration and tubule formation by VECs in the context of angiogenesis (Figure 5a,b) [34]. In this signalosome, PDE3B and EPAC1 reciprocally regulated one another’s activities and subcellular compartmentation. Thus, membrane-associated PDE3B bound EPAC1 directly via protein–protein interactions and tethered this cAMP-effector to membranes. In addition, PDE3B-catalyzed cAMP hydrolysis acted to antagonize EPAC1 binding of cAMP and consequently inhibited EPAC1 activation by cAMP. Interestingly, PDE3B association with EPAC1 increased PDE3B activity through an as yet unknown mechanism. EPAC1 binding of cAMP signals in cells by promoting accumulation of GTP-bound and activated Rap and/or R-Ras www.sciencedirect.com

Subcellular signaling in the endothelium Maurice 661

Figure 5

βY PIP3

PIP2

PDE3B

GTP p110γ R-Ras

p84

EPAC1

GDP

cAMP 5´AMP

R-Ras

Signalling (d) (c)

PDE3B-Catalytic Domain NHR1

NHR2

(b) Pl3KY, p84-Regulatory Subunit DEP

(a)

cBD

REM CDC25HD RA Current Opinion in Pharmacology

(a) A scheme depicting how cAMP hydrolysis by PDE3B limits EPAC1, PI3Kg, and R-Ras activations. (b) Protein–protein interactions between PDE3B, EPAC1, and the regulatory domain of PI3Kg, p84. Peptides which allow protein–protein interactions are shown as red lines. Individually they represent EPAC1 peptides [(a) Thr-218 to His-242], [(b) Glu-398 to Leu-422] and PDE3B peptides [(c) Met-1 to Glu-25] and [(d), Pro-436 to Asn-460]. In PDE3B, NHR1, and NHR2 are N-terminal hydrophobic membrane association regions 1 and 2. In EPAC1 the DEP (dishevelled, Egl-10, and pleckstrin) domain, cBD (cyclic AMP binding), the REM (Ras exchange motif), the RA (Ras-association), and GEF (guanine nucleotide exchange factor) domains are shown.

proteins. In turn, activated Rap or R-Ras signal by activating one of several potential effectors including, phospholipase Ce (PLCe), phospholipase D (PLD), Raf-1, p38-MAPK, and phosphoinositide 3-kinase (PI3K) [55– 58]. Since a previous report had shown that PDE3B could be coimmunoprecipitated with a PI3Kg regulatory subunit, namely p84 [59], and a cardiac phenotype was associated with loss of PI3Kg-catalytic domain (p110g) in mice had been attributed to increases in cAMP-signaling [60], we investigated whether PI3Kg was also tethered via PDE3B and if this interaction was related to the PDE3B–EPAC1 complex. Consistent with the idea that these three enzymes could integrate into a common cAMP-signalosome, PDE3B was found to interact directly with p84 through protein–protein interactions. Since p84 and EPAC1 bound PDE3B via distinct sites (Figure 5b), we surmised that PDE3B could simultaneously tether and anchor both EPAC1 and the www.sciencedirect.com

p84-regulated form of PI3Kg in cells. Consistent with this, PDE3 inhibition with cilostamide or selective PDE3B knockdown in HAECs each promoted EPAC1dependent activation of Rap1 and R-Ras, and of PI3Kg and its downstream activated kinase in VECs. Indeed, actions through this cAMP-signalosome were shown to allow the PDE3B-regulated pool of cAMP to directly promote EPAC1-dependent activation of PI3Kg, PKB, and ERK1/2 [34]. Cell biological experiments confirmed that the PDE3B/ EPAC1/R-Ras/p84-regulated PI3Kg-signalosome controlled cAMP-mediated HAEC adhesion, migration and tubule formation. Thus, while PDE3 inhibition with cilostamide, or PDE3B knockdown, each promoted HAEC adhesions to ECM proteins, and promoted tubule formation, knockdown of EPAC1 or of p84 each abolished tubule formation behavior in these cells. Most strikingly, Current Opinion in Pharmacology 2011, 11:656–664

662 Endocrine and Metabolic Diseases

all of these events occurred without addition of activators of adenylyl cyclases and, as such, occurred without changing global cAMP levels. In a very recent report, the catalytic domain of PI3Kg (p110g) was identified as a potential AKAP that could tether PKA and PDE3B in murine cardiac myocytes and in cells expressing these proteins heterologously [61]. Since p110g was not integrated into the PDE3B-based cAMP-signalosome which we identified when p84 was not present, it is currently unclear how this recent finding relates to the above description. Of potential translational importance, we identified a region in PDE3B and two regions in EPAC1 and showed that these domains coordinated the direct protein– protein interaction between these two proteins. On the basis of this information, we designed a cell permeable PDE3B-based peptide which antagonized binding between these proteins in cells. Consistent with our predictions, the PDE3B-based EPAC-displacing peptide effectively competed binding between these two proteins in HAECs, promoted EPAC1/R-Ras-dependent activation of PI3Kg in these cells and fostered tubule formation. The internal consistency of these data to our model was observed when the displacing peptide rendered cells insensitive to the effects of PDE3B inhibition. These findings, when combined with those obtained using the EPAC1-based PDE4D displacing peptide discussed above, validate the concept that selective regulation of individual cAMP-sensitive cellular events can be achieved with reagents, or drugs, able to selectively disturb the formation of individual cAMPsignalosomes in cells.

Conclusions Studies have shown that VECs form both a barrier, and an entry port, for cells and molecules from the circulation, and that through these activities, and their ability to release active factors can regulate coagulation, fibrinolysis, thrombosis and inflammation, as well as vascular effects including vascular contractility and vascular remodeling in response to trauma or disease. Physiological and pharmacological agents that alter cAMP-signaling impact virtually all these VEC-dependent events with remarkable selectivity. A paradigm is emerging which supports the idea that individual cAMP-signalosomes, composed of various components of the cAMP-signaling system and tethered throughout these cells, are the tools which allow this selectivity. As a corollary, evidence is emerging which supports the notion that interfering with the function or tethering of these signalosomes may provide greater therapeutic specificity that targeting these elements globally within cells.

Acknowledgements Some of the studies described here were supported by Canadian Institutes of Health Research Grant MOP57699 (DHM). Also, Dr. Maurice is a Career Investigator of the Heart and Stroke Foundation of Ontario. Current Opinion in Pharmacology 2011, 11:656–664

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Cavallaro U, Dejana E: Adhesion molecule signalling: not always a sticky business. Nat Rev Mol Cell Biol 2011, 12:189-197.

2.

Dejana E: The role of wnt signaling in physiological and pathological angiogenesis. Circ Res 2010, 107:943-952.

3.

Dejana E, Tournier-Lasserve E, Weinstein BM: The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev Cell 2009, 16:209-221.

4.

Triggle CR, Ding H: The endothelium in compliance and resistance vessels. Front Biosci 2011, 3:730-744.

5.

Gkaliagkousi E, Ferro A: Nitric oxide signalling in the regulation of cardiovascular and platelet function. Front Biosci 2011, 16:1873-1897.

6.

Custodis F, Schirmer SH, Baumha¨kel M, Heusch G, Bo¨hm M, Laufs U: Vascular pathophysiology in response to increased heart rate. J Am Coll Cardiol 2010, 56:1973-1983.

7.

Brevetti G, Giugliano G, Brevetti L, Hiatt WR: Inflammation in peripheral artery disease. Circulation 2010, 122:1862-1875.

8.

Watt SM, Athanassopoulos A, Harris AL, Tsaknakis G: Human endothelial stem/progenitor cells, angiogenic factors and vascular repair. J R Soc Interface 2010, 7(Suppl. 6):S731-S751.

9.

Eilken HM, Adams RH: Dynamics of endothelial cell behavior in sprouting angiogenesis. Curr Opin Cell Biol 2010, 22:617-625.

10. Carmeliet P, Jain RK: Molecular mechanisms and clinical applications of angiogenesis. Nature 2011, 473:298-307. 11. Ebos JM, Kerbel RS: Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat Rev Clin Oncol 2011, 8:210-221. 12. Avogaro A, Albiero M, Menegazzo L, de Kreutzenberg S, Fadini GP: Endothelial dysfunction in diabetes: the role of reparatory mechanisms. Diabetes Care 2011, 34(Suppl. 2):S285-S290. 13. Nakagawa T, Tanabe K, Croker BP, Johnson RJ, Grant MB, Kosugi T, Li Q: Endothelial dysfunction as a potential contributor in diabetic nephropathy. Nat Rev Nephrol 2011, 7:36-44. 14. Sitia S, Tomasoni L, Atzeni F, Ambrosio G, Cordiano C, Catapano A, Tramontana S, Perticone F, Naccarato P, Camici P et al.: From endothelial dysfunction to atherosclerosis. Autoimmun Rev 2010, 9:830-834. 15. Antoni FA: Molecular diversity of cyclic AMP signalling. Front Neuroendocrinol 2000, 21:103-132. 16. Yamamizu K, Yamashita JK: Roles of cyclic adenosine monophosphate signaling in endothelial cell differentiation and arterial-venous specification during vascular development. Circ J 2011, 75:253-260. 17. Sayner SL: Emerging themes of cAMP regulation of the  pulmonary endothelial barrier. Am J Physiol Lung Cell Mol Physiol 2011, 300:L667-L678. A comprehensive review of the role of cAMP, and of cAMP PDEs in regulating vascular endothelial cell permeability. 18. Fantidis P: The role of intracellular 30 50 -cyclic adenosine monophosphate (cAMP) in atherosclerosis. Curr Vasc Pharmacol 2010, 8:464-472. 19. Beavo JA, Brunton LL: Cyclic nucleotide research — still expanding after half a century. Nat Rev Mol Cell Biol 2002, 3:710-718. 20. Cooper DM, Crossthwaite AJ: Higher-order organization and regulation of adenylyl cyclases. Trends Pharmacol Sci 2006, 27:426-431. www.sciencedirect.com

Subcellular signaling in the endothelium Maurice 663

21. Kamenetsky M, Middelhaufe S, Bank EM, Levin LR, Buck J, Steegborn C: Molecular details of cAMP generation in mammalian cells: a tale of two systems. J Mol Biol 2006, 362:623-639. 22. Omori K, Kotera J: Overview of PDEs and their regulation. Circ Res 2007, 100:309-327. 23. Francis SH, Blount MA, Corbin JD: Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 2011, 91:651-690. 24. Taylor SS, Kornev AP: Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci 2011, 36:65-77. 25. Gloerich M, Bos JL: Epac: defining a new mechanism for cAMP action. Annu Rev Pharmacol Toxicol 2010, 50:355-375. 26. Welch EJ, Jones BW, Scott JD: Networking with AKAPs: context-dependent regulation of anchored enzymes. Mol Interv 2010, 10:86-97. 27. Dessauer CW: Adenylyl cyclase-A-kinase anchoring protein complexes: the next dimension in cAMP signaling. Mol Pharmacol 2009, 76:935-941. 28. Netherton SJ, Sutton JA, Wilson LS, Carter RL, Maurice DH: Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells. Circ Res 2007, 101:768-776. 29. Houslay MD, Baillie GS, Maurice DH: cAMP-Specific  phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling. Circ Res 2007, 100:950-966. Review that describes in great detail the various protein-binding partners of PDE4 family gene products. 30. Pawson CT, Scott JD: Signal integration through blending,  bolstering and bifurcating of intracellular information. Nat Struct Mol Biol 2010, 17:653-658. Review that describes in great detail the importance of interactions between domain-based proteins in cell signaling. 31. Scott JD, Pawson T: Cell signaling in space and time: where proteins come together and when they’re apart. Science 2009, 326:1220-1224. 32. Netherton SJ, Maurice DH: Vascular endothelial cell cyclic nucleotide phosphodiesterases and regulated cell migration: implications in angiogenesis. Mol Pharmacol 2005, 67:263-272. 33. Rampersad SN, Ovens JD, Huston E, Umana MB, Wilson LS,  Netherton SJ, Lynch MJ, Baillie GS, Houslay MD, Maurice DH: Cyclic AMP phosphodiesterase 4D (PDE4D) tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability. J Biol Chem 2010, 285:33614-33622. First manuscript to describe how PDE4D tethers EPAC1 within a VECADbased complex and how this cAMP-signalosomes controls VEC permeability. 34. Wilson LS, Baillie GS, Pritchard LM, Umana B, Terrin A, Zaccolo M,  Houslay MD, Maurice DH: A phosphodiesterase 3B-based signaling complex integrates exchange protein activated by cAMP 1 and phosphatidylinositol 3-kinase signals in human arterial endothelial cells. J Biol Chem 2011, 286:16285-16296. First manuscript to describe how PDE3B tethers EPAC1 within a membrane-associated domain and that describes how this cAMP-signalosome regulates PI3Kg-based signaling and tubule formation in human VECs. 35. Sayner SL, Alexeyev M, Dessauer CW, Stevens T: Soluble  adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelial cell barrier. Circ Res 2006, 98:675-681. First mention that compartments are involved in regulating VEC functions. 36. Lorenowicz MJ, Fernandez-Borja M, Kooistra MR, Bos JL, Hordijk PL: PKA and Epac1 regulate endothelial integrity and migration through parallel and independent pathways. Eur J Cell Biol 2008, 87:779-792. 37. Cullere X, Shaw SK, Andersson L, Hirahashi J, Luscinskas FW, Mayadas TN: Regulation of vascular endothelial barrier function by Epac, a cAMP-activated exchange factor for Rap GTPase. Blood 2005, 105:1950-1955.

www.sciencedirect.com

38. Kooistra MR, Corada M, Dejana E, Bos JL: Epac1 regulates integrity of endothelial cell junctions through VE-cadherin. FEBS Lett 2005, 579:4966-4972. 39. Birukova AA, Zagranichnaya T, Fu P, Alekseeva E, Chen W, Jacobson JR, Birukov KG: Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res 2007, 313:2504-2520. 40. Houslay MD: Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci 2010, 35:91-100. 41. Favot L, Keravis T, Holl V, Le Bec A, Lugnier C: VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors. Thromb Haemost 2003, 90:334-343. 42. Zhu B, Kelly J, Vemavarapu L, Thompson WJ, Strada SJ: Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells. Biochem Pharmacol 2004, 68:479-491. 43. Thompson WJ, Ashikaga T, Kelly JJ, Liu L, Zhu B, Vemavarapu L, Strada SJ: Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4). Biochem Pharmacol 2002, 63:797-807. 44. Surapisitchat J, Jeon KI, Yan C, Beavo JA: Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3. Circ Res 2007, 101:811-818. 45. Sadhu K, Hensley K, Florio VA, Wolda SL: Differential expression of the cyclic GMP-stimulated phosphodiesterase PDE2A in human venous and capillary endothelial cells. J Histochem Cytochem 1999, 47:895-906. 46. Keravis T, Komas N, Lugnier C: Cyclic nucleotide hydrolysis in bovine aortic endothelial cells in culture: differential regulation in cobblestone and spindle phenotypes. J Vasc Res 2000, 37:235-249. 47. Lugnier C, Schini VB: Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells. Biochem Pharmacol 1990, 39:75-84. 48. Suttorp N, Ehreiser P, Hippenstiel S, Fuhrmann M, Kru¨ll M, Tenor H, Schudt C: Hyperpermeability of pulmonary endothelial monolayer: protective role of phosphodiesterase isoenzymes 3 and 4. Lung 1996, 174:181-194. 49. Howe AK, Baldor LC, Hogan BP: Spatial regulation of the cAMPdependent protein kinase during chemotactic cell migration. Proc Natl Acad Sci U S A 2005, 102:14320-14325. 50. Howe AK, Juliano RL: Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nat Cell Biol 2000, 2:593-600. 51. Ru¨egg C, Dormond O, Mariotti A: Endothelial cell integrins and COX-2: mediators and therapeutic targets of tumor angiogenesis. Biochim Biophys Acta 2004, 1654:51-67. 52. Sakurai T, Suzuki K, Yoshie M, Hashimoto K, Tachikawa E, Tamura K: Stimulation of tube formation mediated through the prostaglandin EP2 receptor in rat luteal endothelial cells. J Endocrinol 2011, 209:33-43. 53. Jin H, Garmy-Susini B, Avraamides CJ, Stoletov K, Klemke RL, Varner JA: A PKA-Csk-pp60Src signaling pathway regulates the switch between endothelial cell invasion and cell-cell adhesion during vascular sprouting. Blood 2010, 116:5773-5783. 54. Namkoong S, Kim CK, Cho YL, Kim JH, Lee H, Ha KS, Choe J, Kim PH, Won MH, Kwon YG et al.: Forskolin increases angiogenesis through the coordinated cross-talk of PKAdependent VEGF expression and Epac-mediated PI3K/Akt/ eNOS signaling. Cell Signal 2009, 21:906-915. 55. Breckler M, Berthouze M, Laurent AC, Crozatier B, Morel E, Lezoualc’h F: Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications. Cell Signal 2011, 23:1257-1266. 56. Jyaraj SC, Unger NT, Chotani MA: Rap1 GTPases: an emerging role in the cardiovasculature. Life Sci 2011, 88:645-652. Current Opinion in Pharmacology 2011, 11:656–664

664 Endocrine and Metabolic Diseases

57. Chrzanowska-Wodnicka M: Regulation of angiogenesis by a small GTPase Rap1. Vascul Pharmacol 2010, 53:1-10. 58. Frische EW, Zwartkruis F: Rap1, a mercenary among the Raslike GTPases. Dev Biol 2010, 340:1-9.

60. Patrucco E, Notte A, Barberis L, Selvetella G, Maffei A, Brancaccio M, Marengo S, Russo G, Azzolino O, Rybalkin SD et al.: PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and independent effects. Cell 2004, 118:375-387.

59. Voigt P, Dorner MB, Schaefer M: Characterization of p87PIKAP, a novel regulatory subunit of phosphoinositide3-kinase gamma that is highly expressed in heart and interacts with PDE3B. J Biol Chem 2006, 281:9977-9986.

61. Perino A, Ghigo A, Ferrero E, Morello F, Santulli G, Baillie GS, Damilano F, Dunlop AJ, Pawson C, Walser R et al.: Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110g. Mol Cell 2011, 42:84-95.

Current Opinion in Pharmacology 2011, 11:656–664

www.sciencedirect.com