Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease.

MATBIO-01026; No of Pages 10 Matrix Biology xxx (2014) xxx–xxx

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Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease☆ Natasha M. Rogers a,d, Maryam Sharifi-Sanjani a, Gábor Csányi a,c, Patrick J. Pagano a,c, Jeffrey S. Isenberg a,b,c,d,⁎ a

Vascular Medicine Institute, University of Pittsburgh School of Medicine Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine d Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA 15261, United States b c

a r t i c l e

i n f o

Article history: Received 16 November 2013 Received in revised form 24 December 2013 Accepted 2 January 2014 Available online xxxx Keywords: Thrombospondin-1 CD47 Nitric oxide Blood flow Cardiovascular disease ROS

a b s t r a c t Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this crosstalk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and diseaserelated responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health. © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction The clinical manifestation of cardiovascular disease (CVD) are many and include myocardial infarction, pulmonary hypertension (both with subsequent heart failure), peripheral arterial disease and poor wound healing, renal failure requiring dialysis or transplantation, and cerebrovascular events including stroke and hemorrhage (Centers for Disease and Prevention, 2011, 2013; Go et al., 2013). CVD remains the leading cause of morbidity and mortality in the Western world, superseding communicable diseases and malignancy (http://www.cdc.gov/nchs/ fastats/lcod.htm). In CVD multiple pathophysiologic changes have been demonstrated in the macro and microcirculation that combine to decrease tissue blood flow and perfusion (Dinerman et al., 1993). Loss of signaling from the biogas nitric oxide (NO) in vascular cells and blood vessels is a common finding in and contributor to CVD (Napoli and Ignarro, 2009). Decreased NO production or biochemical interactions between preformed NO and other reactive moieties (such as superoxide) account for some of the observed disease-associated ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: BST, Room E1258, 200 Lothrop Street, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States. Tel.: +1 412 383 5424. E-mail address: [email protected] (J.S. Isenberg).

loss of NO signaling (Chirkov and Horowitz, 2007; Heinrich et al., 2013). Thrombospondin-1 (TSP1) is a secreted matricellular protein upregulated by vascular cells after injury and in chronic disease. It alters cell responses through binding to cell receptors (Lawler, 2000). Increasingly, molecular evidence demonstrates a pivotal role for TSP1, through interaction with its high affinity receptor CD47, in controlling vascular cell responses in a NO-dependent (Roberts et al., 2012) and -independent manner, limiting blood flow and tissue perfusion in health and disease. In this review we will highlight the broadening role for the involvement of TSP1 and CD47 in CVD. 1.1. The importance of NO to vascular health NO production is systematically regulated by three nitric oxidase synthases (neuronal, inducible and endothelial) that mediate conversion of L-arginine to L-citrulline (Moncada and Higgs, 1993; Bredt and Snyder, 1994). NO binds many intracellular proteins covalently at cysteine and tyrosine residues modifying their structure and function (Derakhshan et al., 2007a,b). One such interaction with the heme moiety of soluble guanylyl cyclase (sGC) leads to arterial vasodilation (Ignarro et al., 1987). Physiological levels of NO are essential to vessel structural integrity as it promotes endothelial and vascular smooth muscle cell health (Isenberg et al., 2005b, 2006a,b,c, 2009c). Endothelial-derived NO rapidly diffuses into the vessel wall acutely regulating arterial tone. In VSMC, NO reduces calcium availability for

0945-053X/$ – see front matter © 2014 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matbio.2014.01.002

Please cite this article as: Rogers, N.M., et al., Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease, Matrix Biol. (2014), http://dx.doi.org/10.1016/j.matbio.2014.01.002

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N.M. Rogers et al. / Matrix Biology xxx (2014) xxx–xxx

levels of 20 μg/L (Bergseth et al., 2000). Thus the systemic vasculature is chronically exposed to picomolar concentrations of TSP1 which may have significant implications for regulating vascular function in homeostasis. Recently, TSP1 has been found to function as an inhibitor of NO signaling (Fig. 1). TSP1-mediated control of NO signaling is redundant and occurs through inhibition of several key effectors of the canonical NO signaling cascade. Endothelial cell nitric oxide synthase (eNOS), when stimulated by hormones or laminar shear stress increases NO production (Fleming and Busse, 2003). Treatment of endothelial cells with nanomolar concentrations of TSP1 inhibited eNOS-mediated production of NO (Bauer et al., 2010) (Fig. 2), while tissues from TSP1-null mice displayed increased phosphorylation of eNOS, consistent with increased enzyme activity (Bauer et al., 2010). Optimal function of eNOS requires a number of modifications including myristoylation, and this, in turn, is limited by the cellular supply of essential fatty acids (Zhu and Smart, 2005). TSP1, through binding endothelial CD36 and inhibiting essential free fatty acid uptake, limited eNOS activity (Isenberg et al., 2008e). The primary intracellular target of NO in vascular cells is cytoplasmic soluble guanylyl cyclase (sGC) that produces cyclic guanosine monophosphate (cGMP) when stimulated by low physiologic amounts of NO (Isenberg et al., 2005b). Importantly, picomolar concentrations of TSP1 inhibited NO-mediated activation of sGC and limited cGMP production (Isenberg et al., 2005b) (Fig. 2). TSP1−/− endothelial cells demonstrated both higher basal cGMP and a greater NO-stimulated elevation in cGMP compared to wild-type cells. This inhibitory effect is independent of effects on cGMP hydrolysis by phosphodiestereases but limits both heme-dependent and hemeindependent sGC activation (Miller et al., 2010). TSP1-induced

myosin light chain (MLC) kinase-mediated phosphorylation of MLC2 to inhibit vasoconstriction (Lincoln et al., 2001). Diffusion of NO into the vessel lumen decreases αIIbβ3 integrin activation and inhibits platelet aggregation and inflammatory cell adhesion to the vessel wall (Radomski et al., 1991). In addition to an inhibitory effect on inflammatory cell adhesion (Bath, 1993), these in vitro findings are consistent with the inherent anti-atherogenic role of NO (Shabani et al., 1996; Weller and Finnen, 2006; Schaffer et al., 2007; Blecher et al., 2012) (Fig. 1). 1.2. The integral role for TSP1 and CD47 in controlling NO signaling Thrombospondin-1 (TSP1) is a large matricellular glycoprotein stored preformed in platelet α-granules (Lawler, 2000). TSP1 is released from platelets on activation (Baenziger et al., 1971). TSP1 is also produced and secreted by multiple primary cells (vascular smooth muscle (Isenberg et al., 2006a), endothelial (Phelan et al., 1998) and epithelial cells (Rogers et al., 2012), fibroblasts (Dameron et al., 1994) and keratinocytes (Wikner et al., 1987)) in response to stress as well as by innate immune cells (dendritic cells, macrophages (DiPietro and Polverini, 1993) and T cells (Li et al., 2002)) when activated by proinflammatory cytokines. Its large, multi-domain structure determines cell-based interactions via binding to multiple cell surface receptors, including integrins (predominantly β1 and β3), CD47, CD36, heparin sulfate proteoglycans, low density lipoprotein-related protein 1 and very low density lipoprotein receptor (Carlson et al., 2008; Murphy-Ullrich and Iozzo, 2012). TSP1 is detected as early as embryonic day 10 within the heart and intestine, followed by lung (Tooney et al., 1998). In healthy adults, TSP1 is present in minute quantities and is detectable in plasma at

Decreased adhesion of inflammatory cells

Decreased platelet aggregation

Laminar blood flow

Integrin activation

VEGF

NO

NO Angiogenesis, migration, proliferation, permeability

VEGFR2

PI3K

PLCγ

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P

NO

eNOS Akt

cGK-I

cGMP

decreased Ca2+, MLC2 phosphorylation, vasodilation and proliferation

sGC

endothelial cells

vsmc

adventitia

Fig. 1. Nitric oxide (NO) signaling within arteries targets multiple cells types to promote blood flow. Endothelial nitric oxide synthase (eNOS) reacts to hormones, including VEGF, and to the mechanical signal of laminar blood flow to increases production of the reactive nitrogen species NO. As a biogas, NO rapidly crosses cell membranes to activate the intracellular target soluble guanylyl cyclase (sGC) stimulating increased production of cyclic guanosine monophosphate (cGMP) itself a signaling intermediate. NO stimulates endothelial cell angiogenesis and suppresses expression of adhesion proteins on cell membranes. NO also acts distal to the site of production. In the artery lumen, NO decreases production of inflammatory cell cytokines and suppresses integrin signaling and platelet aggregation. In the wall of arteries NO acutely inhibits vascular smooth muscle cell contraction to dilate blood vessels and chronically suppresses cell overgrowth and hypertrophy, thus preserving vascular diameter and blood flow.



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TSP1

CD47 VEGFR2 Cav-1

NOX1 uncoupled eNOS

eNOS s1177

p47phox

NADPH oxidase

NO PDK1

CaM P-S1177-

P–T308- Akt

sGC

eNOS

O 2-

Hsp90 Gαβγ αβγ

cGMP AC

ROS

survival cAMP Fig. 2. TSP1 binds and activates cell receptor CD47 to limit blood flow directly and indirectly. Stationary and circulating vascular cells express CD47. TSP1 at picomolar concentrations binds and activates CD47. In endothelial cells this signal alters calcium flux to limit eNOS activation and NO production. On the endothelial cell surface CD47 also constitutively associates with the VEGF receptor (VEGFR2). Soluble TSP1 on binding to CD47 alters this interaction, inhibits VEGF signaling and thus blocks the NO-mediated and NO-independent effects of VEGF. In other cells, including VSMC and platelets activated CD47 directly inhibits the cytoplasmic NO-target sGC and the downstream signal transducer cGMP-dependent kinases (PKG). TSP1, via CD47, also directly inhibits adenylate cyclase production of second messenger cAMP to alter VSMC growth and promote vasoconstriction. In VSMC TSP1, via CD47, stimulates . NAPDH oxidase production of superoxide (O2−). In hypoxic endothelial cells TSP1, via CD47, dysregulates the caveolin-1-eNOS interaction and promotes increased reactive oxygen species (ROS) production. ROS further limits vasodilation by then scavenging NO. Together these direct and indirect processes promote vasoconstriction and decrease blood flow.

inhibition of sGC persists for some time, as demonstrated in lysates generated after treatment of cells with the TSP1-based CD47 binding recombinant protein E123CaG1 (containing the EGF repeats, the type 3 repeats, and the carboxyl terminal domain of TSP1), and likely involves a covalent phosphorylation modification (Ramanathan et al., 2011). However, additional exploration of this phenomenon is warranted. TSP1 also inhibits cell responses to the membrane-permeant cGMP analog 8-bromo-cGMP. In platelets, this occurred by inhibiting phosphorylation of vasodilator-stimulated phosphoprotein (VASP) via cGMP-dependent protein kinase (cGK) and identified the cGK as an NO-sGC-cGMP-independent downstream target of TSP1 (Butt et al., 1994; Isenberg et al., 2008d) (Fig. 2). Limitations on eNOS-mediated actions of NO subsequently affect paracrine modulation of vascular smooth muscle cell (VSMC) and platelet activity, although TSP1 also exerts independent effects on both of these cell types. The widely expressed cell surface protein CD47 (also known as integrin associated protein, IAP) is a receptor for SIRP-α (Sarfati et al., 2008). The interaction between macrophage CD47 and nonmacrophage SIRP-α inhibits inflammation to promote “self” recognition (Olsson and Oldenborg, 2008; van den Berg and van der Schoot, 2008). The structure of CD47 has been described in detail (Soto Pantoja et al., 2013). In brief it is characterized by a large globular extracellular domain, 5 trans-membrane domains and a short variably-spliced cytoplasmic domain that recognizes only a few intracellular targets. CD47 interacts with several TSP family members, specifically thrombospondin-1, -2 and-4 (Isenberg et al., 2009a). However, binding studies suggest the interactions of TSP2 and TSP4 with CD47 are relatively weak, and TSP1 is now appreciated

to be the high affinity soluble ligand of CD47 (Isenberg et al., 2009a). TSP1 engages CD47 via the C-terminal domain of the protein, though this has only recently been confirmed (Isenberg et al., 2009a). Additionally, a post-translational modification that initiates serine residue phosphorylation of the extracellular domain of CD47 is required for TSP1-mediated inhibition of T cell signaling (Kaur et al., 2011). Both CD36 and CD47 mediate TSP1-induced inhibition of NO-mediated sGC and thus cGMP production (Isenberg et al., 2005a,b, 2006a,c). Consistent with these findings, TSP1-mediated inhibition of NO signaling persists in CD36-null vascular cells but is lost in CD47-null vascular cells indicating CD47 is necessary for this process (Isenberg et al., 2006a). As previously noted, TSP1 inhibits eNOS activation and NO signaling via CD36. However, an independent, and more potent, inhibitory effect is mediated through CD47 (Bauer et al., 2010; Ochoa et al., 2011). Experiments in human endothelial cells employing radiolabelled essential eNOS substrate L-arginine demonstrated that nanomolar TSP1 inhibited acetylcholine (Ach)-stimulated eNOS activation (as determined by quantification of the metabolic byproduct L-citrulline) and inhibited phosphorylation of the key activating eNOS residue serine 1177. Functionally, TSP1-null vessels demonstrated enhanced Ach-stimulated endothelium-dependent relaxation compared to wild type vessels, which was reversed by replenishing TSP1. Conversely CD47-null arteries were resistant to TSP1-mediated inhibition of eNOS-stimulated vasodilation (Bauer et al., 2010). These results have several implications that warrant emphasis. Previous work in situations where NO is not limiting demonstrated that TSP1, through several receptors, has divergent effects including stimulating apoptosis in endothelial cells (Nor et al., 2000) and promoting


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proliferation in VSMC (Patel et al., 1997) (reviewed further in (Adams and Lawler, 2011; Lawler and Lawler, 2012)). In contrast, where NO is limiting TSP1 via interaction with CD47, uniformly inhibits physiologic NO signaling in multiple vascular cells types including endothelial cells (Isenberg et al., 2005b), VSMC (Isenberg et al., 2006a,c) and platelets (Isenberg et al., 2008d). Thus NO synchronizes TSP1-mediated effects on vascular cells. Studies of NO-mediated events have also emphasized that the TSP1CD47 interaction is (1) a potent interaction with picomolar concentrations of TSP1 inhibiting NO signaling and (2) a high affinity interaction with a dissociation constant of 12 pM (Isenberg et al., 2009a) (compared to 227 ± 19.89 nM for CD36 (Frieda et al., 1995) and 10 to 25 nM for low density lipoprotein receptor-related protein (Godyna et al., 1995; Mikhailenko et al., 1997)). Cell culture data supports the first point as TSP1-mediated effects in cells via alternative TSP1 receptors have been noted at significantly higher concentrations (Iruela-Arispe et al., 1999; Simantov et al., 2001; Short et al., 2005). However, it remains to be determined if in vivo tissue TSP1 concentrations mimic levels employed in cell culture experiments. Nonetheless, emerging evidence indicates that plasma TSP1 concentrations, though varying in human health and disease (Smadja et al., 2011; Novelli et al., 2012), are within the range required to inhibit NO signaling, implying that TSP1-CD47 signaling may be an important promoter of diseases characterized by decreased NO. 1.3. TSP1, via CD47, regulates additional non-NO pathways important to vascular function Disturbances in the bioavailability of and sensitivity to NO are not the sole mechanism underlying the initiation and subsequent progression of CVD. Similarly, TSP1 engaging its cognate receptor does not represent a singular signaling event through inhibition of NO signaling elements. New work suggests TSP1, via CD47, is an important regulator of vascular cell calcium levels. Both TSP1 and the signature domain of the protein that binds CD47 (E123CaG1) increased T cell cytosolic calcium in a β1 integrin and Gi protein independent fashion. Conversely, in endothelial cells TSP1 inhibited ionomycin-stimulated calcium flux (Bauer et al., 2010). TSP1 is known to be a critical regulator of angiogenesis via antagonism of VEGF. Direct TSP1-mediated sequestration of VEGF (Laklai et al., 2009), competitive binding of TSP1 to heparin sulfate proteoglycans (Gupta et al., 1999), and endocytosis of this protein complex via LDL-related receptor protein-1 (Greenaway et al., 2007) are contributing factors. However, a recent study suggests that the endothelial cell VEGF receptor, VEGFR2, is constitutively associated with CD47 on the cell surface. TSP1, on binding CD47, alters this constitutive interaction inhibiting VEGFR2 phosphorylation and downstream signaling (Kaur et al., 2010). Thus, TSP1 captures both the NO-dependent and NOindependent protein kinase B (Akt)-mediated signaling stimulated by VEGF. TSP1, via CD47, also inhibits production of and signaling through the essential second messenger 3′-5′-cyclic adenosine monophosphate (cAMP). TSP1 and a peptide derived from the same that presumably binds CD47 have been shown, in a heterotrimeric G protein-dependent manner, to limit cAMP levels (Wang et al., 1999; Rath et al., 2006; Yao et al., 2011). In TSP1-null VMSC and skeletal muscle cAMP levels were constitutively elevated compared to wild type cells and tissues, while treating TSP1-null VSMC with exogenous TSP1 decreased basal and agonist-mediated increases in cAMP. In arterial VSMC, TSP1 attenuates cAMP levels through both direct inhibition of adenylyl cyclase (Fig. 2) and indirectly via cGMP-mediated stimulation of phosphodiesterase 3 activity (Yao et al., 2011). At the level of cell function cAMP regulates morphology and adhesion (Eid, 2012). TSP1 abrogated cAMPstimulated changes in F-actin organization and collagen-mediated adhesion. In arteries, TSP1 inhibited isoproterenol- and forskolin-induced endothelium-independent vasodilatation, in part by blocking cAMPmediated dephosphorylation of myosin light chain-2 (Yao et al., 2011).

Lastly, heart tissue from TSP1- and CD47-null mice demonstrated elevated cAMP levels compared to wild type controls. Accelerated atherosclerosis is well-defined in diabetes, both epidemiologically (Kataoka et al., 2013) and in standard animal models (Hamamdzic and Wilensky, 2013). Exposure to excess glucose (as a mimic of diabetic hyperglycemia) leads to a concomitant rise in TSP1 secretion from VSMC (Raman et al., 2007; Wang et al., 2010). VSMC chemotaxis, proliferation and migration are promoted by TSP1 (Patel et al., 1997). This occurs in a β3 integrin-PKCβ dependent manner that is enhanced by excess glucose (Panchatcharam et al., 2010). TSP1 also modulates multiple growth factors involved in progression of atherosclerotic lesions. TSP1 bound platelet-derived growth factor (PDGF) (Hogg et al., 1997), the latter synthesized by multiple cells within plaques, while TSP1-null VSMC were deficient in PDGF-mediated proliferation and migration (Isenberg et al., 2005a). TSP1 also activates latent transforming growth factor-β (TGF-β) (Schultz-Cherry et al., 1994a,b), while TGF-β promoted diabetic-associated atherosclerotic plaque formation (Yang et al., 2010). Dysregulated matrix metalloproteinase activity contributes to vasculopathy in diabetes, and here too, TSP1 may play a role as VSMC MMP2 (Lee et al., 2003) and endothelial cells MMP9(Qian et al., 1997) are upregulated by TSP1. TSP1 further promotes vasculopathy by increasing inflammatory cell-endothelial cell interactions. TSP1, via CD47, upregulates the adhesion proteins VCAM-1 and ICAM-1 on endothelial cells (Narizhneva et al., 2005) and mitigates NO-signaling in the vasculature (Bauer et al., 2010), enhancing inflammatory cell adhesion to and transmigration through the vascular endothelium. This, in turn, promotes atherosclerotic plaque formation (Koltsova et al., 2013). Alterations in vessel flow dynamics, as frequently is the case in vessel segments with atherosclerotic plaque, upregulate expression of TSP1 and CD47 in the endothelium (Freyberg et al., 2000, 2001), and initiate apoptosis which may accelerate vascular pathology at sites of vessel bifurcations or vessel wall irregularities. 1.4. TSP1, via CD47, maintains vascular tone and blood pressure and modulates cardiac response in vivo TSP1-CD47 signaling acutely modulates artery function in part by inhibiting responses to vasodilators. TSP1 and the signature domain E123CaG1 also increased phenylephrine-mediated arterial vasoconstriction (Bauer et al., 2010). These findings raise the intriguing possibility that TSP1 regulates blood pressure. Long term blood pressure monitoring via telemetric implants demonstrated resting hypotension, with significantly decreased systolic, diastolic and mean blood pressure, in CD47-null mice compared to wild type mice (Bauer et al., 2010). Interestingly, in active CD47-null mice blood pressure returned to levels comparable to wild type animals, suggesting compensatory responses by the sympathetic nervous system. On the other hand, TSP1-null mice did not exhibit a significant difference in basal blood pressure compared to wild type animals (Isenberg et al., 2009b), implying the existence of TSP1-independent CD47 signaling in the cardiovascular system. The reliance on autonomic control of vascular tone as a compensatory mechanism in null animals was highlighted by the rapid cardiovascular collapse seen in TSP1-null animals following autonomic nervous system inhibition via administration of the postganglionic blocker hexamethonium (Isenberg et al., 2009b). Conversely, treatment of wild type and TSP1-null mice with intravascular TSP1 or a CD47 antibody elevated mean arterial blood pressure (Bauer et al., 2010). Not surprisingly, TSP1- and CD47-null mice displayed a greater hypotensive response to systemically administered NO-donor agents compared to wild type mice, regardless of the kinetics of NO-release (Isenberg et al., 2009b). Changes in cardiac function were also found following a NO-challenge, with marked increases in ejection fraction and cardiac output in both TSP1- and CD47-null animals. These features are consistent with increased cGMP and cAMP levels in the heart, in addition to an increased cardiac capillary density in cardiac (and



skeletal) muscle and greater exercise capacity in TSP1-null animals (Malek and Olfert, 2009). 1.5. TSP1-CD47 signaling as a mediator of oxidative stress-induced vascular disease Studies have demonstrated that TSP1 increases systemic blood pressure and attenuates peripheral arterial flow (Csanyi et al., 2012). Strong evidence indicates that acute and chronic overproduction of reactive oxygen species (ROS) is integral in the development of hypertension and other vascular disorders (Harrison et al., 2007; Sedeek et al., 2009), but there are very limited data on the effect of TSP1 on ROS production in vascular cells. A previous study demonstrated that the type 3 repeat/C-terminal domain of TSP1 (at concentrations of 3 μM, following 48 hours incubation) stimulated ROS production in a leukemia cell line (Saumet et al., 2005). Treatment of cortical neurons with TSP1 (at 24 nM, for 3 h) and the purported CD47-activating peptide 4N1K (at 72 μM, for 6 h) stimulated ROS production and led to cell death (Xing et al., 2009). However, the role of CD47 in this process is unclear, as peptide 4N1K (as well as the related 4 N1-1 peptide) exerts effects in CD47-null cells (Tulasne et al., 2001). The ability of these peptides to bind CD47 (and TSP1) has been further questioned by the finding that the relevant Val–Val–Met (VVM) motifs are not surface exposed in the published crystal structure for the C-terminal domain of TSP1 (Kvansakul et al., 2004). However, computational modeling results have predicted a potential conformational change in the C-terminal domain of TSP1 that could expose the VVM motif (Floquet et al., 2008). Further experimental work is required to verify this. TSP1 (48 nM, 70 min) significantly increased phorbol12-myristate-13-acetate (PMA)-stimulated superoxide anion (O•2) production in monocytic cells (Martin-Manso et al., 2008). The identity and enzymatic source of ROS in these and other reports was indeterminate or perhaps linked to mitochondrial membrane potential shifts (Roue et al., 2003; Saumet et al., 2005). It is also important to note that concentrations of TSP1 used in these studies were 10- to 20-fold higher than the plasma concentration of TSP1 under conditions associated with platelet activation (Novelli et al., 2012). Nonetheless, mitochondrial ROS production may be promoted through TSP1 and CD47. In support of this, TSP1-and CD47-null skeletal muscle cells displayed more and larger mitochondria even though levels of electron transport chain enzymes per mitochondrion were similar compared to mitochondria from wild type muscle (Frazier et al., 2011). Mitochondrial ROS production was lower in null mitochondria compared to wild type skeletal muscle derived mitochondria. At a functional level, null mice were capable of physical exercise approximately 25% longer compared to wild type (Frazier et al., 2011). More recent work is consistent with TSP1 promoting ROS in endothelial cells via uncoupled NOS in hypoxic animals. Following chronic hypoxia, nitrotyrosine protein modifications (as a marker of nitrooxidative stress) in lung tissues from TSP1-null mice were significantly less compared to lung samples from hypoxic wild type animals (Bauer et al., 2012). Additionally, an identified constitutive association on endothelial cells of CD47 with caveolin-1 was shown to be disrupted by TSP1, increasing monomeric eNOS activity and subsequent superoxide generation (O•− 2 ) (Bauer et al., 2012). Moreover, treatment of human pulmonary arterial endothelial cells with either a CD47 antagonist antibody or the NOS inhibitor L-NG-nitro-arginine methyl ester (L-NAME) completely abrogated hypoxia-induced O•− 2 production, suggesting that CD47 activation, via eNOS uncoupling, stimulates O•− 2 generation in pulmonary endothelial cells (Bauer et al., 2012). However, it has not been known whether TSP1 and CD47 can stimulate increased ROS production from primary enzymatic sources. A recent report showed that treatment of both human and rodent VSMC with physiologically relevant concentrations of TSP1 (2.2 nmol/L) for 60 min stimulated increased O•− production, without cytotoxic effects (Csanyi et al., 2

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2012). In that study, TSP1 treatment of VSMC activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase (Nox) organizer subunit p47phox and increased Nox1-derived O•− 2 generation (Csanyi et al., 2012) (Fig. 2). Intravenous TSP1 decreased hind limb blood flow following ischemia reperfusion (Smadja et al., 2011). Conversely, treatment with a CD47 blocking antibody or a Nox1 gene silencing oligonucleotide morpholino abrogated TSP1-mediated inhibition of arterial vasodilatation ex vivo and mitigated TSP1-induced impairment of tissue blood flow following IRI (Csanyi et al., 2012). Taken together, these data suggest that TSP1 contributes to impaired NO signaling through multiple mechanisms, including direct inhibition of NO production (eNOS), inhibition of down-stream NO targets (sGC and PKG), and concurrent stimulation of Nox with subsequent generation of the potent NO scavenger O•− 2 . Further, cell experiments indicate that TSP1-mediated inhibition of primary NO targets occurs rapidly within minutes, while TSP1-mediated activation of Nox-derived ROS occurs over 1–1.5 h, suggesting both an acute and chronic role for TSP1 in propagating reactive nitrogen and reactive oxygen species (RNS-ROS) signaling. Future studies are required to define the specific redox-sensitive signaling cascades activated by TSP1. 1.6. Enhanced TSP1-CD47 signaling in pre-clinical models of CVD TSP1 levels are increased in a variety of models of CVD. Diabetic rats demonstrate adventitial expression of TSP1 and reduced vasa vasorum density, as well as TSP1-mediated induction following carotid artery angioplasty (Stenina et al., 2003). This is in keeping with in vitro data that showed incubation of cells with excess glucose led to upregulated TSP1 (Stenina et al., 2003). In apolipoprotein E (ApoE) null mice (characteristically predisposed to atherosclerotic plaque burden), suppression of the TSP1-CD47 signaling axis enhanced tissue blood flow under ischemic stress (Isenberg et al., 2007a,b). Likewise in a model of carotid artery ligation (as a surgical mimic of vascular injury), TSP1null mice had reduced neointimal formation in the injured vessel secondary to delayed VSMC migration and proliferation (Moura et al., 2007). TSP1 expression is induced following ischemia and/or IRI to the heart (Frangogiannis et al., 2005; Sezaki et al., 2005), brain (Lin et al., 2003; Jin et al., 2009), hind limb (Isenberg et al., 2007a,b), liver (Isenberg et al., 2008a) and kidney (Thakar et al., 2005; Rogers et al., 2012), although it is not clear whether parenchymal cells (Rogers et al., 2012) or infiltrating inflammatory cells (Sezaki et al., 2005) are the predominant source of the TSP1 in these scenarios. Some data suggest a biphasic TSP1 release in response to injury, with a secondary stimulus induced by monocytes and macrophages that infiltrate tissue in response to injury (Lin et al., 2003). Indeed, TSP1 induces proinflammatory cytokine release (including IL-6) from mononuclear cells in vitro (Sezaki et al., 2005). It is likely that both circulating and parenchymal TSP1-CD47 signaling contributes to CVD. Pulmonary arterial hypertension (PAH) is a progressive, fatal condition characterized by increased pulmonary vascular resistance with secondary right ventricular hypertension and subsequent heart failure (Rabinovitch, 2012). In both animal models of PAH and human disease NO signaling is significantly attenuated or absent (Scarborough et al., 1998; Xu et al., 2004). Wild type mice exposed to chronic hypoxia (FiO2 10% for 3 weeks) to induce PAH showed upregulation of pulmonary TSP1 concurrent with right ventricular hypertrophy, decreased cardiac output and pulmonary arterial overgrowth (Bauer et al., 2012). Conversely, TSP1-null mice were resistant to hypoxia-mediated PAH with minimal right ventricular hypertrophy, improved cardiac function and minimal pulmonary vascular overgrowth. Additionally, rats treated with a CD47 blocking antibody did not develop PAH following monocrotaline treatment (Bauer et al., 2012). Ageing is appreciated to have deleterious effects on tissue perfusion, especially after injury (Allwood, 1958; Tucker and Lillich, 1977). TSP1 expression was increased in aged animals (Rogers et al., 2013) and is


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associated with decreased tissue levels of cGMP (Isenberg et al., 2007a) and less cutaneous blood flow at baseline and after pharmacologic or thermal challenge (Rogers et al., 2013). Ischemic myocutaneous flaps in senescent wild type mice demonstrated decreased blood flow and poor survival compared to younger mice (Isenberg et al., 2007a). In this study tissue survival was moderately improved by the addition of the NO-donor isosorbide dinitrate to the drinking water. In contrast soft tissue flaps created in senescent TSP1- or CD47-null mice tolerated ischemic stress with minimal tissue loss and maintenance of near normal blood flow (Isenberg et al., 2007a). Though NO-mediated vasodilation accounts for the results seen in the absence of TSP1 and CD47, it is also possible that null tissue units have greater vascular density (Audet et al., 2013). Lastly, NO-stimulated increases in hind limb blood flow were greater in aged (18 months old) null mice compared to young (12 weeks old) wild type animals (Isenberg et al., 2007a). 1.7. The CD47-TSP1 signaling axis in human CVD TSP1 is typically not detectable via Western immunoblots or immunofluorescent histology in normal human vessels (Canfield et al., 2002). However, there is now increasing data demonstrating the pathogenic role of both TSP1 and CD47 in human CVD. Immunohistochemical studies have demonstrated increased TSP1 within the intima of systemic arteries from patients with atherosclerosis, and in renal vessels affected by chronic vascular rejection (Canfield et al., 2002). TSP1 also localized to primary coronary atherosclerotic plaques (Riessen et al., 1998). Not surprisingly, as a secreted protein, only a minor proportion of TSP1 was located intracellularly in diseased arteries (Riessen et al., 1998). TSP1 mRNA has been detected in myocardial biopsies, is increased in biopsies from cardiac allografts, and correlated with allograft vasculopathy and rejection (Zhao et al., 2001). Microarray analysis of peripheral blood samples in patients following myocardial infarction identified TSP1 as a potential biomarker of ensuing left ventricular failure (LVF) (defined as left ventricle ejection fraction b40% by echocardiography) (Devaux et al., 2010). Interestingly, the predictive value of plasma TSP1 in this study was greater than both brain natriuretic peptide and troponin T (Devaux et al., 2010). Conversely, TSP1 mRNA levels were decreased in biopsy samples from patients with end-stage LVF (Batlle et al., 2009). One explanation for this may be that a compensatory response was at play. Familial myocardial infarction (MI) has been associated with single nucleotide polymorphism (SNP) variations in several thrombospondin family members (TSP1, -2, and -4), although the highest risk was associated with the TSP1 variant (Topol et al., 2001). Replacement of an asparagine with a serine at residue 700 of TSP1 led to a protein variant that, in cell culture experiments, promoted pro-thrombotic phenomena, including augmented fibrinogen binding and enhanced platelet aggregation (Narizhneva et al., 2004). The presence of the TSP-1 A2210G variant has been associated with the small for gestational age phenotype (itself a risk factor for coronary artery disease) in newborns from affected parents (Andraweera et al., 2011). Additionally, umbilical cord blood endothelial cells from low birth weight infants (also known to be at risk for cardiovascular disease) were found to overexpress TSP1 and this was associated with decreased angiogenic activity in these cells (Ligi et al., 2011). Together, these results suggest a transgenerational role for TSP1 in controlling vascular responses. New evidence has also linked TSP1 to human PAH. Two distinct TSP1 SNPs have been identified in familial PAH, which are hypothesized to alter transcription factor binding and/or modify pulmonary vascular smooth muscle and endothelial cell growth (Maloney et al., 2012). Though rare in comparison to premature MI, these genetic variations in PAH implicate the importance of TSP1 in promoting pulmonary vascular pathology. Preliminary studies in lungs explanted from patients undergoing transplantation for end-stage PAH found significant upregulation of the TSP1-CD47 signaling axis at both the mRNA

and protein level in whole lung (Bauer et al., 2012) and in the proximal and distal isolated pulmonary artery vasculature (Rogers et al., 2013). Intriguingly, a positive correlation with elevated plasma TSP1 levels and adverse outcomes, including death, in PAH has recently been described (http://scidok.sulb.uni-saarland.de/volltexte/2012/4692/pdf/ Dissertation_Christian_Frantz_2011.pdf). Conversely, decreased plasma TSP1 levels were associated with hypotension in a cohort of febrile patients compared to normotensive controls (Liu et al., 2008). In this clinical cohort the extent of hypotension correlated directly with the degree of decrease in plasma TSP1. Plasma TSP1 levels were also increased in patients with conditions associated with dysregulated RNS-ROS signaling, including vasculitis (McCrohan et al., 1987) and sickle cell disease (SCD) (Novelli et al., 2012). Elevation of plasma TSP1 in SCD positively correlated with vaso-occlusive events and a history of acute chest syndrome (Novelli et al., 2012). In children with SCD, CD47 expression was increased on circulating cells (Odievre et al., 2008), while elevated plasma TSP1 positively correlated with cerebral infarct (Faulcon et al., 2013). Indeed, in at least one study elevation of TSP-1 in systemic blood samples of patients with CVD has also been demonstrated (Smadja et al., 2011). That other thrombospondin family members may contribute to human CVD is suggested by new work reporting elevated plasma TSP2 was correlated with increased cardiovascular mortality (Golledge et al., 2013). This latter finding is particularly interesting in light of our report that TSP2 binds CD47, albeit with lower affinity, and inhibits NO signaling in vascular cells (Isenberg et al., 2009a,b). Immunohistochemical staining of tissue from patients with critical leg ischemia revealed extensive TSP-1 expression in both infiltrating macrophages and arterial endothelial cells (Smadja et al., 2011). This clinical finding correlated with TSP1-mediated inhibition of endothelial colony forming cell proliferation that was CD47-dependent and modulated by the CXCR4/stromal cell derived factor-1 pathway. Flow cytometric analysis of monocytes from patients with carotid artery stenosis has been shown to correlate with surface TSP1 expression, monocyte-platelet association and plaque lucency (Jurk et al., 2010). Speaking to a possible larger role in regulating vascular homeostasis, a recent report has demonstrated upregulation of TSP1 in patients with chronic venous stasis wounds of the lower extremity (Shih et al., 2012). 1.8. Therapeutic implications of targeting CD47 Results from animal models indicate that inhibition of TSP1-CD47 signaling could be an important therapeutic strategy to improve NOsignaling and attenuate or reverse CVD. Beneficial effects were obtained in various models of CVD through several strategies including antibody blockade of CD47 and TSP1, and CD47 gene suppression via antisense morpholino oligonucleotides. CD47-blocking antibodies have elicited beneficial therapeutic effects in a wide variety of conditions, including increasing skin graft healing (Isenberg et al., 2008b), preserving blood flow and tissue/organ survival in ischemia and/or IRI in soft tissues flaps (Isenberg et al., 2007a,b, 2008c), and in the kidney (Rogers et al. 2012), the liver (Isenberg et al., 2008a), and the hind limb. Treatment with a CD47 antagonist antibody also prevented pharmacologicallyinduced PAH (Bauer et al. 2012). Of more relevance to clinical situations, CD47 targeting is also effective at increasing blood flow and promoting tissue protection after reperfusion is established, and thus may be a useful therapeutic strategy under conditions of delayed presentation to health care centers (Maxhimer et al., 2009a). Radiation injury is recognized to promote tissue ischemia and accelerate peripheral vascular disease (McCready et al. 1983; Chin et al. 2013). Blockade of CD47 signaling improved tissue blood flow and healing after high dose radiation injury to the lower extremities of animals (Maxhimer et al., 2009b). CD47 antagonist antibodies and CD47 morpholino oligonucleotides hold promise as therapeutics inhibiting TSP1-CD47 signaling. Development of humanized CD47 antibodies is currently underway for treatment of cancer, transplantation-related IRI and diabetic retinopathy



(see the following NIH funded proposals — Development of a Novel Method for Inhibiting Diabetic Retinopathy; Tumor-Toxic CD47 MAB Therapy for Leukemia: A Proof of Concept Study; and Treatment of Transplant Reperfusion with CD47 Antibody at the following NIH web page link: http://projectreporter.nih.gov/reporter_SearchResults.cfm? icde=18262101). Further, morpholino oligonucleotides are now in clinical trials and represent a proven clinical strategy for treatment of various human disorders (Jearawiriyapaisarn et al., 2010; Moulton and Moulton, 2010), indicating efficacy as a class of drugs and suggesting therapeutic opportunity for a CD47 targeting morpholino. 2. Concluding remarks In the Western world, the greatest disease burden arises from cardiovascular and cerebrovascular disease that comprise the leading global cause of death (http://www.heart.org/HEARTORG). Populations in third-world countries are increasingly affected by CVD, typically in countries that have limited financial resources and healthcare infrastructure to appropriately deal with diagnosis and management. With an ageing population the burden and medical costs of CVD is expected to increase in the coming decades, hence an emphasis must be placed on effective preventative and acute care. Emerging data from cell culture, tissue and animal research, and clinical reports, suggest a role for the TSP1-CD47 signaling axis as a promoter of CVD. In cells, tissues and organs, and animals activation of CD47 by TSP1 inhibits a range of important salutary agents and mechanisms including NO, cyclic nucleotides (cGMP, cAMP), VEGF and mitochondrial function, while concurrently inducing pathologic ROS production. Few ligand-receptor signaling axes have been identified that redundantly and potently regulate such a wide range of important mechanisms to control cardiovascular homeostasis. Results in animals have consistently shown therapeutic benefits from targeting the TSP1-CD47 axis to ameliorate CVD. Further work will determine the etiological contribution of dysregulated TSP1-CD47 to CVD, and the potential clinical utility of agents blocking this pathway. Disclosures J.S.I. is chair of the Scientific Advisory Boards of Vasculox, Inc. (St. Louis, MO) and Radiation Control Technologies, Inc. (NY, NY) and holds equity interests in the same. Funding Sources This work was supported by NIH grant R01 HL-108954 (J.S.I.); and by NIH grant 1R01HL112914-01A1 (P.J.P and J.S.I.); and by R01HL079207 (P.J.P.); and by K99 HL114648 (G.C.); and an American Heart Association award 13POST14520003 and American Transplant Society award (N.M.R.). This work was also supported by the Institute for Transfusion Medicine, the Hemophilia Center of Western Pennsylvania and the Vascular Medicine Institute (J.S.I., P.J.P.). References Adams, J.C., Lawler, J., 2011. The thrombospondins. Cold Spring Harb. Perspect. Biol. 3, a009712. Allwood, M.J., 1958. Blood flow in the foot and calf in the elderly; a comparison with that in young adults. Clin. Sci. (Lond.) 17, 331–338. Andraweera, P.H., Dekker, G.A., Thompson, S.D., North, R.A., McCowan, L.M., Roberts, C.T., Scope, C., 2011. A functional variant in the thrombospondin-1 gene and the risk of small for gestational age infants. J. Thromb. Haemost. 9, 2221–2228. Audet, G.N., Fulks, D., Stricker, J.C., Olfert, I.M., 2013. Chronic delivery of a thrombospondin-1 mimetic decreases skeletal muscle capillarity in mice. PLoS One 8, e55953. Baenziger, N.L., Brodie, G.N., Majerus, P.W., 1971. A thrombin-sensitive protein of human platelet membranes. Proc. Natl. Acad. Sci. U. S. A. 68, 240–243. Bath, P.M., 1993. The effect of nitric oxide-donating vasodilators on monocyte chemotaxis and intracellular cGMP concentrations in vitro. Eur. J. Clin. Pharmacol. 45, 53–58.

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