The role of thrombospondin-1 in cardiovascular health and pathology

International Journal of Cardiology xxx (2013) xxx–xxx

IJCA-16256; No of Pages 15

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International Journal of Cardiology

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Review

The role of thrombospondin-1 in cardiovascular health and pathology

Smriti Murali Krishna 1, Jonathan Golledge ⁎ ,2

The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, School of Medicine and Dentistry, James Cook University, Australia

⁎ Corresponding author. Tel.: +61 7 4781 4986; fax:E-mail address: [email protected] (J. Go

1 This author takes responsibility for all aspects of thbias of the data presented and their discussed interpret

2 This author takes responsibility for the discussedinformation.

0167-5273/$ – see front matter © 2013 Elsevier Irelandhttp://dx.doi.org/10.1016/j.ijcard.2013.04.139

Please cite this article as: Krishna SM, Gollehttp://dx.doi.org/10.1016/j.ijcard.2013.04.13

a b s t r a c t
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Article history:Received 6 October 2012Received in revised form 9 March 2013Accepted 6 April 2013Available online xxxx

Keywords:Thrombospondin-1Cardiovascular diseasesTransforming growth factor-β1ThrombosisMatrix remodelling

Cardiovascular diseases (CVDs) remain a leading cause of morbidity and mortality in the developed worldand are becoming increasingly prevalent in the developing world. Although a range of therapies alreadyexist for established CVDs, there is a significant interest in further understanding disease pathogenesis inorder to improve treatment. Thrombospondin (TSP)-1 is an important extracellular matrix component thatinfluences the function of vascular smooth muscle cells, endothelial cells, fibroblasts and inflammatorycells with important implications for CVDs. TSP-1 regulates matrix production and organisation therebyinfluencing tissue remodelling and promoting the generation of T regulatory cells that control the inflamma-tory response. Reported findings from in vitro and animal studies are conflicting and suggest differing effectsof TSP-1 on various cellular mechanisms, depending on the experimental setting. Vascular cells express anumber of TSP-1 receptors, such as CD36, proteoglycans and several integrins, which are regulated by specificcontextual signals which may explain the varying effects that TSP-1 elicits in different environments. Differ-ent domains of TSP-1 activate distinct signalling pathways eventually resulting in quite different cellular phe-notypes and tissue specific effects. The sum total of the various pathways activated likely determines theoverall effect on angiogenesis or proliferation in a specific tissue. Hence defining a commonmechanism of ac-tion of TSP-1 in CVD is complicated. Increasing the understanding of the role of TSP-1 in various CVDs willpotentially provide new opportunities for therapeutic intervention using peptides derived from its variousdomains currently under evaluation in other diseases.

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Cardiovascular disease (CVD) is a leading cause of death, disabilityand health cost in both the developed and developing worlds. Myocar-dial infarction (MI) and stroke account for approximately 12 milliondeaths worldwide each year. It is expected that by 2030, 7 of 10 deathsworldwide will be due to chronic diseases, and CVD is expected to re-main the commonest cause of death [1]. Cigarette smoking, hyperten-sion, dyslipidemia, diabetes and obesity remain important risk factorsfor CVD, accounting for more than 50% of the variation in disease prev-alence [2]. Genetic factors also contribute to an individual's risk ofdeveloping CVD. A body of evidence has demonstrated that the biolog-ical changes related to CVDs are complicated and involve a myriad ofcellular elements and subcellular signalling pathways. A range of thera-pies already exist for established CVDs, however there is a significantinterest in further understanding disease pathogenesis in order to im-prove primary prevention, diagnosis and therapy.

+61 7 4781 4730.lledge).e reliability and freedom fromation.interpretation of presented

Ltd. All rights reserved.

dge J, The role of thrombosp9

The extracellular matrix (ECM) forms a complex meshwork com-posed of structural proteins (such as type I and III collagen), proteo-glycans, glycosaminoglycans, and a basement membrane, which areresponsible for maintaining tissue strength and structure. Matricellularproteins are non-structural proteins usually present at low concentra-tions within normal ECM but frequently expressed at increased levelsfollowing tissue injury or during pathological changes. Thrombospondin(TSP)-1 is a matricellular protein which was first identified within theECM and subsequently shown to be a major component of α-granulesof platelets which is released upon the activation of these cells [3].TSP-1 is a calcium-binding protein that regulates cellular adhesion andmigration; cytoskeletal organisation; and cell proliferation and apopto-sis [4,5]. TSP-1 is constitutively present within blood vessels and inter-acts with a range of key proteins known to maintain vascular structureand homeostasis. TSP-1 also interacts with multiple cell-surface recep-tors, proteases, growth factors and various bioactivemolecules. The clas-sification of TSP-1 as amatricellular protein is due to the ability of TSP-1to inhibit cell-substrate interactions, and hence it has been groupedalong with genetically and structurally distinct multidomain proteingroups as a counter-adhesive protein [6]. A number of studies have im-plicated TSP-1 in the pathogenesis of a range of CVDs [7–10]. The aim ofthe present review was to summarise previous research assessing therole of TSP-1 in CVDs using data from cell culture, animal models andhuman investigations.

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2. TSP-1 structure

TSPs are a family of five extracellular calcium binding matrix pro-teins and the family is separated into two groups since TSP-1 andTSP-2 are trimers (Group A) and TSP-3, TSP-4 and TSP-5/COMP (car-tilage oligomeric matrix protein) are pentamers (Group B) [5,11,12].Even though the TSP family members show structural similarity,they are differentially expressed in various tissue types suggestingthat their roles are distinct and not redundant. TSP-1 is a 450 kDahomotrimeric glycosylated protein and each TSP-1 subunit consistsof N-terminal and C-terminal globular (G) domains which areconnected by a thin strand (Fig. 1). The N-domain is cleaved by sever-al proteases (such as thrombin, plasmin, cathepsins, elastases, trypsinand chymotrypsin) and thus can either exist in a soluble state or inassociation with activated platelet membranes [11]. The free-thiolsand the calcium modulated C-terminal region enable TSP-1 to formcovalent bonds with other proteins such as thrombin. Individualchains of TSP-1 comprise a globular N-terminal domain, which isfollowed by an oligomerization domain containing the inter-chain di-sulphide bonds, followed by a type I procollagen (PC) homology re-gion also known as von Willebrand C (vWC) repeat. The signaturepiece of TSP-1 is the central repeat region which follows the Type 1PC region and includes 3 type I (TSP or properdin-like) repeats(TSR1), 3 epidermal growth factor (EGF) like repeats called TSP type2 repeats (TSR2) and 7 calcium binding repeats called TSP type 3 re-peats (TSR3), the last of which contains the RGD (Arg-Gly-Asp) se-quence. In addition, there is a \COOH terminal domain that forms

Fig. 1. Schematic diagram of the structure of TSP-1 monomeric subunit. The various domaindifferent domains. Below the receptors are listed some of the functions or activities relatedmains. TSP-1 consists of a globular NH2 (N)-terminal domain also known as the heparin bindisulphide bonds which help in trimer formation. This is followed by a type I procollagen homor properdin-like) repeats (TSR1, 2 & 3). The signature piece of TSP-1 is the 3 type II (epiderlast of which contains the RGD sequence. The globular (G) C-terminal domain is the COOH-signature piece interact extensively to form 3 structural regions termed stalk, wire and globeMMP = matrix metalloprotenase; TIMP = tissue inhibitors of matrix metalloproteinase; ECsmooth muscle cells; NO = nitric oxide; IAP = integrin-associated peptide; ↓ Anti or decre

Please cite this article as: Krishna SM, Golledge J, The role of thrombosphttp://dx.doi.org/10.1016/j.ijcard.2013.04.139

the lectin-like β-sandwich. For additional information, readers are re-quested to refer to previous reviews which have given extensive ac-counts of the structure of the TSP family members [12,13].

3. TSP-1 receptors

The conflicting and often opposing effects of TSP-1 reported invarious cell types may be due to the presence of multiple domainsin the TSP-1 sequence which can interact with an array of specific cel-lular receptors (Figs. 1 and 2). There are a number of cellular recep-tors for TSP-1, including CD36, proteoglycans, several integrins andCD47, which is the integrin associated protein (IAP) (reviewed in[4]). The heparin binding domain present in the N-terminal of TSP-1binds to heparin sulphate proteoglycans [14], whereas the peptide se-quence CSVTCG present in the TSR1 of TSP-1 interacts with CD36 [15].The RGD sequence of the TSR3 interacts with a variety of integrin βsubclass receptors [16] and the C-terminal cell-binding domainbinds to CD47 [17]. Vascular cells have numerous integrins that rec-ognises TSP-1 including α3β1, αIIbβ3, αvβ3, α4β1 and α5β1 [4].TSP-1 also interacts with fibronectin and fibrinogen and binds tocomponents of the fibrinolytic system such as plasminogen, uroki-nase and its inhibitor plasminogen activator inhibitor (PAI)-1 and tocathepsin G and elastase [18].

The scavenger receptor CD36 which is the primary TSP-1 receptoris expressed on a variety of cells and is responsible for most of theanti-angiogenic and inflammatory functions exhibited by TSP-1[15,19]. The G-domain of TSP-1 which is specific to the TSP family is

s of TSP-1 are depicted with the important receptors that have been identified for theto the specific domains. The important peptide sequences are also localised to the do-ding domain (HBD), followed by an oligomerization domain containing the inter-chainology region (PC) also known as vonWillebrand type C (vWC) repeat and 3 type I (TSP

mal growth factor-like) repeats (TSR2), 7 type III (calcium binding) repeats (TSR3), theterminal domain that forms the lectin-like β-sandwich. The various components of theand are further stabilised by disulphide bonds and bound calcium [13]. Abbreviations:s = endothelial cells; VEGF = Vascular endothelial growth factor; VSMCs = vascularase; ↑ Pro or increase.



Fig. 2. Diagram to illustrate the amino acid sequences, the binding regions and the peptides derived from the sequences within the domains of TSP-1. The two main domains ofTSP-1 are depicted along with the amino acid sequences. The numbers above the TSP-1 monomeric subunit indicate the amino acid sequence in the signal peptide. The variousamino acid sequences are marked with coloured boxes and linked to the corresponding major binding regions, receptors or functions. The synthetic peptides derived from the var-ious amino acid sequences are also localised in the schematic diagram. Abbreviations: TSRs = thrombospondin repeats; TGF-β = transforming growth factor β; FGF = fibroblastgrowth factor. LSKL, ABT and DI-TSP are peptides.

3S.M. Krishna, J. Golledge / International Journal of Cardiology xxx (2013) xxx–xxx

involved in platelet aggregation and also enables the adhesion of ECsthrough the CD36 receptor [11]. CD47 is one of the most importantreceptor for TSP-1 and is a member of the immunoglobulin superfamily. CD47 is a component of the β3 integrin complex and isexpressed on T cells and polymorphonuclear cells and is involved inmaintaining immune tolerance [20]. TSP-1 binds CD47 to inhibit therelease of interleukin (IL)-2 [21]. Thus the availability of multiple re-ceptors to the various domains of TSP-1 results in complex cell-typespecific responses in different experimental contexts. These factorsmake it difficult to assign specific pathogenic roles to TSP-1.

4. Relevance of the TSP-1-TGF-β axis

The transforming growth factor (TGF)-β superfamily comprises anarray of multifunctional cytokines which are evolutionarily conservedand are involved in a range of diverse and often contradictory func-tions. TGF-β family members are secreted as inactive complexeswith latency-associated peptide (LAP), a protein derived from theN-terminal region of the TGF-β1 gene product [22]. The inactive com-plex of TGF-β and LAP is termed Small Latent Complex (SLC). SLCforms a complex with latent-TGF-β-binding protein (LTBP) whichplays a crucial role in the secretion and targeting of TGF-β to theECM [23](Fig. 3). The most crucial step in TGF-β regulation in vivo isthe extracellular activation of these complexes. Liberation of TGF-βfrom the latent complex is needed to activate TGF-β which enablesit to bind to its receptor. TGF-β is activated upon release from LAP


through a number of mechanisms and among those the most impor-tant is the binding of SLC to TSP-1 [24].

TSP-1 is a unique member of the TSP family due to its ability to ac-tivate TGF-β1. TSP-1 has 2 binding sites for TGF-β1, the amino acidsequence KRFK is involved in the activation of latent TGF-β1 andthe amino acid sequence GGWSHW binds to active TGF-β [25]. Themotif KRFK is situated between the first and second TSRs and thismotif is unique to TSP-1 (Fig. 2). TSP-1 modulates cytokine responsesand secretion of other growth factors through its interaction withTGF-β [26]. In addition to activating the latent complex of TGF-β,TSP-1 also controls the bioavailability of active TGF-β1 by controllingthe interactions of TGF-β1 with extracellular inactivators, such asα2-macroglobulin and decorin proteoglycans [11]. In addition, theexpression of TSP-1 is induced by several growth factors includingTGF-β1 and TGF-β2, indicating a positive feedback loop in the signal-ling pathway [27–30].

The role of TGF-β in the cardiovascular system is ambiguous. TGF-βhas been attributed a protective role since there is strong evidence of itbeing an anti-atherogenic and plaque-stabilizing factor (protective cyto-kine hypothesis). On the other hand TGF-β has been shown to exertsome pro-inflammatory effects. The relevance of TGF-β to CVD is proba-bly dependent on the exact pathological problem and context of its ex-pression. In some circumstances it is protective, for example within theunstable atherosclerotic plaque the pro-fibrotic and reparative actionsof TGF-βmay promote plaquematuration and stabilisation [31]. Similar-ly in the pressure-overloadedheart TGF-βpromotesmatrix preservation,preventing chamber dilatation, by regulating fibroblast phenotype [32].



Fig. 3. The classical TGF-β pathway. TGF-β is activated upon release from LAP through a number of mechanisms, and among those the most important is TSP-1 binding of SLC. Fol-lowing the binding of TSP-1 to SLC, TGF-β gets activated and complexes with TGF-β receptor (TB-R)-II. The complex of TGF-β-TB-RII heterodimerizes with co-receptor TB-RI forsubsequent phosphorylation of Smads in the cytoplasm. Signal propagation to the nucleus occurs through an array of Smad proteins namely, receptor regulated (R Smads-1, 2, 3,5, 8), common mediators (Co Smad4) and inhibitory Smads (I Smads-6 & 7). Eventually upon activation Smad2/3 forms heteromeric complexes with Smad4 and accumulates inthe nucleus where they interact with other transcription factors, co-activators, or co-repressors and the Smad binding element. When complexed with transcriptionalco-activators target genes are activated, and when associated with co-repressors, transcription is repressed. Abbreviations: TGF-β = transforming growth factor-β; LAP = latencyassociated peptide; SLC = small latent complex; LTBP = latent-TGF-β-binding protein; Smad = Sma and Mad Related Family; JNK = Jun NH2-terminal kinase; MAPK = mitogenactivated protein kinase; PI3K = phosphoinositide 3-kinase; SBE = Smad binding element; TF = transcription factors; COA = co-activators; COR = co-repressors.


In other situations, such as following angioplasty and MI [7,8], TGF-βmay promote restenosis and cardiac fibrosis, favouring development ofheart failure [33]. Experimental evidence also suggests that the TSP-1-TGF-β axis is a significantmediator of fibrotic complications importantin diabetes-induced cardiomyopathy [34,35]. Cell type-specific post-transcriptional regulation of the TSP-1-TGF-β pathway has been sug-gested as a potential mechanism important in diabetes associated car-diomyopathy [36].

5. In vitro and in vivo studies assessing the effect of TSP-1 on vas-cular cells

5.1. Vascular smooth muscle cells (VSMCs)

TSP-1 has been shown to be an autocrine growth factor for VSMCssince it augments the mitogenic effects of oestrogen growth factor orplatelet derived growth factor (PDGF) to promote proliferation ofVSMCs. Several in vitro and in vivo studies have suggested that TSP-1 in-duces VSMC proliferation (Fig. 4) [31,37–44] (Table 1). Elevated TSP-1levels have been associated with cardiac allograft vasculopathy inwhich the accumulation of VSMCs in the intima of large epicardial aswell as small intra-myocardial coronary arteries has been noted [43].

Isenberg JS and colleagues showed that the addition of exogenousTSP-1 at low dose stimulates growth of TSP-1 null VSMCs, where ashigher doses of exogenous TSP-1 are inhibitory [39]. It was also


shown that the C-terminal domain of TSP-1 and a CD47-binding pep-tide from this domain mediate an anti-proliferative effect on VSMC[45]. These results suggest that TSP-1 has both stimulatory and inhib-itory effects on specific VSMC responses through distinct receptors.Another elegant study by the same group showed that both exoge-nous and endogenous TSP-1 inhibit nitric oxide (NO) signalling inVSMC [46]. However, in the presence of NO, TSP-1 acts as a potent in-hibitor of VSMC responses through the CD36-dependent cGMP sig-nalling pathway [46].

Based on these observations it can be suggested that whenexpressed at normal low levels TSP-1 might be acting as a VSMC prolif-eration inducer, whereas in pathological conditions such as atheroscle-rosis where TSP-1 is upregulated, excessive TSP-1 expression could beacting as an anti-proliferative agent reducing VSMC density. Since pro-cesses such as atheroma formation and restenosis following percutane-ous vascular intervention involve the growth and migration of VSMCsinto neointimal lesions, the proliferation inducing effect of TSP-1 onVSMCs may contribute to restenosis and atherosclerosis progression.The role of TSP-1 in promoting VSMC proliferation may be of particularrelevance in diseases involving VSMC deficiency such as abdominal aor-tic aneurysm (AAA). AAA is typically associatedwith a reduction in me-dial VSMCdensity due to reduced proliferation and increased apoptosis,suggesting that activating the proliferation machinery using TSP-1might restore more normal aortic wall architecture. Thus the effects ofTSP-1 are likely to depend on the vascular pathology concerned.



Fig. 4. Overview of TSP-1 receptors, cellular pathways and cell types influenced by TSP-1. A schematic diagram showing the major signalling pathways and cell types activated byTSP-1. Major effects of TSP-1 include: (i) TSP-1 maintains the balance between MMPs and TIMPS maintaining matrix repair; (ii) TSP-1 releases Latent TGF-β from LAP and LTBP andactivates the TGF-β/Smad signalling pathway through the receptors TGFBR1 and TGFBR2; (iii) TSP-1 activates the CRT/LRP1 complex which activates cGMP and phosphoinositide3-kinase (PI 3-kinase) signalling pathways; (iv) TSP-1 activates various integrins and downstream pathways which affects the response of multiple cell types; (v) TSP-1 binds toone of its major receptor CD47 and activates various cells through cyclic guanosine monophosphate (cGMP) and nitric oxide (NO) signalling; (vi) TSP-1 binds to another majorreceptor CD36 which elicits various cellular response through Src family kinase (SFK) or Jun NH2-terminal kinase (JNK)-dependent pathways; (vii) TSP-1 inhibits angiogenesisthough VEGF and its receptor. The multiple signalling pathways influenced by TSP-1 affect multiple cell types in the vasculature which indicates the context dependent roleexhibited by TSP-1. Abbreviations: MMP = matrix metalloproteinase; TIMP = tissue inhibitor of matrix metalloproteinase; LAP = latency associated peptide; LTBP = latentTGF binding protein; TGF-β = transforming growth factor-β; CRT = calreticulin; LRP1 = low density lipoprotein receptor-related protein-1; NO = Nitric oxide; VEGF = vascularendothelial growth factor; VSMCs = vascular smooth muscle cells; Smad = Sma and Mad Related Family; ECs = endothelial cells; ECM = extracellular matrix.


In vitro studies suggest that VSMC proliferation induced by highglucose is dependent on TSP-1 upregulation [38]. It was reportedthat the activation of the hexosamine pathway by glucose catabolismin VSMCs upregulates TSP-1 production [38,41]. Increased TSP-1 ex-pression due to hyperglycaemia could activate integrin signallingleading to VSMC proliferation and migration to areas of vascular inju-ry, suggesting its role in restenosis [47]. A recent microarray study inVSMC indicated that hyperglycaemia amplifies the expression ofgenes induced by TSP-1 [48]. The results suggest that in hyper-glycaemic conditions, TSP-1 upregulates several genes and activatesmultiple canonical pathways associated with atherosclerosis and dia-betic vascular disease.

Increased migration of aortic VSMCs induced by TSP-1 has beenidentified both in vivo [49–51] and in vitro [31]. Studies indicate thatthe chemotaxis of VSMC depends on the interaction of TSP-1C-terminuswithα4β1 andα5β1 integrinswhich leads to the activationof an array of downstream signal transducers [49,52,53]. Most of theVSMC inhibitory effect of TSP-1 currently reported can be attributedto its role in inducing NO mediated VSMC relaxation [54–58]. Expres-sion of TSP-1 was found to occur as an early response to injury inVSMCs and deficiency of TSP-1 modified the VSMC phenotype. In vari-ous animalmodels, elevated TSP-1 expressionwas found to be associat-ed with the medial and neointimal VSMCs that also showed enhancedexpression of collagen and osteopontin, and decreased smooth muscleactin expression [31,40–42] suggesting an altered phenotype.


5.2. Endothelial cells (ECs)

ECs maintain a vasodilator, anti-thrombotic and anti-inflammatoryenvironment under normal conditions and are crucial in determiningvascular tone, leukocyte adhesion and VSMC proliferation [59]. Animalstudies suggest that TSP-1 is selectively upregulated in the microvascu-lar endothelium after ischaemia induced by TGF-β and basic fibroblastgrowth factor (FGF)-β [8] (Table 1). Frangogiannis NG and colleagues at-tribute the primary role of TSP-1 in the infarcted heart to its effects onthe inflammatory response [8]. However, the selective localisation ofTSP-1 in the infarct border zone suggests that microvascular endotheli-um may be capable of modulating the healing process throughdifferential TSP-1 expression. TSP-1 has been identified in early stageatherosclerotic lesions of hyper-cholesterolemic rabbits [40,42]. Neu-tralisation of TSP-1 by antibodies has been reported to acceleratere-endothelialisation and reduce the development of neointima aftercarotid artery denudation in rats [54].

TSP-1 has been shown to induce adhesion and chemotaxis of mu-rine lung capillary and bovine aortic ECs [44]. Cytoskeletal changesfollowing inflammation promotes the adhesion of immune cells toECs and leads to disruption of EC junctions eventually resulting in in-creased vascular permeability. Adhesion of leucocytes to ECs is aug-mented by TSP-1 inducing the cell surface expression of variousadhesion molecules [60]. Animal models with reduced TSP-1 expres-sion display leaky vessels with profound reduction of basement



Table 1Summary of studies assessing the association of TSP-1 with CVDs and the effects of TSP-1 on relevant cell types.

Cell types relevantto CVD formation

Model system studied Changes induced by TSP-1

Vascular smoothmuscle cells(VSMCs)

TSP-1−/− mice; Carotid artery ligation model TSP-1 deficiency lead to alteration of VSMC to a more fibrotic phenotype [31]Human aortic VSMCs High glucose concentration activates TSP-1 and promotes proliferation of

VSMCs [38]Endomyocardial biopsy samples of human cardiac allografts Higher TSP-1 mRNA levels were observed in cardiac myocytes and neointimal

VSMCs; Elevated expression of TSP-1 promotes proliferation [43]Allogeneic stimulation of T cells Allogeneic stimulation induces angiogenic growth factors andTSP-1 in T cells [43]Primary atherosclerotic tissue TSP-1 was identified in tissue sections; Expression of TSP-1 promotes prolif-

eration [42]Rat carotid artery after balloon angioplasty Elevated TSP-1 expression in neointima and media after injury; Elevated

TSP-1 expression promotes proliferation [37]Hypercholesterolemic lesions in rabbits TSP-1 promotes proliferation and intimal hyperplasia in hypercholesterolemic

lesions [40]Modified Boyden chamber Exogenous TSP-1 promotes chemotaxis [47]Quiescent bovine aortic VSMCs Exogenous TSP-1 induces MMP-2 activation and promotes VSMCmigration [51]VSMCs from TSP-1−/− mice Exogenous TSP-1 promoted proliferation in low doses and inhibited prolifer-

ation at high doses [39]Diabetic aorta of Zucker rats Glucose induced increased TSP-1 expression results in inhibition of prolifer-

ation and angiogenesis; Controls density of vasa vasorum [41]Endothelial cells(ECs)

Balloon-injured rat carotid artery Inhibition of EC proliferation and migration [54]TSP-1−/− mice and canine models of reperfusion infarction Ischemia induced an increase in TSP-1 protein and mRNA expression in mi-

crovascular endothelium [8]Human lung microvascular ECs Exogenous TSP-1 disrupts the endothelial barrier through EGFR/ErbB2 acti-

vation in a dose dependent manner [63]Carotid artery denudation in rats Neutralisation of TSP-1 accelerated re-endothelialisation [54]Murine lung capillary and Bovine aorta ECs TSP-1 inhibits proliferation [44]Human cardiac allografts Lower TSP-1 mRNA levels in ECs; Inhibits proliferation [43]Cultured postconfluent EC monolayers Exogenous TSP-1 induces increased EC permeability on the movement of al-

bumin across EC monolayers [62]Murine lung capillary ECs and Bovine aorta ECs Addition of TSP-1 induced adhesion and chemotaxis [44]Ocular pigment epithelial cells TSP-1 produced by T-regs is essential for immune regulation in the eye [83]

Fibroblasts TSP-1−/− mice; pressure overload heart TSP-1 regulates fibroblast phenotype and prevents left ventricularhypertrophy [32]

Age-associated heart failure in mice Age associated increase in TSP-1 expression [71]Human dermal fibroblasts Ultraviolet irradiation induced TSP-1 expression regulates type I pro-collagen

expression [70]Human fibroblasts in culture Fibroblasts secrete TSP-1 in response to Epac/Rap1 activation resulting in in-

hibition of angiogenesis [69]Transmembrane assay TSP-1 stimulated the migration of human fibroblast in a TGF-β1-independent

manner [67]Collagen gel contraction assay TSP-1 promoted fibroblast-mediated collagen gel contraction [66]

Leucocytes TSP-1−/− mice TSP-1 deficiency causes prolonged inflammation [80]Ischemia studies in TSP-1−/− mice and canine MIs TSP-1 deficiency results in enhanced post-infarction inflammatory response [8]Course of wound healing studies in TSP-1−/− mice TSP-1 deficiency results in prolonged persistence of inflammation and delayed

scab loss [84]TSP-1−/− mice TSP-1 deficiency results in enhanced pneumonia with macrophages and

neutrophil infiltration [16]Human primary CD4+ T cells and Jurkat T cells Addition of TSP-1 promotes T cell stimulation, migration and leukocytes re-

cruitment to the site of injury [82]T cell clones and lines from rheumatoid arthritis patients TSP-1 provides the costimulatory signal that is necessary for the activation of

autoreactive T cells [81]Human promyelocytic leukaemia cell line HL-60 Addition of TSP-1 stimulates chemotaxis and haptotaxis of polymorphonu-

clear leukocytes [86,93]Human monocyte cell line U937 TSP-1 enhances IL-6 release from macrophages by interaction with CD36 [19]Umbilical cord blood mononuclear cells and peripheral bloodmononuclear cells from normal healthy volunteers maintained in culture

TSP-1-CD47 interaction generates T-regs in response to inflammation [20]

TSP-1−/− mice Lack of TSP-1 resulted in reduced inflammatory cell infiltration [87]Human platelets or platelets from wild-type and CD47 null mice CD36/TSP-1 interaction could trigger the CD47-dependent pathway in platelet

adhesion on inflamed vascular endothelium under flow [72]Platelets Platelet interaction studies in TSP-1−/− mice Injection of human TSP-1 corrected the defective platelet activation [73]

Human platelets Exogenous TSP-1 induces platelet activation through CD36-dependent inhi-bition of cAMP/protein kinase A signalling cascade [74]

Platelets from integrin-associated protein (IAP) deficient mice TSP-1 acts via Integrin-associated protein (IAP)/CD47 to synergize with col-lagen in platelet activation [75]

Human platelets and mouse platelets deficient in FcR gamma chain TSP-1 induces platelet aggregation through the Fc receptorgamma-chain-associated signalling pathway and by agglutination [76]

Mouse platelets TSP-1 C-terminal peptide induces activation-independent agglutination ofplatelets [78]

Platelets from patients with severe asymptomatic and symptomatic CAD Higher surface expression of TSP-1 was found in circulating single as well asmonocyte-bound platelets from symptomatic CAD patients [79]

TSP-1 — thrombospondin-1; Tsp-1−/− — thrombospondin-1 knock out; VSMC — vascular smooth muscle cell; EC — endothelial cell; CAD — Coronary Artery Diseases.


membrane thickness, laminin content and recruitment of mural cells;indicating that TSP-1 regulates vascular integrity through collagen ma-trix assembly [61]. Cell culture studies using human and bovine


pulmonary ECs suggest that TSP-1maintains EC permeability and influ-ences trans-endothelial flux of macromolecules [6,62] (Table 1). A re-cent report indicates that TSP-1 disrupts endothelial barrier integrity




through the activation of EGFR signalling pathways in humanmicrovas-cular ECs in a dose, time and protein tyrosine kinase (PTK)-dependentmanner [63]. This is supported by the recent observations that TSP-1 in-hibits cell–matrix as well as cell–cell interactions in ECs, which are me-diated by various factors such as EGFR, FGF and vascular endothelialgrowth factor (VEGF), suggesting the involvement of multiple signal-ling pathways activated by TSP-1 [6,64]. The opposing effect of TSP-1in modulating permeability may be because ECs can produce as wellas respond to TSP-1 and since it can recognise multiple EC receptors.Thus at times TSP-1 induces opposing biological responses (Fig. 4).

5.3. Fibroblasts

Fibroblasts are important matrix producing cells which are stronglyregulated by their environment with effects on the healing process invascular tissues. Collagen, which gives tensile strength to tissues, is de-posited by fibroblasts upon stimulation by TGF-β1 [65]. Myofibroblastsdifferentiate from fibroblasts and are associated with scar formationand previous studies indicate the role of TGF-β1 in stimulatingmyofibroblast differentiation (Fig. 4). An interesting study by Xia Y etal. [32] suggests that TSP-1 expression is an essential regulator of fibro-blast phenotype and prevents left ventricular hypertrophy in pressureoverloaded hearts. TSP-1−/− mice have cardiac hypertrophy andcardiomyocyte injury which is accompanied by an increase in themyofibroblast density and the presence of functionally defective fibro-blasts that exhibit impaired differentiation and concomitant reductionin collagen content [32]. Thus TSP-1 appears crucial in regulating thephenotype of fibroblasts by modulating TGF-β1 pathway signallingbased on some in vivo [32] and in vitro [66] experimental studies. How-ever, TSP-1 induced fibroblast migration and collagen expression havebeen suggested to be independent of TGF-β1 [67,68] (Table-1). In trans-membrane experiments, the effects of TSP-1 on fibroblast migration areTGF-β1-independent since blocking antibodies to TGF-β1 had no effecton TSP-1-stimulated migration of fibroblast [67]. Furthermore, TSP-1has also been shown to bind to calumenin in the presence of calciumand calumenin has been shown tomodulate the patterns of protein ex-pression and cell cycle of fibroblasts further supporting the TGF-β1 in-dependent role of TSP-1 in the modulation of fibroblast phenotype[68]. In addition to responding to TSP-1,fibroblasts have also been iden-tified to secrete TSP-1 in response to activation through the cyclic aden-osine 5′-monophosphate (cAMP) pathway leading to the inhibition ofangiogenesis [69].

TSP-1 has been identified to play a major role during cardiac fibro-sis and diabetic cardiomyopathy, mainly by stimulating an increase incollagen type III expression [35]. A direct effect of TSP-1 in increasingthe expression of type I procollagen was reported in human dermalfibroblasts exposed to ultraviolet irradiation [70]. Increased TSP-1 ex-pression has been identified in age-related heart failure in mice dueto the decrease in expression of their targeting micro RNAs [71].These expression changes occurred specifically in cardiomyocytesand were absent in cardiac fibroblast supporting the cell type specific-ity of TSP-1 expression [71]. Reports from various animal models,such as a rat MI model, suggest that TSP-1 expression correlateswith the area of fibrosis [8,9] (Table 1). These observations in myocar-dium are very interesting and further research is needed to establishwhether TSP-1 could be playing a similar role at other sites in the car-diovascular system.

5.4. Platelets

TSP-1 is secreted from α-granules of platelets upon their activationand plays a major role in platelet–endothelium interactions. It hasbeen shown that both free and endothelium bound TSP-1 can mediateplatelet–EC association involving receptors such as CD47 and CD36(Fig. 4) [72]. TSP-1 also protects endothelium-bound vWF fromA-disintegrin and metalloproteinase with thrombospondin motifs


(ADAMTS)-mediated degradation, enhancing the dynamic recruitmentof platelets into developing thrombi [73]. TSP-1–CD36 interaction leadsto the activation of the Src family kinase-dependent pathway thatmod-ulates cAMP generation resulting in an increased platelet aggregationand arrest under flow [74]. This observation was supported by studiesshowing that synthetic peptides based on the C-terminal of TSP-1 stim-ulate platelet spreading on immobilised fibrinogen, and induce plateletaggregation through signalling complexes involving an array of tyrosinekinases [75–77]. TSP-1 also modulates platelet inhibitory pathwaysstimulated by endothelium-derived mediators. Under physiologic con-ditions, the release of TSP-1 upon platelet activation blunts variousinhibitory cyclic nucleotide pathways, which in turn reduce the thresh-old for platelet activation, facilitating the rapid capture of platelets atsites of vascular injury. This is achieved by increasing the availabilityof TSP-1 binding sites by the augmented surface expression of CD36 fol-lowing platelet activation by thrombin [78]. Enhanced TSP-1 expressionhas also been identified in circulating monocyte bound plateletssuggesting the role of TSP-1 in platelet andmonocyte rosette formationduring the progression of atherothrombotic cerebral ischaemia [79].The potential pathophysiologic importance of TSP-1 signalling path-ways in modulating platelet activity at the sites of vascular injuryneeds further investigation in the context of various CVDs.

5.5. Leukocytes

TSP-1 is believed to play an important role in reducing inflamma-tion by interacting with the CD47 receptor mainly expressed on Tcells and polymorphonuclear cells (Fig. 4) [80]. CD47 has a crucialrole in T cell stimulation, migration and leukocyte recruitment tothe site of injury [81,82]. TSP-1 promotes the activation of thymus de-rived T regulatory cells (T-regs), which are crucial in maintaining im-mune tolerance and maintain a suppressive phenotype when theCD47 receptor is stimulated [20]. TSP-1 is produced in the lymphnodes mainly by mature dendritic cells and induces conversion ofnaive T cells into T-regs that downregulate the inflammatory process[20]. In the presence of neutralising antibodies against TSP-1, T-regsfail to suppress activation of bystander T cells indicating that TSP-1has a crucial role in T-reg generation and function [83]. Thus, itseems that TSP-1 when released in the context of an inflammatory re-sponse promotes the generation of peripheral T-regs that control theinflammatory process and avoid collateral damage induced by self orforeign antigen. The anti-inflammatory role played by TSP-1 is highlyrelevant in CVDs such as atherosclerosis and AAA, since inflammationis believed to play an important role in the pathogenesis of thesediseases.

Several studies have shown that TSP-1 plays an important role inthe recruitment of monocyte and macrophages to sites of tissue inju-ry or inflammation [84–87]. TSP-1 also provides the co-stimulatorysignal that is necessary for the activation of antigen presenting cells(APCs) which is mediated by the interaction of TSP-1 with CD36[88]. The characteristic focal expression of TSP-1 on APCs such asmacrophages and fibroblast-like synoviocytes also supports the roleof TSP-1 in the expansion of tissue-infiltrating T-regs [81,88]. TSP-1modulates various functions of monocytes such as the enhanced ex-pression of IL-6 [19] and promotes haptotaxis and chemotaxis inmonocytes and neutrophils [85,89,90].

6. Studies assessing the effect of TSP-1 on mechanisms relevant toCVDs

6.1. Thrombus formation

TSP-1 promotes the formation of platelet and monocyte rosettes inpatients with severe coronary artery stenosis and atherothrombotic ce-rebral ischaemia [79]. Localisation of TSP-1 protein was reported in cal-cified atherosclerosis lesions [91]. A hallmark of the TSPs is their ability



Table 2Studies assessing the role of TSP-1 in various mechanisms implicated in the initiation and progression of CVDs.

Mechanismsrelevant to CVDformation

Model system studied Effect of TSP-1

Thrombosis Tsp-1−/− mice; vascular injury model TSP-1 deficiency promotes thrombus formation [93]Tsp-1−/− mice Injection of human TSP-1 restored embolization of thrombus in a shear field showing normal

platelet aggregation but defective thrombus adherence [73]Thrombocytes TSP-1 binds to Calumenin and is involved in the pathophysiology of thrombosis [91]Patients with severe coronary artery stenosis andprogressive atherothrombotic cerebral ischemia

Circulating and monocyte bound platelets showed significantly enhanced expression of TSP-1followed by formation of platelet and monocytes rosettes [79]

Tsp-1−/− mice TSP-1 deficiency leads to failure of platelet aggregation in response to thrombin in thepresence of exogenous NO [112]

Serum and intraluminal thrombus from AAA patients Serum TSP-1 levels were negatively associated with AAA [99]Matrix remodelling Human breast and prostate cancer cell lines Exogenous TSP-1 increased TIMP-1 protein expression [105]

Tsp-1−/− mice, canine and murine models ofreperfused infarction

Tsp-1−/− mice had more extensive post-infarction remodelling with increased macrophageand myofibroblast density [8]

Binding assays using Bovine aortic VSMCs TSP-1 induced activation of MMP-2 through transcriptional and post-translational mechanisms [51]Renal ablation model Enhanced TSP-1 expression in a fibrotic remodelling process [101]Epithelial cells in the proximal renal tubules TSP-1 induces matrix synthesis by high glucose concentrations [30]Bovine aortic EC collagen gel cultures TSP-1 stimulates the activity of MMP-9 [102]

Endothelialdysfunction

Tsp-1−/− mice;Aortic VSMCs

TSP-1 inhibits NO-stimulated cGMP synthesis in VSMCs and downstream effects on cell ad-hesion [57]

Tsp-1−/− mice skin grafts Antibody blockade of TSP-1 and antisense morpholino oligonucleotide suppression of CD47increased graft survival [56]

Cutaneous porcine flaps Blocking TSP-1 and CD47 signalling increases ischaemic tissue survival [112]Human vascular ECs and human aortic VSMCs TSP-1 peptide ABT-510 modulates fatty acid uptake into vascular cells and downstream NO/

cGMP signalling [108]Tsp-1−/− mice TSP-1 deficiency promotes elevated basal tissue cGMP levels; increase in regional blood flow

in response to NO challenge [55]Tsp-1−/− mice TSP-1 deficiency causes smaller plasma vWF multimer size [98]

Angiogenesis ECs TSP-1 and CD36 expression were downregulated under in vitro shear stress in a force- andtime-dependent manner [117]

Mammary tumour-prone mice that either lack orspecifically overexpress TSP-1 in the mammary gland

Absence of TSP-1 resulted in increased association of vascular endothelial growth factor(VEGF) with its receptor and higher levels of active MMP-9 [119]

Human microvascular ECs TSP-1 transcription is upregulated after balloon injury [138]Peripheral arterial disease patients (PAD);Matrigel-plug assay in Tsp-1−/− mice

TSP-1 is a potential biomarker of PAD and human endothelial colony-forming cell -inducedangiogenesis [103]

Tsp-1−/− mice;Carotid artery ligation model

TSP-1 deficiency results in delayed and dysregulated VSMC activation and reduced neointimaformation on mild vascular injury [31]

Invasion assays using bovine aortic VSMCs TSP-1 induces MMP-2 activity; TSP-1 is critical in VSMC migration [51]Boyden chamber and transwell assays COOH domain of TSP-1 molecule is responsible for VSMC chemotaxis and migration [50]Modified Boyden Chamber Assays TSP-1 increased migration of Rat aortic VSMC [49]Antibody blockade of TSP1 in VSMCs and p21-deficientmouse embryonic fibroblasts

Blockade of TSP-1 facilitated re-endothelialization and reduced neointimal lesion formationafter balloon denudation [54]

Tsp-1−/− mice TSP-1 deficiency leads to reduced apoptosis in ECs through Fas or FasL pathway [123]Apoptosis Tsp-1−/− mice TSP-1 deficiency leads to defective CD47 signalling mediated T cell apoptosis [80]

Apc(Min/+) mice; multiple intestinal neoplasia model Absence of TSP-1 results in reduced apoptosis leading to increased intestinal adenoma growth [126]Nude mice with B16/F10 melanoma tumours Mice treated with TSP-1 showed increased EC apoptosis [5]Human dermal microvascular ECs;SCID mice

TSP-1 mediates apoptosis and inhibits angiogenesis with increased Bax expression anddecreased Bcl-2 expression and processing of Caspase-3 into proapoptotic forms [122]

Cultured ECs TSP-1 antagonises VEGF-driven Akt survival signalling [130]

TSP-1 — thrombospondin 1; NO — nitric oxide; AAA — abdominal aortic aneurysm; TIMP-1 — tissue inhibitor of matrix metalloproteinase, MMP — matrix metalloproteinase; VSMC —

vascular smooth muscle cell; EC— endothelial cell; VEGF— vascular endothelial growth factor; vWF — von Willebrand Factor; SCID mice — severe combined immunodeficient mice.


to bind large numbers of calcium ions and each molecule of TSP-1 bindsan average of 35 calcium ions [92]. Genetic analysis suggests that a mis-sense polymorphism in TSP-1 at amino acid 700, causing an asparagine-to-serine substitution (N700S), alters its calcium binding properties andenhances platelet aggregation and thrombus formation [12]. Tsp-1 defi-cient (Tsp-1−/−) mice develop significant thrombus at the site of arteri-al balloon injury [93]. This suggests the involvement of TSP-1 incontrolling thrombus growth following intra-arterial injury in areas ofpredicted high turbulent flow (Table 2). Tsp1−/− mice with vascular in-jury show normal platelet aggregation but defective adherence ofthrombus which gets detached and flows in the direction of bloodflow [73]. After being released from activated platelets, TSP-1 is incorpo-rated into the fibrin network of clots and enhances platelet aggregationby facilitating a bridge between aggregating platelets via binding to fi-brinogen [94–96]. Photochemical injury of intestinal blood vessels hasbeen reported to induce massive thrombus embolization in Tsp1−/−

mice compared to wild type mice suggesting an important role forTSP-1 in thrombus anchorage to the vessel wall [73]. This suggeststhat TSP-1 restores embolization of thrombus in a shear field, and


TSP-1 can protect vWF from degradation, thus securing platelet tether-ing and thrombus adherence to inflamed and injured endothelium.Themodulation of vWF by TSP-1 also plays a role inmaintaining the ho-meostasis of the vascular system, since high levels of vWF promote en-dothelial dysfunction and hyper-coagulability [97]. TSP-1 has beenshown to reduce the activity of vWF by reducing its multimer size bycleaving the disulphide bonds that holds the multimers together[11,97]. The average vWF multimer size of Tsp-1−/− mice was foundto be significantly smaller than that of wild type mice and TSP-1 wasalso observed to slow the rate of vWF proteolysis [98].

Thrombocytes also express TSP-1 which functions as both an auto-crine and paracrine activator [91]. Expression of Tsp-1 is highly rele-vant in the context of resting platelet adhesion to inflammatoryvascular endothelium since they additionally play a role in secondarythrombus formation [72]. A recent report from our group identifiedTSP-1 in thrombus conditioned media [99]. The result suggestedthat the intraluminal thrombus selectively sequesters proteins ratherthan actively releasing them and serum TSP-1 levels were negativelyassociated with AAA. Increased TSP-1 levels could be a compensatory




mechanism and acts as a limiting factor for the immune response thusplaying a role as a biomarker of the restorative process taking placeduring disease development.

6.2. Matrix remodelling

TSP-1 was found to be upregulated following MI in experimentalmodels and has been implicated in regulating cellular interactionsand promoting matrix reorganisation (Table 2) [8,100,101]. TSP-1also plays an important role in the induction of matrix synthesis byhigh glucose concentrations in epithelial cells in the proximal renaltubules [30]. TSP-1 influences the activation of several proteolytic en-zyme families, including MMP-9 production in bovine aortic ECs[102] (Table 2). TSP-1 also upregulates MMP-2 by transcriptionaland non-transcriptional mechanisms and in vitro data suggests thatMMP-2 activation by TSP-1 is crucial for VSMCmigration [51]. Animalmodel studies also show defective upregulation of MMP-2 activity inthe absence of TSP-1 and reduced neointima formation on mild vas-cular injury [31,103].

Contrasting reports from in vitro assays suggest that the presence ofintact TSP-1 and its TSR domain inhibits MMP activity [104]. Thefunction of MMP is maintained by the balance between the enzyme andits physiologic inhibitor TIMP, and it is reported that TSP-1 up-regulatesTIMP-1 expression [105]. The expression of TIMP-1 has been reportedto be inhibited by antibodies against the TSR1 domain of TSP-1 furthersupporting that TSP-1 promotes TIMP-1 secretion. The heparin bindingdomain of TSP-1 increases MMP-1 andMMP-9 activities and down regu-lates TIMP-2 levels. On the other hand, the carboxyterminal fragment ofTSP-1 is reported to inhibit MMP-2 andMMP-9 activities and upregulateTIMP-2 expression [106]. These observations in rabbit corneal ECs, werelater on confirmed in ECs isolated from bovine heart indicating that thecontradictory results of TSP-1 on MMP regulation is due to the involve-ment of different domains of TSP-1 [107]. The various fragments originat-ing from the respective TSP-1 domain switch the EC phenotype bytargeting themitogen activated protein kinase (MAPK) signalling cascadeexerting opposing effects onMMP/TIMP balance thereby modulating thematrix reparative process [107]. Recently it was shown that theN-terminal domain of TSP-1 binds to the cell surface via calreticulin(CRT) and regulates collagen expression and fibroblast organisation invitro during tissue remodelling [68]. TSP-1 stimulates collagen synthesisvia the TSP-1/CRT/low density lipoprotein receptor-related protein 1(LRP1) co-complex through the PI3-Akt pathway [68], supporting theTGF-β independent role of TSP-1 in matrix remodelling in CVDs. Thus itis evident that bymaintaining theMMP–TIMPbalance and through inter-action with multiple mediators, TSP-1 plays a crucial role in maintainingmatrix repair and ECM turnover through multiple pathways in variousCVDs.

Table 3Summary of human studies reporting TSP-1 expression levels in CVDs.

Disease condition Study design

Atherosclerosis TSP-1 level is elevated in plasma of patients with CAD andCalcified aortic valve disease TSP-1 expression is elevated in human aortic valvesIschaemic heart diseases TSP-1 level is elevated in serum within 24 h after the onsetChronic heart failure Circulating TSP-1 concentration is lowMicroarray analysis ofblood cells of MI patients

TSP-1 was identified as a candidate gene in predicting leftdysfunction

PAD patients TSP-1 is expressed at higher plasma concentrations in patiinadequate neovascularization in PAD

Intraluminal thrombus inAAA

Serum TSP-1 levels were negatively associated with diseas

TSP-1 — thrombospondin-1; CAD — coronary artery disease; DM — diabetes mellitus; MIaneurysm.


6.3. Endothelial dysfunction

During endothelial dysfunction the synthesis of vasodilators is re-duced and the level of vasoconstrictors is elevated. Endothelial dys-function in CVDs is characterised by the activation of variouspathways in ECs which alters the production/activity of vasodilatormediators such as NO, prostacyclin, endothelium-derived hyperpo-larizing factor (EDHF), and bradykinin, as well as vaso-constrictivemediators such as endothelin, angiotensin II and thromboxane [59].Direct studies assessing the role of TSP-1 in endothelial dysfunctionare limited; however several reports indicate the role of TSP-1 inNO modulation [55,57,108,109] (Table 2). At low levels, NO isanti-inflammatory, but in inflamed tissues the levels of NO increasewith a concomitant increase in blood flow, vascular permeabilityand angiogenesis [110]. The attenuating effect of TSP-1 on NO medi-ated blood vessel dilatation is attributed to its binding to CD47[56,111]. Gene silencing studies using CD47 antisense morpholino-oligonucleotides as well as antibody blockade of TSP-1 suggest thatthe down regulation of TSP-1 dramatically increases survival of softtissue subjected to fixed ischaemia [55,112]. TSP-1 has been identifiedas a crucial inhibitor of the NO-cyclic GMP signalling pathway in vas-cular cells which support the negative effect of TSP-1 on the NO path-way in vascular cells [46,113,114].

TSP-1 increases regional blood flow in ischaemic tissues therebyregulating blood pressure and cardiac response to vasoactive stress.Tsp-1−/− mice have increased shear-dependent blood flow andgreater endothelium dependent vasorelaxation compared to wildtype animals, suggesting that TSP-1 is crucial in maintaining arterialtone [115]. Tsp-1−/− mice demonstrate elevated basal tissue cGMPlevels and increased regional blood flow in response to NO challenge[111]. Thus, in vascular cells, endogenous TSP-1 limits cGMP levelsconsistently and antagonises NO to maintain blood pressure andplatelet homeostasis.

6.4. Angiogenesis

TSP-1 is upregulated after vascular injury in experimental models[7] and within human restenotic arteries [37,40,54,116]. Severaltranswell assay studies have shown that TSP-1 inhibited migrationand chemotaxis of ECs (Table 2) [49–51]. TSP-1 has however been at-tributed an anti-angiogenesis role in the vascular wall. The angiogen-esis inhibition by TSP-1 is brought about by the interaction of TSP-1and CD36 on ECs which leads to their apoptosis (Fig. 4) [117]. Studiesshow that circulating levels of TSP-1 are sufficient to limit angiogenicresponses by antagonising signals from various angiogenic factors[113]. The pro-collagen domain and the TSR sequences are identifiedas the sites with anti-angiogenesis function and the TSR was found to

Association ofTSP-1 with CVD

TSP-1 expression in diseasecondition

DM Positive Elevated TSP-1 levels in aortic tissues [133]Positive Elevated TSP-1 in calcified tissues [131,132]

of MI in humans Positive Elevated serum TSP-1 levels [134]Negative Decreased TSP-1 expression in circulation [135]

ventricular Positive Elevated Tsp-1 expression in blood cells [136]

ents suggesting Positive Higher levels in plasma [103]

e Negative Lower levels in serum [99]

— myocardial infarction; PAD — peripheral arterial disease; AAA — abdominal aortic




block VEGF inducedmigration of humanumbilical vein ECs [118]. TSP-1is also reported to attenuate the binding of VEGF with its receptor(VEGFR2), and thereby inhibits angiogenesis (Fig. 4) [119]. Interactionof TSP-1 with the CD47 receptor additionally inhibits angiogenesis byantagonizing NO signalling which was observed in various vascularcells [58] and animal models [56].

6.5. Apoptosis

Studies assessing human atherosclerotic plaques and plaques fromcholesterol-fed rabbits show a defective intra-plaque phagocytosis of ap-optotic cells [120]. Enhanced necrotic core formation in plaques alongwith elevated inflammation, accelerated atherosclerotic plaque matura-tion and vessel wall degeneration have been observed in Tsp-1−/− mice[121]. TSP-1has a significant role in EC apoptosis by increasing expressionof apoptotic proteins Bax and also downregulating VEGF mediated Bcl-2expression (Table 2) [122]. Tsp-1−/− mice have been reported to havesignificantly reduced apoptosis of ECs through alterations in the Fas orFasL pathways [123]. An increase in the rate of microvascular EC apopto-sis was observed in mice injected with human TSP-1 [5] and it has previ-ously been shown that TSP-1 and its mimetic peptide ABT-510 increaseFasL and induce apoptosis [124,125]. Mice deficient in adenomatouspolyposis coli (Apc-Min/+ mice) which also lack TSP-1, show reducedapoptosis further supporting the role of TSP-1 in maintaining normal ar-chitecture of the vascular system [126].

The majority of the apoptosis inducing roles of TSP-1 are attribut-ed to CD36 receptor binding (Fig. 4) which leads to the activation ofsignalling pathways including p59fyn, caspase-3-like proteases, andp38 MAPK [5]. However, TSP-1 signalling via CD47 is reported to in-duce caspase-independent type III cell death in leukocytes [80,127].A recent novel finding indicates that TSP-1 induces apoptosis mediat-ed by tumour necrosis factor (TNF) receptor-1 in microvascular ECsfrom human brain [128]. Another recent report by Ren and col-leagues, shows that the TSR3 domain of TSP-1 induces apoptosis inprimary ECs by up-regulating the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 in a CD36 and Jun NH2-terminal kinase (JNK)-dependent manner leading to the activation of

Table 4Summary of human genetic studies assessing TSP-1 polymorphisms in CVDs.

Disease condition Study population Study design

CAD Case−control SNP study in WhiteAmericans; b45 years in men or>50 years in women

398 families; cases = 352 & c

Case–control study in SouthIndians

Cases = 511 & controls = 522

MI Case–control SNP study in ChineseHan population

Cases = 302 MI patients (228& Controls = 337 (232 men &

Case–control SNP study of MI inItalians;b45 years

Cases = 1425 individuals withfirst episode & controls = 142

SNP study of acute MI, Age b 60 inChinese Han population


Case–control SNP study in WhiteAmericans; b45 years in men or>50 years in women

398 families; cases = 352 & c

Case–control study in SouthIndians


Meta analysis of case–controlstudies; Southern Germans

Cases = 3657; age 64 years &age 60 years

CVD risk throughoutlife course

Risk of CVDs across the life coursein Adelaide and Auckland

Cases = 2123 Caucasian womendelivered a small for gestationalcontrols = 1185 Uncomplicated

Abbreviations: SNP — Single nucleotide polymorphism, CAD — Coronary Artery Disease; M


both intrinsic and extrinsic apoptotic machineries [129]. TSR3 wasalso found to attenuate the Akt survival pathway, indicating that TSP-1plays a critical role in regulating the pro-apoptotic signalling pathwaysthat control growth and death of ECs. Recently it was shown that TSP-1antagonises VEGF-driven Akt survival signalling mediated by the re-cruitment of Fyn to membrane domains containing CD36 in EC, butwhen TSP-1 is absent, an opposing Src recruitment contributes toVEGF-driven Akt phosphorylation and capillary survival [130]. ThusTSP-1 induces apoptosis in cells of various lineages by virtue of its inter-action withmultiple receptors leading to the activation of cell type spe-cific signalling pathways.

7. Evidence of TSP-1 as a biomarker in human CVDs

Table 3 summarises previous studies assessing TSP-1 expressionlevels in CVDs. TSP-1 expression is constitutively present in aorticvalves [131] and higher levels of TSP-1 have been reported in largeblood vessels with atherosclerotic lesions [132] and also in the presenceof diabetes mellitus [133]. Studies of ischaemic heart disease indicatethat after the onset of MI, TSP-1 appears in the serum within 24 h[134]. A study in chronic heart failure patients to assess the relationshipamong circulating markers of inflammation, endothelial dysfunctionand angiogenesis, suggested that decreased TSP-1 concentrationswere associated with endothelial repair [135]. Moreover, a novelstudy to identify new prognostic biomarkers in MI patients, using acombined protein interaction networks and microarray analysis ap-proach using blood cells, identified TSP-1 as a candidate genewith a po-tential to significantly predict left ventricular dysfunction supportingthe potential of TSP-1 as a biomarker in CVDs [136]. A recent report sug-gests that TSP-1 is expressed at higher plasma concentrations in periph-eral arterial disease (PAD) patients suggesting elevated plasma TSP-1expressionmodulates endothelial progenitor cell angiogenic propertiesand contribute to the inadequate neovascularisation in PAD [103]. Fur-thermore, elevated TSP-1 levels have been associated with cardiacallograft vasculopathy [43] as well as with the fibrotic response inhuman cardiac allografts [137]. Even though TSP-1 expression wasupregulated in the various disease conditions mentioned above, the

THSB1, SNP status

ontrols = 418 r228262; A > G;OR = 7.32 (95% CI 0.88–61.2);p b 0.05 [139]r228262,OR = 1.68 (95% CI 0.927–3.055);p = 0.087 [142]

men & 74 women)105 women)

A2210G, OR = 2.82(95% CI 0.54–14.64); p = 0.37Unrelated to MIG1678A, AA genotype, OR = 1.53 (95% CI 1.08 − 2.16); p = 0.016AA genotype was a significant risk factor for MI [143]

MI who survived a5

r228262; A > G;Homozygous, OR = 1.9 (95% CI), p b 0.05,Heterozygous, OR = 1.4 (95% CI), p b 0.01 [140]N700S; GA vs. AA; OR = 2.48 (95% CI, 0.48–12.86);p = 0.46 unrelated to MI [144]

ontrols = 418 r228262; A > G;OR = 8.66 (95% CI, 0.96–78.1),p b 0.05 [139]Patients who were homozygous for the THBS1 variant (SS) hadthe highest OR for MI (8.16) and a significantly lower plasmalevel of THBS1 than other genotypes

; r228262, A > G; OR = 1.84 (95% CI 0.846–4.007); p = 0.124[142] Genotypes had no impact on circulating TSP-1 levels

controls = 1211; Adj OR = 1.12 (95% CI, 0.93–1.35) No significant risk for theminor allele carriers [141]

, 216 (10.2%)age infant &pregnancies

A2210G associates with small for gestational age infants;Paternal adjOR, 1.4 (95% CI 1.0–2.0) & neonatal adjOR, 1.8 (95%CI, 1.1–2.7) [145]

I — Myocardial Infarction, CVD — Cardiovascular diseases.




exact mechanism of the upregulation of expression is not yet well un-derstood. As evident from the available literature, TSP-1 expressioncould originate from thrombin-induced platelet activation due to its re-lease from α-granules and in addition, many vascular cell types alsoconstitutively secrete and express TSP-1 [138]. The elevated TSP-1 ex-pressionmight be amarker for the reparative process happening duringdisease progression.

8. Studies assessing the associations of human TSP-1 polymor-phisms and CVDs

Only 6 studies assessing single nucleotide polymorphisms (SNPs)in the THSB1 gene (Enterez Gene ID: 7057; Chromosome 15q15) inpatients with CVDs have been previously reported. A single codingSNP (rs2228262; 2210A > G; Asn > 700 > Ser) has been associatedwith coronary artery disease (CAD) and MI [96] (Table 4). The studyby Topol et al., included 352 cases and 418 controls and showed astrong association of the SNP rs2228262 with CAD (OR = 7.32, 95%CI, p b 0.05) and MI (OR = 8.66, 95% CI, p b 0.05) in their patient co-hort [139]. Lower plasma TSP-1 levels observed in the patients corre-lated with the highest risk genotype and support a potentialbiological link between TSP-1 variation and early-onset CAD. Anotherlarge case-control study involving 1425 individuals with prematureMI showed that the rs2228262 SNP in TSP-1 was a significant risk fac-tor for MI in both homozygous (OR = 1.9, 95% CI 1.1–3.3; p = 0.01)and heterozygous carriers of the S700 allele (OR = 1.4, 95% CI 1.1–3.3; p = 0.01) [140]. However, a subsequent meta-analysis usinglarger number of cases (n = 6388) did not reflect any strong associ-ations with MI [141]. Similar analysis in other ethnic populationsalso revealed conflicting evidence regarding the role of THSB1 inCVDs [142–144]. Overall, the two well designed studies identified astrong association of the SNP with CAD and MI, which was notreflected in the isolated studies in various ethnic populations involv-ing smaller patient numbers. The variation in outcomes by the geneticstudies assessing the THSB1 gene polymorphisms could be due to theintrinsic differences between the populations assessed, since it hasbeen shown that sample biases and confounders affect the results gen-erated by genetic association studies and may account for some of thediscrepant results. A recent report on the THSB1-2210A > G SNPshowed its association with small for gestational age pregnancies,

Table 5Table showing the peptides derived based on the sequences of various functional domains

Peptide Targeted functional domain ofTSP-1 sequence

Function

ABT-526 TSR1 Anti-angiogenic

ABT-510 TSR2 Pro-apoptotic

Anti-angiogenicAnti-angiogenic

Short portion of N-terminal end Anti-angiogenicAnti-apoptotic

Wispostatin-1 (WISP-1) TSR1 Anti-angiogenicVasculostatin (BAI-1) 5 TSR1 Anti-angiogenic

Lexatumimab (agonistantibody against TRIAL)

TSR1 Anti-angiogenic Anti-apop

D1-TSP D-isoleucylenantiomer Anti-angiogenicPre-procollagen homologyregion & propidine repeats

Anti-angiogenic

D1-TSPa Soluble D-isoleucylenantiomer Anti-angiogenicReduced microvessel densIncreased apoptotic index

Angiocidin Peptide that binds to TSP-1 Anti-tumour activityLSKL Peptide antagonist of TSP-1 Selectively blocks glucose

mediated TGF-β signalling

TSR1 — TSP-1 type specific repeats; EC — Endothelial cell; AngII — Angiotensin II; TGF-β; T


further suggesting that this SNP may be a risk factor for CVDs[145]. Hence, systematic studies assessing the role of various func-tional variants in the THSB1 gene in larger cohorts are necessaryto identify clearly whether genetic variations in this gene predis-pose to CVDs.

9. Therapeutic relevance of TSP-1

A number of TSP-1 peptide analogues have been generated (Fig. 2)and a lot of experimental data back up the potential of TSP-1 mediatedtreatments in various pathological conditions (Table 5) [2,146–152].Most of the therapeutic effects of the TSP-1 peptides appear to be dueto the anti-angiogenic, anti-proliferative and pro-apoptotic effects of var-ious TSP-1 domains which are of immense potential in diseases such ascancer [18,153,154]. A study using a peptide antagonist of TSP-1, LSKLwhich selectively blocks glucose and angiotensin II-stimulated TGF-βsignalling suggests the use of TSP-1 antagonists as a therapeutic strategytomodifyfibrotic complications in diabetes [34]. Studies in a ratmodel oftype 1 diabetes using the LSKL peptide demonstrated reduced cardiac fi-brosis, myocyte hypertrophy, and improved left ventricular functionsupporting the hypothesis that the blockade of TSP-1-dependent TGF-βactivationmay be a novel therapeutic strategy to prevent or reverse car-diac fibrosis [34]. Increased expression of TSP-1 was identified in mousemodels ofMarfan syndrome (MFS) [155]. Experimental studies assessingthe potential effect of TSP-1 antagonists are currently being carried out inmousemodels ofMFS [156]. However, in diseases inwhich TGF-β plays aprotective role, these therapeutic strategies have to be considered withcaution, since alterations in these mechanisms may have detrimentaleffects.

10. Future directions

The multitude of effects induced by TSP-1 in various cellular mech-anisms crucial in the maintenance of normal vasculature and its signif-icant role as a TGF-β1 regulator, makes TSP-1 a complex candidate fortherapeutic intervention. The role of TGF-β1 is still an area of debate,i.e. whether it plays a protective or detrimental role in various CVDs[157], however, the recent observations of the TGF-β independentrole of TSP-1 suggest an alternative mechanism for TSP-1 related inter-ventions in CVD. The recently revealed role for TSP-1/CD47 signalling in

of TSP-1, the functions of the peptides and the assay system studied.

Assay system

Phase I study- malignant tumour and NonHodgkins lymphoma in dogs [146]Inhibition of neovascularisation in rat cornea model [147]Induction of apoptosis of syngenic mouse epithelial cancer cells inC57BL6 mice [148]Advanced soft tissue sarcomas [152]Reduced growth of syngeneic lewis lung sarcoma in mice [147]Phase I and II clinical studies in lymphoma and osteosarcoma [18]Reduces proliferation, angiogenesis and increases apoptosis; Used intherapy of brain tumours [154]Inhibited migration and proliferation of ECs [149]Suppressed growth of malignant gliomas in rats and inhibitsregeneration of human dermal microvascular Ecs [150]

totic Inhibits progression of human colon cancer in nude mice and inducesapoptosis in human microvascular ECs [129]Inhibits lung metastasis in mouse models [151]Human microvascular ECs in culture [15]

Primary human bladder tumours [151]ity; Cancer cell lines [151]

Human colorectal xenograft models [153]and AngII Modified fibrotic complications of Type 1 diabetes in mouse models and

improved cardiac fibrosis [155]

ransforming growth factor-β.




limiting the NO pathway, and the role of calreticulin binding sequenceof TSP-1 in regulating collagen expression, cell migration, matrix degra-dation and reorganisation during tissue remodelling support the TGF-βindependent roles of TSP-1 and provide a novel target for CVD treat-ment. Given that TSP-1 has conflicting effects on the various celltypes, understanding the in vivo significance of TSP-1 signalling path-ways in specific tissues will aid in dissecting out the role of specificcell types in various CVDs. Because TSP-1 plays a role in controlling in-flammation and a basal level of TSP-1 is necessary for promoting ananti-inflammatory environment, optimal timing of therapeutic inter-ventions should also be identified to target the TSP-1-TGF-β axis inCVD patients. Hence more mechanistic studies using the Tsp-1−/−

mouse model and an array of Tsp-1 peptides are warranted. Systematicclinical studies assessing the relevance of TSP-1 gene variations are alsoneeded in an effort to understand the role of TSP-1 in the pathogenesisof CVDs.

Abbreviations used

CVD Cardio vascular diseasesMI Myocardial infractionECM Extra cellular matrixTSP/THSB ThrombospondinTSR TSP-1 type specific repeatsvWF von Willebrand FactorT-regs T regulatory cellsIL InterleukinsVSMCs Vascular smooth muscle cellsEGFR Epidermal growth factor receptorPDGF Platelet derived growth factorAAA Abdominal aortic aneurysmNO Nitric oxideECs Endothelial cellsTGF-β Transforming growth factor-βFGF-β Fibroblast growth factor-βVEGF Vascular endothelial growth factorMMP Matrix metalloproteinasesTIMP Tissue inhibitor of matrix metalloproteinasesPAD Peripheral arterial diseasesSNP Single nucleotide polymorphismsMFS Marfan syndromeCAD Coronary Artery Diseases

Acknowledgements

J.G. is supported by grants from the National Health andMedical Re-search Council [project grants 540404, 1003707, 1021416, 1020955;Centre of Research Excellence; fellowship 1019921], BUPA and Queens-land Government.

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The role of thrombospondin-1 in cardiovascular health and pathology

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