Human expression patterns: qualitative and quantitative analysis of thrombospondin‐1 under physiological and pathological conditions

Abstract Thrombospondin‐1 (TSP‐1), a matricellular protein and one of the first endogenous anti‐angiogenic molecules identified, has long been considered a potent modulator of human diseases. While the therapeutic effect of TSP‐1 to suppress cancer was investigated in both research and clinical settings, the mechanisms of how TSP‐1 is regulated in cancer remain elusive, and the scientific answers to the question of whether TSP‐1 expressions can be utilized as diagnostic or prognostic marker for patients with cancer are largely inconsistent. Moreover, TSP‐1 plays crucial functions in angiogenesis, inflammation and tissue remodelling, which are essential biological processes in the progression of many cardiovascular diseases, and therefore, its dysregulated expressions in such conditions may have therapeutic significance. Herein, we critically analysed the literature pertaining to TSP‐1 expression in circulating blood and pathological tissues in various types of cancer as well as cardiovascular and inflammation‐related diseases in humans. We compare the secretion rates of TSP‐1 by different cancer and non‐cancer cells and discuss the potential connection between the expression changes of TSP‐1 and vascular endothelial growth factor (VEGF) observed in patients with cancer. Moreover, the pattern and emerging significance of TSP‐1 profiles in cardiovascular disease, such as peripheral arterial disease, diabetes and other related non‐cancer disorders, are highlighted. The analysis of published TSP‐1 data presented in this review may have implications for the future exploration of novel TSP‐1‐based treatment strategies for cancer and cardiovascular‐related diseases.

has been termed matricellular proteins, and it represents an expanding group of molecules that includes over nine subfamilies of proteins that are increasingly recognized as playing important roles in homoeostasis and recently in diseases. 2 As the name implies matricellular proteins function to span the area between the cell surface and the matrix scaffold. Also important to the understanding of these molecules is that they do not impart strength to the matrix themselves, instead they can regulate matrix metabolism to alter the biomechanical properties of the extracellular matrix.
A quintessential and founding member of this group is thrombospondin-1 (TSP-1), a protein first identified in the particulate fraction of thrombin-activated platelets 3 and this fact being incorporated in its name. Like many secreted proteins post-development, TSP-1 is minimally detectable in health but rapidly up-regulated with injury and persists in chronic diseases, being found in the parenchyma as well as fluid compartments including the blood, 4 urine 5 and cerebrospinal fluid. 6 TSP-1 is trimeric, with each monomer about 130-150 kD, but the secreted protein is heavily modified by glycosylation and weighs over 450 kD ( Figure 1). 7 Secreted TSP-1 directly transduces signals through binding via discrete domains to cell-surface receptors including but not limited to CD47, CD36 and integrins. 8 Indirectly, TSP-1 regulates cell signalling through binding to other molecules such as enzymes and growth factors. 9,10 Consequently, the manifold roles of TSP-1 in modulating cell functions are concentration-and cell type-specific. Nonetheless, some trends have emerged. From the perspective of pharmacology, high concentrations of TSP-1 check the cell cycle in primary cells to impede selfrenewal and proliferation 11 and can, at certain concentrations, induce cell death. 12,13 These effects arise, in part, from the ability of TSP-1 to limit pro-growth signals and several key elements of metabolism. 14 Another essential feature of TSP-1 is its ability to control tissue repair and remodelling in response to injury and stress, a property enhanced by its important function to increase transforming growth factor beta (TGF-beta) activity. 15 In the central nervous system, TSP-1 is expressed and secreted by astrocytes and is a promoter of synapse formation as well as neuronal proliferation and differentiation. 16 During immune activation, TSP-1 has a supportive role and can increase the activation of inflammatory cells including monocytes, 17 dendritic cells, 18 macrophages 19 and T cells. 20 Conversely, in other settings such as during the resolution of inflammation, TSP-1 may act to suppress inflammation. 21,22 A fundamental and well-established property of TSP-1 is to limit endothelial cell (EC)-mediated angiogenesis by inhibiting the activity of vascular endothelial growth factor (VEGF) 23 and the pleiotropic signals of the gasotransmitter nitric oxide (NO). 24 In this way, TSP-1 adversely impacts angiogenesis and blood flow 25 and modulates blood pressure by limiting NO-mediated vasorelaxation. 26 In this latter capacity, TSP-1 is now recognized to alter cardiovascular responses in general. 25,27 In the light of its role to retard angiogenesis, TSP-1 is shown to suppress tumour growth and is often found down-regulated in the tumour microenvironment coincident with accelerated tumour invasiveness. 28 Increases in circulating TSP-1 expression have also been found to be positively correlated with patient survival in some cancers. 29,30 Expectantly, TSP-1-derived drugs have been developed in the interest of inhibiting angiogenesis and treating cancer. 31,32 However, the effect of TSP-1 in cancer is not simple, and it has been reported that TSP-1 can also support tumour growth and spread. 33 Interest has been growing in TSP-1 as a possible biomarker and an important contributor to human diseases, particularly in age-related and metabolic diseases. 34,35 At the same time, TSP-1 and several of its cell-surface receptors, notably CD36 and CD47, have and continue to be pursued as therapeutic targets. 36,37 Renewed appreciation of TSP-1 in the pathophysiology of diseases has encouraged research into the expression of this protein in different cells and body compartments. However, a systematic characterization and quantification of published TSP-1 expression data in diseases, both amount and rate of production, has not been previously approached. Such an analysis is important, and it can provide insight into the sometimes conflicting activity of this molecule and serve to guide therapeutic interventions that target pathways mediated by TSP-1; it is also important for systems biology computational studies of TSP-1. [38][39][40] Herein, we present the results of a systematic multilevel (cell, fluid, tissue) characterization of TSP-1 concentrations in people in health and disease, specifically in cancer and cardiovascular diseases, derived from analysis of the current literature. Interestingly, strong patterns emerge. In cancer, TSP-1 expression is surprisingly heterogeneous to the point of forestalling prediction as to its role in many of these cases. Conversely, in inflammatory and cardiovascular diseases, TSP-1 expression levels show a persistent trend to be significantly elevated compared to non-diseased subjects.
The correlation between dysregulated TSP-1 expressions in diseases

List of Main Topics
Thrombospondin-1 (TSP-1), a matricellular protein, is often induced at sites of injury and tissue remodelling, and it is secreted by circulating, stromal and parenchymal cells at very different quantities.
The major cell-surface receptors for TSP-1, namely CD36, CD47 and integrins, participate in important cellular processes including apoptosis, angiogenesis, blood flow, phagocytosis, migration and immune regulation.
TSP-1 is known to inhibit angiogenesis and endothelial cell survival.
Cancer patient data on TSP-1 in blood and cancerous tissues suggest that the patterns of TSP-1 expression compared to non-cancer controls are highly heterogeneous across different cancer types and that the correlations between TSP-1 levels in patients and survival are also cancer type-specific.   Beginning at the cell level, a literature search was conducted and TSP-1 secretion rates from different types of cells were analysed (Table 1). [44][45][46][47][48][49][50][51][52][53][54] Interestingly, secretion rates of TSP-1 protein from the stromal components (eg fibroblasts, ECs) are at least one to two orders of magnitude greater than those rates from cancer cells.
Among different types of stromal cells, ECs (represented by human umbilical vein ECs) produce and secrete TSP-1 proteins at very high rates. ECs usually occupy a fairly small portion (1%-2%) of the total cells in a tissue, but this percentage can be significantly higher in some highly vascularized tissues such as in certain tumours, lungs and hearts. 55,56 In this sense, modulating EC-specific pathways (eg via transcription factors, receptor activation and microRNAs) that  Table 1. 59 Still, production and secretion rates of TSP-1 would depend on various factors including the density of cultured cells and also the appropriate stimuli which are present in tissue environments in vivo but may not be contained in the culture media; therefore, the differences between cell-specific secretion rates outlined here should be interpreted in both quantitative and qualitative manners.
Further studies and measurements are needed to elucidate the potential correlations between in vitro (summarized in this review) and in vivo TSP-1 production capacities in the different cell types.

| Human cancers
The potential of circulating and tissue TSP-1 protein as diagnostic or prognostic markers for cancers, given its anti-angiogenic and proapoptotic properties, has been studied extensively. Table 2 summarizes the quantitatively measured TSP-1 protein levels in the plasma, serum, platelet and tissue of individuals with various cancer conditions.  Most measured values of plasma TSP-1 protein levels are in the range of a few hundred to a few thousand ng/mL, indicating that physiological TSP-1 concentrations are relatively low in the circulation. 86 It should be noted that data on soluble plasma and serum TSP-1 may be confounded by varying degrees of platelet activation that serve as a reservoir of pre-formed TSP-1 in alpha granules during sample acquisition and processing. 42 Nonetheless, it is worth noting that a large portion of the plasma TSP-1 data actually indicate an up-regulation of TSP-1 in patients with cancer compared to normal controls, especially in breast cancer, which might be considered counterintuitive to the well-established anti-angiogenic property of TSP-1. In the case of breast cancer (general disease and not in the context of any specific subtypes), four separate studies have found a consistent, significant increase in plasma or tissue TSP-1 protein in patients with cancer compared to healthy controls. 67,71,83,87 A strong positive correlation between plasma and intratumoural TSP-1 is observed, and patients with lymph node metastasis have significantly higher plasma TSP-1 compared to lymph node-negative patients.

| Cardiovascular diseases
In contrast to cancer, angiogenesis is often impaired and therefore desired in many age-related and cardiovascular diseases, especially in ischaemic vascular diseases such as coronary artery disease (CAD) and peripheral arterial disease (PAD). One hypothesis offered to explain the pathophysiology of CAD and PAD is that anti-angiogenic factors (eg TSP-1) may be highly up-regulated in the ischaemic tissue, in addition to the insufficient induction of pro-angiogenic factors (eg VEGF, NO). 39 (Table 3). Marked upregulations of TSP-1 have been observed in the various organs and tissues of patients with diabetes and also in animal models of diabetes. 99 This may be in part secondary to the known effects of high glucose on TSP-1 production. 100 In terms of disease outcome, strong negative correlations between plasma TSP-1 protein levels and patient survival have been observed for pulmonary hypertension, acute ischaemic stroke and end-stage renal disease, all conditions characterized by vasculopathy. [101][102][103] Interestingly, on the other hand, thrombospondin proteins including TSP-1 are involved in the unfolded protein response (also known as the endoplasmic reticulum stress response), and they are found to be induced and exert protective effects following myocardial injury in animal models, which adds another layer of complexity to the functions of the up-regulated TSP-1 in cardiovascular diseases. 104

| Correlations between TSP-1 and VEGF
Paired data on quantitative TSP-1 and VEGF protein expressions in cancers and PAD are shown in Table 4. While the trend of TSP-1 expression in cancers remains elusive, VEGF levels (plasma, serum, platelet, tissue) tend to be up-regulated in most cases. 105 Published data so far have not suggested any correlation between circulating levels of VEGF and TSP-1 in patients with cancer (  The cell-surface receptors that TSP-1 interacts with also have important therapeutic value in cancer and cardiovascular diseases. CD36 is a low-affinity receptor which recognizes the type I repeats of TSP-1 and is reported to be an activator of the apoptotic pathways in ECs and some cancer cells. [114][115][116] The first TSP-1-based therapeutic (ABT-510) was developed based on this interaction more than a decade ago. However, it failed to demonstrate clinical efficacy against metastatic cancers and did not move forward beyond phase II trials. 117,118 Similar to ABT-510, several other peptides that are derived from the type I repeats were shown to be anti-angiogenic in vitro. 119,120 In recent years, CD47, a high-affinity receptor which interacts with the C-terminal domain of TSP-1, has garnered attention. 121 Accumulating evidence has suggested that the TSP-1/CD47 axis could be a promising therapeutic target in cancer as well as in cardiovascular diseases. 32,36 Besides its well-established anti-angiogenic and anti-proliferative effects when engaged with TSP-1, 23 CD47 also participates in immune suppression of macrophages and is found widely overexpressed in different types of cancer. 122  has also been suggested that TSP-1 may trigger pro-survival and pro-migratory functions in cells through binding with some of its receptors. 9,45,126 Indeed, few studies have tracked TSP-1 protein expression concurrent with its receptor level expression. In terms of studying how TSP-1-mediated signal transduction contributes to diseases, it will be important to track both the ligand TSP-1 as well as its specific receptors in cell and tissue compartments.
Besides diseases, many natural biological processes can also contribute to the endogenous regulation of TSP-1. Preclinical studies in mice have demonstrated age-associated up-regulation of TSP-1 in kidney, 127 heart 128 and skin. 129 The fact that both TSP-1 and CD47 are significantly induced in the skin of aged mice and negatively act on blood flow may imply a deleterious role of TSP-1/CD47 axis in ageing and ageing-related complications. 95 In addition, diabetesinduced up-regulation of TSP-1 may contribute to ageing-related vascular rarefaction in the hearts of leptin-resistant mice, and loss of TSP-1 expression can attenuate this pattern. 130 Exercise is another factor that seems to control TSP-1 dynamics. Confirmed in both mice and humans, TSP-1 expression is greatly increased in skeletal muscles following active training and this is accompanied by an increase in VEGF expression. 131,132 Interestingly, a delay is observed between TSP-1 induction and VEGF induction (VEGF induction preceding TSP-1), which suggests endogenous feedback mechanisms through timely regulation of pro-and anti-angiogenic factors to sustain adequate but not excessive angiogenesis and blood flow during and after exercise. 133 Gestation status can also affect TSP-1 expression in the uterus, in which TSP-1 expression was shown to increase over the last few weeks before labour and peak during labour, 134 putatively where its role to promote platelet activation by resisting NO-mediated effects on platelets 135 and blood vessels 26 may have beneficial effects to limit haemorrhage.
The inconsistent pattern of TSP-1 expression observed in different types of cancer is reflected in the equally controversial role of tumour TSP-1 expression as a survival predictor. In accordance to its anti-angiogenic property, high tumour expression of TSP-1 is correlated with increased patient survival in colon, 136 lung, 137 bladder, 138 ovarian, 139 cervical 140 and gastric cancer. 109 However, high tumour tissue TSP-1 is also associated with decreased survival in patients with hepatocellular carcinoma, 81 breast cancer 141 and melanoma. 142 Along with the observation that high VEGF in cancer is usually associated with worse prognosis and increased metastasis, 106