DEFINITION
Thrombospondin 1, also known as THBS1 or TSP-1, is an homotrimeric glycoprotein with disulfide-linked subunits by 180 kD that in humans is encoded by the THBS1 gene. Thrombospondin 1 is a subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. This protein can bind to fibrinogen, fibronectin, laminin, type V collagen and integrins alpha-V/beta-1.
THE GENE
By in situ hybridization, Jaffe et al. mapped the THBS1 gene to human 15q15 (Fig. 1) and the cognate gene to mouse chromosome 2 (region F). Wolf et al. localized the THBS1 gene to 15q11-qter by Southern analysis of human-rodent somatic cell hybrids.
Figure 1. In chromosome 15 is located the THBS1 gene (15q15 region).
Wolf et al. showed that the type I repeating subunits of thrombospondin are encoded by symmetrical exons and that the heparin-binding domain is encoded by a single exon. The THBS1 message is encoded by 21 exons.
CHEMICAL STRUCTURE AND IMAGES
TSP-1 is a large, homotrimeric molecule (420 kDa). Each monomer consists of an interchain disulfide bond (S=S), procollagen homology domain (PC), Type I, II, and III repeats, first discovered in activated platelets. Each subunit comprises a 1152 amino acid residue polypeptide, post-translationally modified by N-linked glycosylation and fi-hydroxylation of asparagine Residues. TSP-1 is the best-studied member of the thrombospondin (TSP) family, which consists of five extracellular calcium-binding multifunctional proteins: TSP-1, TSP-2, TSP-3, TSP-4, and TSP-5. TSP-1 and TSP-2 are structurally similar, and they are expressed on the cell surface during physiological events. A variety of normal cells, including endothelial cells, fibroblasts, adipocytes, smooth muscle cells, monocytes, macrophages, and transformed cells such as malignant glioma cells, secrete TSP-1. TSP-1 binds to protein components of the extracellular matrix, such as fibronectin. By this way, TSP-1 is stored in the extracellular matrix where it folds and changes its conformation. TSP-1-specific domains bind to proteoglycans, membrane proteins such as integrins, and other matrix proteins expressed by a variety of cells.
TSP-1 contains an N-terminal globular domain that binds heparin, the type I , type II, and type III repeats , and a terminal globular domain. The structure of TSP-1 is schematically shown in Figure 1. The NH2-terminal, heparin-binding domain of TSP-1 interacts with low-density lipoprotein receptor-related protein (LRP1). LRP1 releases any metalloproteases already bounded to TSP-1, modulating the protease activity. This TSP-1 domain also binds heparin sulfate proteoglycans and a number of integrins that have an important function in angiogenesis, chemotaxis adhesion, and cell motility. All five members of the TSP family have the repeat domains type II and III, but only TSP-1 and TSP-2 contain the type I repeats. Type I repeats, also called thrombospondin structural homology repeats (TSRs), inhibit angiogenesis by activating CD36 and inducing apoptosis in endothelial cells. CD36 (also known as fatty acid translocase, FAT) is a glycosylated protein member of the class B scavenger receptor family. It plays an important role in multiple processes such as fatty acid and glucose metabolism. CD36 is found on the surface of diverse cell types and binds to many ligands, including TSP-1. It has been reported that upon binding with TSP-1, CD36 dimerizes, becoming actively involved in signal transduction. However, activation of CD36 as a monomer has also been reported. The adhesive and antiangiogenic functions of TSP-1 have been mainly attributed to its interaction with CD36.
TSP-1 is a major activator of transforming growth factor (TGFβ1). Indeed, it is the only member of the thrombospondin family that activates TGFβ1. TGFβ1 mediates wound healing, cell proliferation, extracellular matrix formation, and the immune response. This multifunctional cytokine is secreted to the extracellular matrix in its inactive form, by virtue of its noncovalent association with the latency-associated peptide (LAP).
The activating function of TSP-1 is due to the amino acid sequence RFK located in the TSR. TSP-1 releases TGFβ1 from its latent form when it interacts with the N-terminal region of LAP and binds the mature TGFβ1. This interaction results in the formation of a complex that involves conformational changes in TGFβ1, making it accessible to its receptor. LAP is crucial for TGFβ1 activation and regulates many of its functions; additionally, LAP has functions in inflammation independently of TGFβ1, such as the induction of chemotaxis of monocytes to injured tissues.
The type 3 repeats of TSP-1 are calcium-binding domains. They contain amino acid sequences that interact with the neutrophil elastase, and upon this binding these repeats activate neutrophils. These type 3 repeats also inhibit the binding of fibroblast growth factor to endothelial cells, reducing angiogenesis.
The COOH-terminal domain of TSP-1 binds to CD47, also known as integrin-associated protein. This domain also interacts with integrins such as β1 and βv6 integrins and actively binds to proteoglycans allowing cell adhesion and spreading. These and other interactions significantly affect angiogenesis, cell proliferation, and immune responses. TSP-1 binding with CD47 also regulates nitric oxide (NO), a biogas, quite important in both normal and pathological events. By modulating the effects of NO, the carboxy-terminal domain of TSP-1 has important function in vasodilation and chemotaxis. CD47 impacts angiogenesis to a large extent. This receptor inhibits NO as well as all its vascular functions even when TSP-1 is present at very low (physiological) concentrations. Analysis of wound bed vascularity at 72 hours after skin grafting from TSP-1 and CD47 null mice shows significant increased numbers of blood vessels. Most recently it has been reported that CD47 associates with the receptor of vascular endothelial growth factor (VEGFR2). However, the binding of CD47 with TSP1 or other ligands inhibits VEGFR2 phosphorylation and further angiogenesis (Thrombospondin-1: multiple paths to inflammation, 2011), (Current understanding of the thrombospondin-1 interactome, 2014).
The largest classes are extracellular matrix molecules, cell receptors, growth regulatory factors and proteases (Fig. 2). TSP-1 interacts with proteins and non-protein ligands (Fig. 3). Among the latter, TSP-1 characteristically binds calcium and glycosaminoglycans, including heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate.
Figure 2. Schematic representation of the main classes of TSP-1 interacting molecules.
Figure 3. Interaction of each domain of TSP-1 (monomer) with receptors and ligands. For many ligands, the binding site on TSP-1 has not yet been identified. *In parenthesis: number of bound calciums.
Protein Aminoacids Percentage
Figure 4. Bar graph showing relative content (percentage) of each amino acid expressed in Thrombospondin-1 protein.
CELLULAR FUNCTIONS
Extracellular and intracellular interactions
Most TSP-1 interactions occur in the extracellular space, within the extracellular matrix or in proximity of the plasma membrane. Recently however, intracellular interactions have also been described, opening up a completely new picture of TSP activity. TSP-1 forms complexes with ATF6α and calumenin in the ER, where TSP-1 resides before being secreted and, depending on the cell type or calcium levels, can be retained for variable periods. These studies provocatively propose a role for TSP-1 in the ER stress response and in disease, by assisting protein processing and secretion and contributing to reconstruction of the extracellular
matrix after injury. Notably, TSP-1 has been reported to bind – on the plasmamembrane – to proteins typically associated with intracellular vesicular compartments, such as calreticulin, LIMPII, and STIM1. Although no intracellular interactions between TSP-1 and these proteins have been reported so far, it would be interesting to investigate the formation and role of such complexes in intracellular compartments.
Even more striking is the recent report of an interaction of TSP-1 with ERK, in the cytoplasm. TSP-1 complexes with pERK, though not with inactive ERK, and tethers it to the cytosol, preventing its translocation to the nucleus and activity on nuclear substrates, ultimately mediating oncogenic Ras-induced senescence and functioning as a tumor suppressor.
Both these intracellular activities are due to endogenous TSP-1, and are not reproduced by exogenous, extracellular TSP-1 (Current understanding of the thrombospondin-1 interactome, 2014)..
Thrombospondin-1 and angiogenesis
The antiangiogenic effects of TSP-1 have been most extensively characterized in the tumor microenvironment (Fig. 5). TSP-1 antagonizes VEGF in several important ways, via inhibition of VEGF release from the extracellular matrix, direct interaction, and inhibition of VEGF signal transduction. The level of active matrix metalloproteinase-9 (MMP-9) is inversely correlated with the level of TSP-1. TSP-1 also binds directly to VEGF, and this interaction can mediate the uptake and clearance of VEGF from the extracellular space. TSP-1 binds to low-density receptor-related protein (LRP) in a glycosaminoglycan-dependent manner. This interaction mediates the uptake and clearance of TSP-1 along with its associated proteins. To date, MMPs and VEGF have been shown to be taken up by cells through this mechanism, which represents a second strategy for suppression of active MMPs by TSP-1. VEGF has, however, been reported to bind to the TSRs of other proteins, including pleiotrophin and connective tissue growth factor TSP-1 binds to fibroblast growth factor-2 (FGF-2) through its type 3 repeats, and this interaction has also been proposed to inhibit angiogenesis.
The three TSRs of TSP-1 have also been shown to inhibit VEGF signal transduction by decreasing VEGF-induced phosphorylation of VEGFR2 at tyrosine-1175 in a dose-dependent fashion. Inhibition of VEGFR2 phosphorylation appears to lead to decreased activation of the Akt pathway. Treatment of endothelial cells with 3TSR suppresses the activation of Akt in response to VEGF (Molecular Basis for the Regulation of Angiogenesis by Thrombospondin-1 and -2, 2012).
Figure 5. Schematic representation of the role of TSP-1 in the tumor microenvironment. TSP-1 affects angiogenesis through direct effects on endothelial cells and by antagonizing VEGF function. TSP-1 also suppresses the level of circulating endothelial cells (CEC).
Thrombospondin-1 and apoptosis
In 2000, it was reported that TSP-1 induces apoptosis of endothelial cells in culture.
In the presence of TSP-1, Fyn is recruited to CD36, and mitochondrial-dependent and -independent pathways are activated. In the absence of TSP-1, Src becomes the principal Src family kinase to associate with CD36. The induction of endothelial cell apoptosis by TSP-1 is mediated by the binding of the TSRs to CD36. Multiple non-overlapping peptide sequences in the TSRs of TSP-1 have been shown to be active.
A schematic representation of the signaling pathway that mediates TSP-1-induced apoptosis of endothelial cells is shown in Figure 6. Several studies have shown that activation of Jun amino-terminal kinase (JNK) occurs rapidly after TSP-1 binds to CD36. TSP-1 induces apoptosis through release of cytochrome c and sequential activation of caspase-9 and caspase-3.
Treatment of endothelial cells with TSP-1 reportedly up-regulates the Fas/FasL receptor/ligand pair. In another study, death receptors 4 and 5 were reported to be increased after treatment of endothelial cells with the TSRs. Whereas endothelial cells are usually resistant to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis, they become sensitized to TRAIL after treatment with 3TSR (Molecular Basis for the Regulation of Angiogenesis by Thrombospondin-1 and -2, 2012).
Figure 6. Signaling pathways that mediate the induction of endothelial apoptosis by TSP-1.
Thrombospondin-1 involvement in endothelial cell migration and adhesion
Endothelial cell migration is important to the formation of sprouting capillaries, and TSP-1 antagonizes this process. The inhibition of capillary endothelial cell migration involves the binding of the TSRs to CD36. In large vessel endothelial cells, which express little or no CD36, β1 integrins mediate the inhibition of migration through a process that involves PI3K, but not Akt. The TSRs of TSP-1 reportedly bind multiple β1 integrins. Consistent with the close association of CD36 with β1 integrins, an antibody to the β1 integrin subunit also suppressed the ability of TSP-1 to inhibit the migration of CD36-positive small vessel endothelial cells (Molecular Basis for the Regulation of Angiogenesis by Thrombospondin-1 and -2, 2012).
Thrombospondin-1 in chronic inflammation and fibrosis progression
Acute inflammation could advance to a resolution, progress to the formation of an abscess, walling off by fibrotic capsule, or evolve as scar upon tissue destruction, fibrin and collagen deposition. In many instances, this process continues as chronic inflammation. Chronic inflammation is characterized by infiltration of mononuclear cells, macrophages, lymphocytes, and plasma cells. Chronically inflamed tissues have fibroblast proliferation, angiogenesis, tissue destruction, and fibrosis.
Monocytes and macrophages are key elements of chronic inflammation. They invade the injured area during the acute process but, if the cause is not eliminated, infiltration by macrophages persists for long periods of time. The continued secretion of chemotactic factors allows the constant supply of monocytes from the blood and their conversion to macrophages. These cells are a key for further lymphocyte infiltration, fibroblast proliferation, tissue destruction, and fibrosis. Lymphocytes arise from the hemoblasts of the bone marrow, and later they develop immunocompentence and self-tolerance. T lymphocytes mature in the thymus and confer cell-mediated immunity. Plasma cells or B lymphocytes produce antibodies against antigens persisting in the area and therefore provide humoral immunity. They become immunocompetent when antigen-specific receptors appear on their surface. Plasmatic cells and macrophages are called antigen-presenting cells (APCs). Included in this group are dendritic cells (DCs), which internalize antigens and present antigenic determinants on their surface for recognition by T lymphocytes. They are part of the adaptive immune system that recognizes something as foreign and acts to immobilize and remove it. During the early stages of injury and inflammation, high levels of TSP-1 increase the tolerance of DC to antigens, ending the inflammatory response. TSP-1 can modulate inflammation by inhibiting or enhancing the secretion of the cytokine interleukin 10 (IL10), by this way, TSP-1 can also regulate the functions of DC. In addition, after adding IL-6, IL-10, or TGFβ1 to cultured DC, they become immune tolerant and show upregulation of intracellular TSP-1. TSP-1 also inhibits the function of APC by suppressing their capacity to sensitize T-cells in the host.
CD47 has also a crucial role in T-cell activation. Interaction of TSP-1 with CD47 promotes the activation of thymus-derived CD4+ CD25+ T regulatory cells (Tregs). Through this mechanism, CD47 helps to maintain self-tolerance inducing a suppressive phenotype.
It has been recently reported that bacterial pathogenesis may be mediated by CD47. Suppression of CD47 or TSP-1 expression in DC by using small interfering RNA (siRNA) technique actually protects newborn mice against bacterial (Escherichia coli) meningitis. Again, the loss of CD47 activity prevents the maturation of the DCs and the production of inflammatory cytokines. In conclusion, CD47 seems to have pivotal functions in inflammation and provides a major mechanistic pathway for the functions of TSP-1 in that process.
Finally, the deficiency of CD36 enhances the severity of bacterial and malaria infection. Cd36−/− mice exhibit an impaired early pro-inflammatory response to infection, elevation of cytokines, and higher mortality. These findings suggest that CD36 is quite critical for the recognition and clearance of pathogens in acute and chronic infections (Fig. 7). By binding to this receptor, TSP-1 could modulate the inflammatory process by activating macrophages and favouring phagocytosis. During chronic inflammation, these adaptive immune mechanisms provide defense against disease and are regulated by cellular interactions and cytokines. B lymphocytes secrete antibodies that bind to infectious agents and label them for destruction or elimination. Once inside a cell, a pathogen becomes inaccessible to those antibodies and cytotoxic T cells could destroy them by inducing apoptosis of the cell host. Regulatory T cells can modulate the secretion of cytokines enhancing the functions of macrophages and B-lymphocytes. TSP-1 has been reported to decrease immune responses by inhibition of T-cell effectors, or by directly inducing T cell apoptosis. In addition, by binding to α4β1 integrin TSP-1 promotes T-cell adhesion and chemotaxis.
TGFβ1 activation is a crucial element in intestinal homeostasis. In the intestinal tract, an abnormal response to the normal gut flora is a characteristic of the pathogenesis of inflammatory bowel disease (IBD). Mucosal T cells from patients with IBD express high levels of Smad7, an inhibitor of TGFβ1 signalling. By this mechanism, TGFβ1 mediates intestinal healing and susceptibility to injury. However, by activating TGFβ1, TSP-1 also enhances fibrosis and compromises organ function (Fig. 8). During the immune response, leukocytes produce reactive oxygen species (ROS) that include free radicals and peroxides. ROS are quite important for the killing of pathogens, but they can also produce cell damage. TGFβ1 favours the formation of ROS, and, as a cycle, ROS can also activate TGFβ1 promoting apoptosis and fibrosis (Thrombospondin-1: multiple paths to inflammation, 2011)..
Figure 7. Dual role of CD-36—TSP-1 interaction in inflammation. Expression of CD36 is positively regulated by PPARγ and negatively regulated by TGFβ. As an integral membrane protein, CD36 binds many ligands including TSP-1. The CD36-TSP-1 interaction involves conformational changes in TSP-1. This interaction mediates apoptotic effects via the CD47-dependent pathway, which has multiple effects. The CD36-TSP-1 interaction can also activate macrophage TGFβ1 and the NFκB pathway. Blue shading indicates pathways occurring in endothelial cells, pink shading in macrophages, and yellow shading in epithelial cells.
Figure 8. The homotrimeric TSP-1 activates latent TGFβ1 by binding the N-terminal propeptide LAP and the mature TGFβ1. The binding results in conformational changes in TGFβ1 and allows TGFβ1 to be recognized by its receptor. Mature, active TGFβ1 has been reported to decrease dendritic cell maturation and activate T cells. An opposing role of TGFβ1 results in fibrosis.
REGULATION
Genetic regulation
TSP-1 is synthesised by many cell types in vitro, probably because of the presence of aserum-response element in the promoter.
In a few reports the loss of heterozygocity (LOH) indicates the role of TSP1 as a classical type 2 tumor suppressor. Typically TSP1 expression is promoted by tumor suppressors including p53, phosphatase and tensin homologue (PTEN) and small and mothers against decapentaplegic 4 (SMAD-4). In contrast, TSP1 expression can be repressed by oncogenes such as K-Ras, c-Myc, c-Jun, c-Fos, v-src and c-Met (Therapies using anti-angiogenic peptide mimetics of thrombospondin-1, 2011).
Epigenetic control
Epigenetic modifiers also control TSP1. In multiple tumor types including malignant glioma, hypermethylation of the TSP1 promoter fragments found in serum predicts lower overall survival. Likewise, hypermethylation reduces TSP1 expression in gastric carcinoma and in aggressive neuroblastoma cell lines. Long-term ischemia also increases TSP1 promoter methylation and attenuates its expression in brain endothelial cells. Thus it is possible that hypemethylation of the TSP1 promoter in hypoxic tumors facilitates angiogenesis. Another example of TSP1 epigenetic regulation is repression by arginine methyltransferase 6 (PRMT6) in osteosarcoma cells, where it reduces lysine methylation of histone H3K4 and forces an inactive chromatin conformation on the TSP1 promoter (Therapies using anti-angiogenic peptide mimetics of thrombospondin-1, 2011).
Environmental factors and chemokines
In serum deprived cells, TSP-1 mRNA levels are low but are upregulated rapidly in response to serum or PDGF treatment. TSP-1 thus falls into the category of ‘immediate early’ response genes. TSP-1 expression decreases as cell density increases and is upregulated by stress conditions such as heat shock or hypoxia. TSP-1 mRNA and protein levels are upregulated by polypeptide growth factors including PDGF, TGF-β, and bFGF. TSP-1 protein levels are down-regulated by IL-1β or TNF-α: this does not involve an alteration in mRNA levels, suggesting that post-translational processing is in some way altered. In fact, IL-1β, IL-6 and TNFα downregulate TSP1 post-transcriptionally in endothelial cells and platelet activating factor (PAF) promotes angiogenesis by attenuating TSP1 expression (Thrombospondin-1, 1997).
TSP1 is regulated by oxygen and glucose and the effect of oxygen is cell type-dependent. In the endothelial cells TSP1 is upregulated by hypoxia at the transcriptional level, via hypoxia inducible factor 1α (HIF-1α) whereby it promotes VSMCs recruitment. There are classical hypoxia response elements in the TSP1 promoter. However, in tumor cells TSP1 can be decreased in hypoxic and anoxic conditions via HIF-independent mechanisms as is the case in astrocytes. As was mentioned above, hypoxia leads to TSP1 promoter hypermethylation and lower TSP1 expression. In VSMCs and endothelial cells, TSP1 is upregulated by glucose, via transcription by aryl hydrocarbon receptor (AhR). TSP1 secretion by adipocytes is also stimulated by glucose, albeit posttranscriptionally. In renal mesangial cells, high glucose concentrations block PKG and thereby alleviate TSP1 transcriptional repression. On the other hand, TSP1 is decreased by high glucose in the diabetic eye, suggesting that the regulation is context-dependent.
Shear stress (steady laminar flow) reversibly represses endothelial TSP-1 and CD36 in vitro and in vivo in a force- and time-dependent manner. On the other hand, TSP1 is induced by the lack of flow or by disturbed (turbulent) flow and this increase causes proatherogenic changes including endothelial cell apoptosis and increased VSMC proliferation mediated by αvβ3/CD47.
TSP1 can be upregulated by chemokines including fractalkine (CX3CL1) in human and mouse macrophages. Similarly, IL-8 and EGF decrease TSP1 production in mammary carcinoma and macrophage migration inhibitory factor (MIF-1) decreases it in melanoma
(Therapies using anti-angiogenic peptide mimetics of thrombospondin-1, 2011).
mRNA stability and translation
The 3′-untranslated region (3′UTR) of the TSP1 mRNA contains eight conserved AU-rich elements (AREs) whose mutations increase mRNA half-life by 9 h, whereby mRNA stability is controlled by the RNA-binding protein AU-rich element RNA-binding factor 1 (AUF1). In agreement, another RNA-binding protein, Hu antigen R (HuR), increases stability and elevates TSP1 mRNA and protein levels in MDA-MB231 breast cancer cells.
Recent studies reveal the importance of microRNA (miR) in the regulation of angiogenesis. To date, several miRNA have been found to bind the 3′UTR of the TSP1 mRNA and control its stability and translational activity. In the age-related heart failure, increases in miR-18 and miR-19 are concordant with decreased TSP1. In cancer, miRNAs from the miR-17-92 cluster act downstream of c-Myc, where they downregulate TSP1. Five-fluorouracil, upregulates TSP1 in colon cancer also by blocking the miR-17-92 cluster (Therapies using anti-angiogenic peptide mimetics of thrombospondin-1, 2011).
Signal transduction pathways
TSP1 is influenced by intracellular signals. It is repressed by PI3K and Akt, via mammalian target of rapamycin (mTOR), or via the MAPK/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade. Hepatocyte growth factor (HGF) represses TSP1 in OvCa cells via MAPK and AF1 transcription factor. Recent findings indicate that β1C integrin upregulates TSP1 in PCa cells by blocking the focal adhesion kinase.
In thyroid carcinoma, Doxorubicin blocks TSP1 production via ceramide and JNKs, which phosphorylate transcription factor . E2F-1 transcription factor activates TSP1 by binding –144/–137 motifs and this activation is reversed by phosphorylated retinoblastoma protein (Rb). Five-fluorouracil, a chemotherapy agent induces TSP1 production in gastric carcinoma by blocking thymidine phosphorylase. In breast cancer cells, S-adenosylmethionine decarboxylase, a key enzyme in polyamine synthesis, causes profound TSP1 downregulation and intense angiogenesis.
TSP1 can be controlled by steroid hormone receptors: whereby androgen receptor upregulates TSP1 and attenuates angiogenesis by PCa cells and estrogen receptor maintains TSP production in breast cancer and in the endothelial cells. Progesterone receptor upregulates TSP1 in endothelioma and breast cancer cells (Therapies using anti-angiogenic peptide mimetics of thrombospondin-1, 2011).
Degradation
TSP-I can be degraded both extracellularly or intracellularly. In the cardiovascular system. TSP-1 released from platelets and incorporated into the fibrin clot is a substrate for thrombin and Factor XIIIa. In tissues and in cell culture systems, TSP-1 is detected as small, granular patches within the matrix or in association with cell surfaces. The appearance of these patches is quite distinct from the network of fibrils formed by fibronectin, collagens or elastin microfibrils . TSP-1 is added to growing cultures of fibroblasts. endothelial cells or smooth muscle cells, a portion is retained in the matrix but most of the protein is endocytosed and released within 30 min after degradation in lysosomes. This process is mediated by binding of the aminoterminal domain of TSP-1 to proteoglycans and the low density lipoprotein receptor-related protein (Thrombospondin-1, 1997).
PATHWAYS INVOLVED IN DIABETIC COMPLICATIONS
Diabetes and its complications are rapidly becoming the world's most significant cause of morbidity and mortality. The adverse effects of persistently elevated plasma glucose levels on the different body parts vary according to the cell types. The cells expressing high levels of the glucose transporter 1 (GLUT 1), such as vascular endothelial cells, are unable to regulate intracellular glucose concentrations and are more susceptible to hyperglycaemia-induced damage. Renal mesangial cells overexpressing GLUT 1 acquire characteristics of the diabetic phenotype, including activation of the polyol pathway and increased extracellular matrix (ECM) synthesis. The complex cascade of events which leads to cellular malfunction in response to high levels of glucose is not fully understood. One of these events is formation of advanced glycation end products (AGEs). The elevated levels of glucose starts forming covalent adducts with plasma proteins through a non-enzymatic process known as glycation.
Protein glycation reactions leading to AGEs are thought to be the major causes of different diabetic complications. High glucose levels may induce glycation of various structural and functional proteins including plasma proteins and collagen. The non-enzymatic modification of plasma proteins such as albumin, fibrinogen and globulins may be produce various deleterious effects including alteration in drug binding in the plasma, platelet activation, generation of oxygen free radicals, impaired fibrinolysis and impairment in immune system regulation (Fig. 9). On the other hand, the structural impairment in collagen alters the osteoblast differentiation leading to bone remodeling and skeletal fragility.
Figure 9. Persistently elevated glucose levels during long standing diabetes induce structural and functional changes in different protein in the body including albumin, globulins, fibrinogen and collagens. Glycation of these proteins is associated with induction.
Advanced glycation is one of the major pathways involved in the development and progression of different diabetic complications including nephropathy, retinopathy and neuropathy. Tissue and circulating AGE levels are higher in smokers with concurrent increase in inflammatory markers. There is evidence from animal studies that exposure to high levels of exogenous AGEs contributes to renal and vascular complications. AGEs often accumulate intracellularly as a result of their generation from glucose-derived dicarbonyl precursors. These intracellular AGEs play important roles as stimuli for activating intracellular signaling pathways as well as modifying the function of intracellular proteins. AGEs accumulate in most sites of diabetes complications, including the kidney, retina, and atherosclerotic plaques. Glycation of proteins interferes with their normal functions by disrupting molecular conformation, altering enzymatic activity, reducing degradation capacity, and interfering with receptor recognition. The mechanism by which glycation alters the cell functions include denaturation and functional decline of the target protein and lipid, organopathy due to accumulation of AGEs in tissue, activation of receptor-mediated signal pathway in cells, generation of oxidative stress and carbonyl stress. The intermolecular collagen cross-linking caused by AGEs leads to diminished arterial and myocardial compliance, increased vascular stiffness increase, increase in diastolic dysfunction and systolic hypertension. Turk and his co-workers reported that the presence of autoantibodies against serum AGEs are capable of forming AGE-immune complexes in diabetic patients and may play a role in atherogenesis. Glycation-derived free radicals can cause protein fragmentation and oxidation of nucleic acids and lipids. The amino groups of adenine and guanine bases in DNA are also susceptible to glycation and AGE formation (Advanced Glycation End Products and Diabetic Complications, 2014).
RECEPTORS FOR ADVANCED GLYCATION END PRODUCTS (RAGE)
Interaction of AGEs with their cellular receptors has an important role in the pathogenesis of diabetic complications. RAGE was first described as receptor for AGEs. Many receptors for AGE have been identified, such as lactoferrin, scavenger receptors types I and II, oligosaccharyl transferase-48 (OST-48), 80K-H phosphoprotein, galectin-3, and CD36. RAGE is a multiligand receptor and a member of the immunoglobulin superfamily of cell surface molecules and found on smooth muscle cells, macrophages, endothelial cells and astrocytes. RAGE has been identified as receptor for amyloid-beta peptide (Aβ) and β-sheet fibrils S100/calgranulins; amphoterin and Mac-1.
AGEs bind only to the V domain of RAGE and a sustained period of cellular activation mediated by receptor-dependent signaling, leads to inflammation. It is proposed that RAGE activation is largely responsible for the pathogenicity associated with AGEs. RAGE can be stimulated not only by AGEs, but other ligands including S100-calgranulins which are a group of pro-inflammatory cytokines, amphoterin, amyloid-β and other fibrillar proteins. Expression of RAGE is enhanced in certain cells during diabetes and inflammation. Interaction of AGEs with RAGE on macrophages causes oxidative stress and activation of nuclear factor-κB (NF-κB) via activation of the p21ras and the mitogen-activated protein (MAP) kinase signaling pathway. NF-κB modulates gene transcription for endothelin-1, tissue factor and thrombomodulin and generation of pro-inflammatory cytokines such as interleukin-1 α (IL-1α), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). There is also enhanced expression of adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), (Fig. 10) in addition to other effects such as increased vascular permeability. A study showed that NF-κB and heme oxygenase mRNA, the markers of oxidative stress, are activated, following binding of AGEs with RAGE in endothelial cells. During glycoxidative stress, NF-κB activates the production of TNF-α which in turn leads to enhanced ROS production. ROS plays a crucial role in the pathogenesis of type II diabetes, neurodegenerative and cardiovascular diseases. It has been reported that direct exposure of endothelial cells to hyperglycaemic concentrations of glucose increases the formation of ROS, which in turn activates the enzyme NADPH oxidase. The inhibition of AGEs formation is another mode for diabetes treatment, which is not dependent on the control of blood glucose, and would be useful in prevention of certain diabetic complications (Advanced Glycation End Products and Diabetic Complications, 2014).
Figure 10. Interaction of AGE with RAGE leading to oxidative stress and initiation of inflammation cascade involving activation of MAPK pathway, NF-kB, IL-6, TNF-α, expression of ICAM-1 and VCAM-2 which ultimately leads to diabetic complications.
CORRELATION BETWEEN TSP-1 EXPRESSION AND DIABETIC COMPLICATIONS
In diabetes mellitus, glucose reduces key factors of islet angiogenesis which might exacerbate beta-cell failure; antiangiogenic TSP-1 was upregulated in diabetic mice, regardless of the presence of RAGE, both in wild-type and RAGE mice (Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes, 2006).
Several articles demonstrate the role of TSP-1 in diabetic nephropaty: TSP-1 enhances fibrosis and renal damage by its interaction with TGFβ1, while LSKL, a peptide antagonist of TSP-1, reduces renal interstitial fibrosis in a rat experimental model of kidney disease. This effect is attributed to the competitive properties of LSKL that prevents TSP-1-mediated activation of TGFβ1 (Blockade of TSP1-dependent TGF-β activity reduces renal injury and proteinuria in a murine model of diabetic nephropathy, 2011).
In particular, Yang et al. demonstrated that AGEs dose-dependently increased both intracellular and extracellular levels of TSP-1 in both glomerular and tubuloepithelial elements. Anti-TSP-1 neutralizing antibodies attenuated the AGE-induced increase in TGF-beta1 bioactivity and hypertrophy. They demonstrated also that TSP-1 might mediate AGE-induced distal renal tubule hypertrophy (Thrombospondin-1 mediates distal tubule hypertrophy induced by glycated albumin, 2004). Thus, regulation of TSP-1 might be critical for hyperglycaemic distal tubule hypertrophy. The development of diabetic nephropathy is attenuated in TSP-1-deficient mice as demonstrated by a significant reduction of glomerulosclerosis, glomerular matrix accumulation, podocyte injury, renal infiltration with inflammatory cells, and alterations of renal functional parameters. It was demonstrated that blockade of TSP1-dependent TGF-β activity reduces renal injury and proteinuria in a murine model of diabetic nephropathy. Furthermore, TSP-1 TFD might be a potential approach to ameliorate diabetic renal hypertrophy (Glucose downregulation of PKG-I protein mediates increased thrombospondin1-dependent TGF-{beta} activity in vascular smooth muscle cells, 2010).
TSP-1 could modulate the functions of TGFβ1 in cardiovascular diseases, atherosclerosis, and obesity. Inflammatory cells secrete TSP-1 during the acute phase of the healing process in myocardial infarction. Infarcted murine hearts show marked upregulation of TGFβ1. In addition, TSP-1 is selectively expressed in the infarcted border suggesting that TSP-1 might inhibit the expansion of the inflammation by activating TGFβ1.
However, TSP-1 expression is increased in response to high glucose in the wall of large vessels and accelerates atherosclerosis and other pathological events observed in diabetes (A novel transcriptional mechanism of cell type-specific regulation of vascular gene expression by glucose, 2011) , (Glucose regulation of thrombospondin and its role in the modulation of smooth muscle cell proliferation, 2010).
TSP-1 mRNA is significantly associated with obesity and insulin resistance in nondiabetic patients. This correlation is explained by the TSP-1-dependent TGFβ1 activation that leads to upregulation of plasminogen activator inhibitor 1 (PAI-1) gene expression. Elevated circulating PAI-1 levels are detected in insulin resistance and metabolic syndrome.
TSP-1 is closely associated with obesity-related mechanisms. TSP-1, an antiangiogenic factor and transforming growth factor (TGF)-β activity regulator, has been recently recognized as an adipokine that correlates with obesity, inflammation and insulin resistance processes. Thrombospondin-1 regulates adiposity and metabolic dysfunction in diet-induced obesity enhancing adipose inflammation and stimulating adipocyte proliferation (Glucose and insulin modify thrombospondin 1 expression and secretion in primary adipocytes from diet-induced obese rats, 2011) , (Thrombospondin-1 regulates adiposity and metabolic dysfunction in diet-induced obesity enhancing adipose inflammation and stimulating adipocyte proliferation, 2013).
Moreover, TSP-1/CD36 pathway contributes to angiogenesis dysfunction by inducing circulating endothelial progenitor cell apoptosis in diabetes (Thrombospondin-1/CD36 pathway contributes to bone marrow-derived angiogenic cell dysfunction in type 1 diabetes via Sonic hedgehog pathway suppression, 2013).
CONCLUSIONS
TSP-1 is a protein that interacts with different ligands and receptors, and it is known to be involved in different biological mechanisms, such as: inhibition of angiogenesis, impairment of inflammation and progression of fibrosis.
These mechanisms are particularly important in progression of diabetic complications targeting several organs such as kidney, brain, eye and cardiovascular system. In this context, a particular role in the evolution of pathology is played by high levels of glucose that stimulate the formation of AGEs by signaling pathways activated by their receptors, RAGEs. Interestingly, some AGEs activate TSP-1-receptor CD36 contributing to angiogenesis dysfunction.
Moreover, TSP-1 expression is up-regulated by high levels of glucose in different cell types and circadian oscillation of TSP-1 is controlled by insulin (Circadian oscillation of circulating prothrombotic thrombospondin-1: ex vivo and in vivo regulation by insulin, 2008). and by AGE (Age-related expression of renal thrombospondin 1 mRNA in F344 rats: resemblance to diabetes-induced expression in obese Zucker rats, 1999)
TSP-1 gene expression is inhibited by kinases involved in signaling pathways such as PI3K/Akt, mTOR, MAPK, ERK and it’s activated by p53.
So, I suppose that insulin could inhibit TSP-1 expression by modulating activation of these intracellular mediators. Moreover, TSP-1 has been recently recognized as an adipokine that correlates with obesity, inflammation and insulin resistance processes. In fact, TSP-1 regulates adiposity and metabolic dysfunction in diet-induced obesity enhancing adipose inflammation and stimulating adipocyte proliferation (Thrombospondin-1 regulates adiposity and metabolic dysfunction in diet-induced obesity enhancing adipose inflammation and stimulating adipocyte proliferation, 2013).
A therapy that could target specifically TSP-1 expression or activity could slow progression of diabetic complications by stimulating angiogenesis, inhibiting the inflammatory process of atherosclerosis and calcification in blood vessels, blocking TGF-β-mediated fibrosis.
