Thrombospondin 2 (TSP2) can inhibit angiogenesis in vitro by limiting proliferation and inducing apoptosis of endothelial cells (ECs). TSP2 can also modulate the extracellular levels of gelatinases (matrix metalloproteases, MMPs) and potentially influence the remodeling of the extracellular matrix (ECM). Here, we tested the hypothesis that by regulating MMPs, TSP2 could alter EC-ECM interactions. By using a three-dimensional angiogenesis assay, we show that TSP2, but not TSP1, limited angiogenesis by decreasing gelatinolytic activity in situ. Furthermore, TSP2-null fibroblast-derived ECM, which contains irregular collagen fibrils, was more permissive for EC migration. Investigation of the role of TSP2 in physiological angiogenesis in vivo, using excision of the left femoral artery in both TSP2-null and wild-type mice, revealed that TSP2-null mice displayed accelerated recovery of blood flow. This increase was attributable, in part, to an enhanced arterial network in TSP2-null muscles of the upper limb. Angiogenesis in the lower limb was also increased and was associated with increased MMP-9 deposition and gelatinolytic activity. The observed changes correlated with the temporal expression of TSP2 in the ischemic muscle of wild-type mice. Taken together, our observations implicate the matrix-modulating activity of TSP2 as a mechanism by which physiological angiogenesis is inhibited.

BACKGROUND

Obesity is prevalent worldwide and is associated with insulin resistance. Advanced studies suggest that obesity-associated low-grade chronic inflammation contributes to the development of insulin resistance and other metabolic complications. Thrombospondin 1 (TSP1) is a multifunctional extracellular matrix protein that is up-regulated in inflamed adipose tissue. A recent study suggests a positive correlation of TSP1 with obesity, adipose inflammation, and insulin resistance. However, the direct effect of TSP1 on obesity and insulin resistance is not known. Therefore, we investigated the role of TSP1 in mediating obesity-associated inflammation and insulin resistance by using TSP1 knockout mice.

RESULTS

Male TSP1-/- mice and wild type littermate controls were fed a low-fat (LF) or a high-fat (HF) diet for 16 weeks. Throughout the study, body weight and fat mass increased similarly between the TSP1-/- mice and WT mice under HF feeding conditions, suggesting that TSP1 deficiency does not affect the development of obesity. However, obese TSP1-/- mice had improved glucose tolerance and increased insulin sensitivity compared to the obese wild type mice. Macrophage accumulation and inflammatory cytokine expression in adipose tissue were reduced in obese TSP1-/- mice. Consistent with the local decrease in pro-inflammatory cytokine levels, systemic inflammation was also decreased in the obese TSP1-/- mice. Furthermore, in vitro data demonstrated that TSP1 deficient macrophages had decreased mobility and a reduced inflammatory phenotype.

CONCLUSIONS

TSP1 deficiency did not affect the development of high-fat diet induced obesity. However, TSP1 deficiency reduced macrophage accumulation in adipose tissue and protected against obesity related inflammation and insulin resistance. Our data demonstrate that TSP1 may play an important role in regulating macrophage function and mediating obesity-induced inflammation and insulin resistance. These data suggest that TSP1 may serve as a potential therapeutic target to improve the inflammatory and metabolic complications of obesity.

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre-treatment of the cells with either heparitinase, chondroitinase or beta-D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.

In addition to the three known beta(1) integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that beta(1) integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-beta(1)-specific. Adhesion of cells that express one dominant beta(1) integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several beta(1) integrins is partially inhibited by each alpha-subunit-specific antagonist and completely inhibited by combining the antagonists. beta(1) integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-beta(1) specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to alpha-subunit antagonists, but a beta(1) blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. beta(1) integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and beta(1) integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits beta(1) integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4.1 is also an antagonist of CD36 binding to TSP1, these data suggest that some biological activities of A4.1 result from antagonism of these novel beta(1) integrin binding sites.

Renal and cardiac fibrosis leading to organ failure are complications of both diabetes and hypertension. These disease processes, when combined, exacerbate development of fibrotic complications. Control of latent transforming growth factor (TGF)-beta activation is a potential determinant of fibrotic progression. Both glucose and angiotensin II (Ang II) upregulate thrombospondin-1 (TSP1), a major activator of latent TGF-beta, and stimulate increased TGF-beta activity. We previously showed that high glucose stimulated TSP1-dependent TGF-beta activation in rat mesangial cells (RMCs). In this paper, we examined whether Ang II similarly upregulates TSP1 production and TSP1-dependent TGF-beta activation alone or in combination with high glucose concentrations. Ang II and high glucose stimulated increases in TSP1 protein levels in the conditioned media of both rat cardiac fibroblasts (RCFs) and rat mesangial cells (RMCs). Meanwhile, Ang II stimulated increases in both TGF-beta activity and protein by RMCs, whereas, RCFs responded to both Ang II and high glucose with increased TGF-beta activity in the absence of altered TGF-beta protein levels. A combination of Ang II and high glucose induced synergistic TGF-beta activation by RCFs. Moreover, Ang II induction of TSP1 and increased TGF-beta activity were blocked by losartan, an antagonist of the Ang II type 1 (AT1) receptor. The increase in TSP1 expression leads to increased TGF-beta activity upon Ang II and/or glucose treatment, since peptide antagonists of TSP1-mediated TGF-beta activation blocked Ang II and glucose-induced TGF-beta activation. Our data support a role for TSP1 in the development and progression of renal and cardiac fibrosis in hypertension and diabetes.

Thrombospondin (TSP) 1 and TSP2 have been implicated in the regulation of several processes during tissue repair. Due to their matricellular nature, these proteins are thought to modulate cell-matrix interactions through a variety of mechanisms specific to the spatio-temporal context of their expression. Most notably, TSP1 and TSP2 appear to play distinct, non-overlapping roles in the healing of skin wounds. In contrast, both proteins have been implicated as regulators of ischemia-induced angiogenesis. Moreover, TSP2 has been shown to be a critical regulator of angiogenesis in the foreign body response (FBR). In this review, we discuss the role of TSPs in tissue repair and examine the mechanistic data regarding the ability of the thrombospondins to modulate cell-matrix interactions in this context.

Thrombospondin 1 (TSP1), an oligomeric matrix protein, is known for its antiangiogenic activity. Recently, TSP1 has been shown to regulate synaptogenesis in the developing brain. In this study, we examine another role of TSP1 in the CNS, namely, in proliferation and differentiation of neural progenitor cells (NPCs). We found that adult mice deficient in TSP1 exhibit reduced proliferation of NPCs in vivo [13,330+/-826 vs. 4914+/-455 (mean+/-se wt vs. TSP1(-/-)); P<0.001, Student's t test] and impaired neuronal differentiation (1382+/-83 vs. 879+/-79; P<0.001). In vitro, NPC obtained from adult TSP1(-/-) mice display decreased proliferation in BrdU assay (48+/-8 vs. 24+/-3.5%; P<0.01) and decreased neuronal fate commitment (8+/-0.85 vs. 4.6+/-0.5%; P<0.05) in contrast to wild-type NPCs. Both proliferation and neuronal differentiation deficits are remediable in vitro by exogenous TSP1. Notably, conditioned medium from TSP1(-/-) astrocytes, unlike that from control astrocytes, fails to promote neurogenesis in wild-type NPCs, suggesting that TSP1 is one of the key molecules responsible for astrocyte-induced neurogenesis. Our data demonstrate that TSP1 is a critical participant in maintenance of the adult NPC pool and in neuronal differentiation.

OBJECTIVE

Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH.

RESULTS

We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH.

CONCLUSIONS

Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

OBJECTIVE

Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress.

RESULTS

Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O(2)(•-)) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O(2)(•-) in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47(phox) and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion.

CONCLUSIONS

Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.

In the apicomplexan protozoans motility and cell invasion are mediated by the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex. Here we characterize a new member of the TRAP/MIC2 family, named TRAP-Like Protein (TLP), that is highly conserved within the Plasmodium genus. Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain. Comparison of the vWA domain of TLP genes from multiple Plasmodium falciparum isolates showed relative low sequence diversity, suggesting that the protein is not under selective pressures of the host immune system. Analysis of transcript levels by quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that TLP is predominantly expressed in salivary gland sporozoites of P. falciparum and P. berghei. Targeted disruption of P. berghei TLP resulted in a decreased capacity for cell traversal by sporozoites, and reduced infectivity of sporozoites in vivo, whereas in vitro sporozoite motility and hepatocyte invasion were unaffected. These results indicate a role of TLP in cell traversal by sporozoites.

BACKGROUND

Insufficient tissue perfusion underlies many acute and chronic diseases. Tissue perfusion in turn requires adequate blood flow, determined in large part by the relative state of relaxation or constriction of arterial vessels. Nitric oxide (NO) produced by vascular cells modulates blood flow and tissue perfusion by relaxing and dilating arteries. Recently, we reported that the secreted protein thrombospondin-1 (TSP1), through its cell surface receptor CD47, limits the ability of NO to relax and dilate blood vessels and thus decreases tissue perfusion. In the present study, we tested the hypothesis that blocking TSP1-CD47 signaling increases ischemic tissue survival in random cutaneous porcine flaps.

METHODS

Random cutaneous flaps 2 x 10 cm2 were raised in white hairless Yucatan miniature pigs and were treated with a monoclonal antibody to TSP1, an antisense morpholino oligonucleotide to CD47 or control agents and tissue survival assessed. Primary vascular smooth muscle cell cultured from Yucatan pigs were also treated with the same agents +/- and an NO donor (DEA/NO) and cGMP quantified.

RESULTS

Antibody blockade of TSP1 or morpholino suppression of CD47 dramatically enhanced survival of random tissue flaps. These responses correlated with increased blood vessel patency and tissue blood flow on vessel injection studies. NO-stimulated cGMP flux in Yucatan vascular smooth muscle cell was abrogated after antibody or morpholino treatment.

CONCLUSIONS

Antibody ligation of TSP1 or antisense morpholino knock down of CD47 greatly increased tissue survival to ischemia. Given the similarity between porcine and human soft tissues these results suggest significant therapeutic potential for people.

Perivascular supporting cells, including vascular smooth muscle cells (VSMCs) and pericytes (PCs), provide instructive signals to adjacent endothelial cells helping to maintain vascular homeostasis. These signals are provided through direct contact and by the release of soluble factors by these cells. Thrombospondin (TSP)1 is a matricellular protein and an autocrine factor for VSMCs. TSP1 activity, along with that of PDGF, regulates VSMC proliferation and migration. However, the manner in which TSP1 and PDGF impact retinal PC function requires further investigation. In the present study, we describe, for the first time, the isolation and culture of retinal PCs from wild-type (TSP1(+/+)) and TSP1-deficient (TSP1(-/-)) immortomice. We showed that these cells express early and mature markers of PCs, including NG2, PDGF receptor-beta, and smooth muscle actin as well as desmin, calbindin, and mesenchymal stem cell markers. These cells were successfully passaged and maintained in culture for several months without significant loss of expression of these markers. TSP1(+/+) PCs proliferated at a faster rate compared with TSP1(-/-) PCs. In addition, TSP1(+/+) PCs, like VSMCs, responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1(-/-) PCs failed to respond to the promigratory and proliferative activity of PDGF-BB. This may be attributed, at least in part, to the limited interaction of PDGF-BB with TSP1 in null cells, which is essential for PDGF proliferative and migratory action. We observed no significant differences in the rates of apoptosis in these cells. TSP1(-/-) PCs were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and alpha(v)beta(3)-integrin on their surface. Thus, TSP1 plays a significant role in retinal PC proliferation and migration impacting retinal vascular development and homeostasis.

BACKGROUND

Paracrine secretions seem to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo).

OBJECTIVE

Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake.

RESULTS

When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1, simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level.

CONCLUSIONS

Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.

Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N-terminal domain of TSP1 binds to the calreticulin-LRP1 receptor co-complex to signal down-regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin-LRP1 activates cell survival signals such as PI3-kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion-independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N-terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin-LRP1 binding site [amino acids 17-35 (hep I)] in the N-terminal domain promotes fibroblast survival under anchorage-independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1-mediated anchorage-independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage-independent survival. These data define a novel role for TSP1 signaling through the calreticulin/LRP1 co-complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.-Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy-Ullrich, J. E. Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.

Thrombospondins belong to a family of extracellular matrix (ECM) proteins widely found from embryonic to adult tissues. The modular structure of thrombospondins contains a series of peptide sequences implicated in a multiplicity of biological functions. Extracellular matrix undergoes important alterations under proteolysis that occurs in pathological processes like tumorigenesis. An elevated secretion of thrombospondin 1 (TSP1) is often observed in tumors and is sometimes considered as a predictive factor. However, the role of TSP1 in cancer progression remains controversial and must be carefully apprehended. The regulation of cell adhesion, proliferation, apoptosis by TSP1 is examined in the present review and it is clear from the literature and from our investigations that TSP1 presents both stimulatory and inhibitory effects. The exposition of cryptic sites upon conformational changes can partially explain this contradiction. More interestingly, the analysis of TSP1-directed intracellular signaling pathways activated through specific receptors or supramolecular receptors docking systems may be useful to discriminate the precise function of TSP1 in tumor progression. The central role played by TSP1 in the control of matrix-degrading enzyme activation and catabolism reveals attractive tracks of research and highlights the involvement of the lipoprotein receptor-related protein (LRP) receptor in these events. Therefore, TSP1-derived peptides constitute a source of potentially active matrikins which could provide essential tools in cancer therapy.

Cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP1) were purified in a native form from normal bovine articular cartilage. The key step in the purification scheme was selective extraction with EDTA-containing buffer. Final separation of these two molecules was achieved by heparin affinity chromatography. Particles viewed by electron microscopy after rotary shadowing and negative staining revealed structures similar to their prototype molecules; from the Swarm rat chondrosarcoma for COMP, or from platelets for TSP1. Attachment of primary bovine chondrocytes to purified matrix proteins was investigated. Cells attached to COMP but not to the structurally related TSP1 indicating separate functions for these proteins in cartilage.

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.

BACKGROUND

Ischemia-reperfusion (I/R) injury remains a primary complication of transplant surgery, accounting for about 80% of liver transplant failures, and is a major source of morbidity in other pathologic conditions. Activation of endothelium and inflammatory cell recruitment are central to the initiation and promulgation of I/R injury, which can be limited by the bioactive gas nitric oxide (NO). The discovery that thrombsospondin-1 (TSP1), via CD47, limits NO signaling in vascular cells and ischemic injuries in vivo suggested that I/R injury could be another important target of this signaling pathway.

METHODS

Wild-type, TSP1-null, and CD47-null mice underwent liver I/R injury. Wild-type animals were pretreated with CD47 or control antibodies before liver I/R injury. Tissue perfusion via laser Doppler imaging, serum enzymes, histology, and immunohistology were assessed.

RESULTS

TSP1-null and CD47-null mice subjected to subtotal liver I/R injury showed improved perfusion relative to wild-type mice. Null mice subjected to liver I/R had decreased liver enzyme release and less histologic evidence of injury. Elevated TSP1 expression in liver tissue after I/R injury suggested that preventing its interaction with CD47 could be protective. Thus, pretreatment of wild-type mice using a blocking CD47 antibody improved recovery of tissue perfusion and preserved liver integrity after I/R injury.

CONCLUSIONS

Tissue survival and perfusion after liver I/R injury are limited by TSP1 and CD47. Targeting CD47 before I/R injury enhances tissue survival and perfusion in a model of liver I/R injury and suggests therapeutics for enhancing organ survival in transplantation surgery.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

Integrin-associated protein (IAP/CD47) augments the function of alpha2beta1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP(-/-) animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti-alpha2beta1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in cAMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates alpha2beta1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity and suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.

BACKGROUND

Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura.

METHODS

Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins.

RESULTS

Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients' samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients' type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients' platelet counts on admission.

CONCLUSIONS

This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.

A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator. Phosphorylation appears to regulate these interactions. The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability. The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4.

CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

Biological activities of the matrix glycoprotein thrombospondin-1 (TSP1) are cell type specific and depend on the relative expression or activation of several TSP1 receptors. Although engaging individual TSP1 receptors in T lymphocytes can elicit costimulating signals, in this study we show that intact TSP1 inhibits TCR-mediated T cell activation, assessed globally using cDNA microarrays. TSP1 signaling suppressed expression of several genes induced in Jurkat T cells, including the T cell activation markers CD69, early growth response gene-1 (Egr-1), and phosphatase of activated cells (PAC-1). TCR-stimulated and CD47-costimulated IL-2 secretion and cell surface CD69 expression were also inhibited by TSP1. The specific inhibitory effect of TSP1 was verified in freshly isolated human PBMCs. TSP1 inhibited TCR-mediated but not protein kinase C-mediated T cell activation. Using CD69 expression as a marker, we demonstrated that the inhibitory activity of TSP1 depended on two TSP1 receptors, CD47 and integrin-associated protein heparan sulfate proteoglycans. Signals from these receptors inhibited TCR signaling downstream of ZAP70, but upstream of NF-AT. Therefore, the expression of TSP1 induced during wound repair and in tumor stroma may limit T cell activation at these sites.