Maria J. Calzada

Interactions of thrombospondins with α4β1 integrin and CD47 differentially modulate T cell behavior

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin α4β1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of α4β1 integrin, and TSP1 inhibited interaction of activated α4β1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The α4β1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the α4β1 integrin–dependent activities of TSP1. The β1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

α4β1 Integrin Mediates Selective Endothelial Cell Responses to Thrombospondins 1 and 2 In Vitro and Modulates Angiogenesis In Vivo

Abstract— We examined the function of &agr;4&bgr;1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. &agr;4&bgr;1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by &agr;4&bgr;1, whereas antagonism of fibroblast growth factor-2–stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an &agr;4&bgr;1-dependent manner. Soluble &agr;4&bgr;1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1–dependent and not observed in explants from thrombospondin-1−/− mice. Antagonizing &agr;4&bgr;1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the &agr;4&bgr;1 binding sequence stimulate angiogenesis in vivo. Therefore, &agr;4&bgr;1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Recognition of the N-terminal Modules of Thrombospondin-1 and Thrombospondin-2 by α6β1 Integrin

In addition to its recognition by α3β1 and α4β1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for α6β1 integrin. α6β1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. α6β1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an α6β1-binding sequence in thrombospondin-1. This peptide inhibited α6β1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by α6β1, but not by α4β1. α6β1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of α6β1 integrin in microvascular endothelial cells by the anti-β1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of α4β1 and α6β1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

HIF-2α-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction.

Aims Hypoxic conditions stimulate pulmonary vasoconstriction and vascular remodelling, both pathognomonic changes in pulmonary arterial hypertension (PAH). The secreted protein thrombospondin-1 (TSP1) is involved in the maintenance of lung homeostasis. New work identified a role for TSP1 in promoting PAH. Nonetheless, it is largely unknown how hypoxia regulates TSP1 in the lung and whether this contributes to pathological events during PAH. Methods and results In cell and animal experiments, we found that hypoxia induces TSP1 in lungs, pulmonary artery smooth muscle cells and endothelial cells, and pulmonary fibroblasts. Using a murine model of constitutive hypoxia, gene silencing, and luciferase reporter experiments, we found that hypoxia-mediated induction of pulmonary TSP1 is a hypoxia-inducible factor (HIF)-2α-dependent process. Additionally, hypoxic tsp1−/− pulmonary fibroblasts and pulmonary artery smooth muscle cell displayed decreased migration compared with wild-type (WT) cells. Furthermore, hypoxia-mediated induction of TSP1 destabilized endothelial cell–cell interactions. This provides genetic evidence that TSP1 contributes to vascular remodelling during PAH. Expanding cell data to whole tissues, we found that, under hypoxia, pulmonary arteries (PAs) from WT mice had significantly decreased sensitivity to acetylcholine (Ach)-stimulated endothelial-dependent vasodilation. In contrast, hypoxic tsp1−/− PAs retained sensitivity to Ach, mediated in part by TSP1 regulation of pulmonary Kv channels. Translating these preclinical studies, we find in the lungs from individuals with end-stage PAH, both TSP1 and HIF-2α protein expression increased in the pulmonary vasculature compared with non-PAH controls. Conclusions These findings demonstrate that HIF-2α is clearly implicated in the TSP1 pulmonary regulation and provide new insights on its contribution to PAH-driven vascular remodelling and vasoconstriction.

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration.

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K(d)=48nM and the related sFRP-2 with a K(d)=95nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3β1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.

Novel Integrin Antagonists Derived from Thrombospondins

Specific antagonists have been successfully developed for several different integrins. Clinical trials have been initiated to study therapeutic uses of these inhibitors in cancer, thrombosis, and inflammatory diseases. Most efforts to date have focused on the platelet integrin alphaIIbbeta3, endothelial alphavbeta3, and the leukocyte integrin alpha4beta1. However, the integrin family contains additional members with interesting tissue specificities and functional properties that could also be useful molecular targets for disease intervention. In many cases, specific recognition motifs for these integrins have not been identified, which has precluded development of specific antagonists. Our recent studies of thrombospondin-1 and thrombospondin-2 recognition by integrins have revealed novel motifs for alpha3beta1 and alpha6beta1 integrins as well as new motifs recognized by the well studied alpha4beta1 integrin. These three integrins play distinct roles in angiogenesis and its modulation by thrombospondins. This review will discuss recent insights into the specificities of alpha3beta1 and alpha6beta1 integrins, their functions in angiogenesis, and potential applications for antagonists of these integrins and of alpha4beta1 to control pathological angiogenesis and other diseases.

Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1

Conformational changes induced in thrombospondin-1 by removal of calcium regulate interactions with some ligands of its N-modules. Because calcium binds primarily to elements of the C-terminal signature domain of thrombospondin-1, which are distant from the N-modules, such regulation was unexpected. To clarify the mechanism for this regulation, we compared ligand binding to the N-modules of thrombospondin-1 in the full-length protein and recombinant trimeric thrombospondin-1 truncated prior to the signature domain. Three monoclonal antibodies were identified that recognize the N-modules, two of which exhibit calcium-dependent binding to native thrombospondin-1 but not to the truncated trimeric protein. These antibodies or calcium selectively modulate interactions of fibronectin, heparin, sulfatide, alpha3beta1 integrin, tumor necrosis factor-alpha-stimulated gene-6 protein, and, to a lesser extent, alpha4beta1 integrin with native thrombospondin-1 but not with the truncated protein. These results indicate connectivity between calcium binding sites in the C-terminal signature domain and the N-modules of thrombospondin-1 that regulates ligand binding and functional activities of the N-modules.

TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

Aims Thrombospondin-1 (TSP1) is a ligand for CD47 and TSP1−/− mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1–CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy. Methods and results We analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1–CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47−/− mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47−/− pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1–CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters. Conclusions In pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1–CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

Inadequate Activation of the GTPase RhoA Contributes to the Lack of Fibronectin Matrix Assembly in von Hippel-Lindau Protein-defective Renal Cancer Cells

The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL(+) cells. We have previously reported that VHL(-) cells fail to form β1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.

Matricellular protein thrombospondin-1 in pulmonary hypertension: multiple pathways to disease

Matricellular proteins are secreted molecules that have affinities for both extracellular matrix and cell surface receptors. Through interaction with structural proteins and the cells that maintain the matrix these proteins can alter matrix strength. Matricellular proteins exert control on cell activity primarily through engagement of membrane receptors that mediate outside-in signaling. An example of this group is thrombospondin-1 (TSP1), first identified as a component of the secreted product of activated platelets. As a result, TSP1 was initially studied in relation to coagulation, growth factor signaling and angiogenesis. More recently, TSP1 has been found to alter the effects of the gaseous transmitter nitric oxide (NO). This latter capacity has provided motivation to study TSP1 in diseases associated with loss of NO signaling as observed in cardiovascular disease and pulmonary hypertension (PH). PH is characterized by progressive changes in the pulmonary vasculature leading to increased resistance to blood flow and subsequent right heart failure. Studies have linked TSP1 to pre-clinical animal models of PH and more recently to clinical PH. This review will provide analysis of the vascular and non-vascular effects of TSP1 that contribute to PH, the experimental and translational studies that support a role for TSP1 in disease promotion and frame the relevance of these findings to therapeutic strategies.