Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration.
ABSTRACT: Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.
Project description:Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.
Project description:OBJECTIVE: Loss of thrombospondin (TSP)-1 in pancreatic islets has been shown to cause islet hyperplasia. This study tested the hypothesis that endothelial-derived TSP-1 is important for ?-cell function. RESEARCH DESIGN AND METHODS: Islet function was evaluated both in vivo and in vitro. Messenger RNA and protein expression were measured by real-time PCR and Western blot, respectively. The role of endothelial-derived TSP-1 for ?-cell function was determined using a transplantation design in which recipient blood vessels either were allowed to grow or not into the transplanted islets. RESULTS: TSP-1-deficient mice were glucose intolerant, despite having an increased ?-cell mass. Moreover, their islets had decreased glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate, as well as increased expression of uncoupling protein-2 and lactate dehydrogenase-A when compared with control islets. Almost all TSP-1 in normal islets were found to be derived from the endothelium. Transplantation of free and encapsulated neonatal wild-type and TSP-1-deficient islets was performed in order to selectively reconstitute with TSP-1-positive or -negative blood vessels in the islets and supported that the ?-cell defects occurring in TSP-1-deficient islets reflected postnatal loss of the glycoprotein in the islet endothelial cells. Treatment of neonatal TSP-1-deficient mice with the transforming growth factor (TGF)?-1-activating sequence of TSP-1 showed that reconstitution of TGF?-1 activation prevented the development of decreased glucose tolerance in these mice. Thus, endothelial-derived TSP-1 activates islet TGF?-1 of importance for ?-cells. CONCLUSIONS: Our study indicates a novel role for endothelial cells as functional paracrine support for pancreatic ?-cells.
Project description:Thrombospondin-4 (TSP-4) is 1 of the 5 members of the thrombospondin protein family. TSP-1 and TSP-2 are potent antiangiogenic proteins. However, angiogenic properties of the 3 other TSPs, which do not contain the domains associated with the antiangiogeneic activity of TSP-1 and TSP-2, have not been explored. In our previous studies, we found that TSP-4 is expressed in the vascular matrix of blood vessels of various sizes and is especially abundant in capillaries. We sought to identify the function of TSP-4 in the regulation of angiogenesis.The effect of TSP-4 in in vivo angiogenesis models and its effect on angiogenesis-related properties in cultured cells were assessed using Thbs4(-/-) mice, endothelial cells (EC) derived from these mice, and recombinant TSP-4. Angiogenesis was decreased in Thbs4(-/-) mice compared with wild-type mice. TSP-4 was detected in the lumen of the growing blood vessels. Mice expressing the P387 TSP-4 variant, which was previously associated with coronary artery disease and found to be more active in its cellular interactions, displayed greater angiogenesis compared with A387 form. Lung EC from Thbs4(-/-) mice exhibited decreased adhesion, migration, and proliferation capacities compared with EC from wild-type mice. Recombinant TSP-4 promoted proliferation and the migration of EC. Integrin ?2 and gabapentin receptor ?2?-1 were identified as receptors involved in regulation of EC adhesion, migration, and proliferation by TSP-4.TSP-4, an extracellular matrix protein previously associated with tissue remodeling, is now demonstrated to possess proangiogenic activity.
Project description:Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and beta1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP-1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP-1-null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP-1, designated 3TSR (for three TSP-1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild-type mice with 3TSR significantly decreased VEGF-induced permeability. Consistent with this result, VEGF-stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR-treated mice. Moreover, 3TSR significantly decreased VEGF-stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP-1 and 3TSR modulate the function of VEGFR2.
Project description:RATIONALE:Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown. OBJECTIVE:We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE(-/-) mice. METHODS AND RESULTS:Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4(-/-)/ApoE(-/-) mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE(-/-) mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ? 2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on ?(2) and ?(3) integrins, which recognize TSP-4 as a ligand. CONCLUSIONS:TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.
Project description:This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.
Project description:Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.
Project description:Angiogenesis is critical for the growth and proliferation of tumors as well as for normal development. We now describe a novel role for histidine-rich glycoprotein (HRGP) in the modulation of angiogenesis. HRGP is a plasma protein that circulates in relatively high concentrations (1.5 microM), but has no known function in vivo. We have shown previously that HRGP binds with high affinity to thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that is a potent inhibitor of angiogenesis. The antiangiogenic activity of TSP-1 is mediated by the binding of properdin-like type I repeats to the receptor CD36. We found that binding of HRGP to TSP-1 was similarly mediated by TSP type I repeats. HRGP colocalized with TSP-1 in the stroma of human breast cancer specimens, and this interaction masked the antiangiogenic epitope of TSP-1. In assays performed in vitro of endothelial cell migration and tube formation, and in vivo corneal angiogenesis assays, HRGP inhibited the antiangiogenic effect of TSP-1. These studies suggest that HRGP can modulate the antiangiogenic activity of TSP-1, and identify a potential mechanism of resistance to the antiangiogenic effect of TSP-1.
Project description:TGF-? is a multifunctional cytokine affecting many cell types and implicated in tissue remodeling processes. Due to its many functions and cell-specific effects, the consequences of TGF-? signaling are process-and stage-dependent, and it is not uncommon that TGF-? exerts distinct and sometimes opposing effects on a disease progression depending on the stage and on the pathological changes associated with the stage. The mechanisms underlying cell- and process-specific effects of TGF-? are poorly understood. We are describing a novel pathway that mediates induction of angiogenesis in response to TGF-?1. We found that in endothelial cells (EC) thrombospondin-4 (TSP-4), a secreted extracellular matrix (ECM) protein, is upregulated in response to TGF-?1 and mediates the effects of TGF-?1 on angiogenesis. Upregulation of TSP-4 does not require the synthesis of new protein, is not caused by decreased secretion of TSP-4, and is mediated by activation of SMAD3. Using Thbs4-/- mice and TSP-4 shRNA, we found that TSP-4 mediated pro-angiogenic functions in cultured EC and angiogenesis in vivo in response to TGF-?1. We observed~3-fold increases in tumor mass and levels of angiogenesis markers in animals injected with TGF-?1, and these effects did not occur in Thbs4-/- animals. Injections of an inhibitor of TGF-?1 signaling SB-431542 also decreased the weights of tumors and cancer angiogenesis. Our results from in vivo angiogenesis models and cultured EC document that TSP-4 mediates upregulation of angiogenesis by TGF-?1. Upregulation of pro-angiogenic TSP-4 and selective effects of TSP-4 on EC may contribute to stimulation of tumor growth by TGF-? despite the inhibition of cancer cell proliferation.
Project description:Thrombospondin-1 (TSP-1), a matricellular protein widely acclaimed to be involved in the inhibition of angiogenesis and tumorigenesis, is synthesized and secreted by many cell types, including osteoblast and cancer cells. TSP-1 is highly upregulated during early stage of osteogenesis, whereas it inhibits terminal osteoblast differentiation. Expression of TSP-1 is downregulated in cancer cells, and its ectopic expression has been shown to restrain tumor growth. Transcriptional regulation of TSP-1 in osteogenesis and cancer is poorly understood; this prompted us to study its regulation by the two key regulators of the aforementioned processes: Runx2 and Runx3. Through a PCR-based cDNA subtraction technique, we identified and cloned a cDNA fragment for mouse TSP-1, whose expression was dramatically upregulated in response to Runx2 expression in mesenchymal stem cells. Moreover, TSP-1 expression was considerably reduced in the lung of Runx2 knockout mouse. On the other hand, TSP-1 gene expression drastically increased at both the transcriptional and translational levels in response to Runx3 expression in B16-F10 melanoma cells. In line with this, Runx2 and Runx3 bound to the TSP-1 promoter and stimulated its activity. Hence, these results provide first line of evidence that TSP-1 is a transcriptional target gene of Runx2 and Runx3.