AbstractMechanisms of hypoxia-related angiogenesis are important for uterine smooth muscle tumors. Factors that are related to angiogenesis during hypoxia include vascular endothelial growth factor (VEGF), hypoxia inducible factor 1α (HIF1α), T-cell intracellular antigen1 (TIA1), eukaryotic translation initiation factor 2α (eIF2α) and thrombospondin 1 (TSP1). We investigated immunoreactivities of VEGF, HIF1α, TIA1, eIF2α and TSP1 using an indirect immunoperoxidase method for formalin fixed, paraffin embedded tumors that had been diagnosed as leiomyoma (LMY), cellular leiomyoma (CLM) or leiomyosarcoma (LMS). TSP1 immunoreactivity was scored as moderate, mild or minimal, while VEGF, eIF2α and TIA1 immunoreactivities were scored as mild, moderate and strong in LMY, CLM and LMS samples, respectively. HIF1α immunoreactivity was scored as mild to minimal in LMY, CLM and LMS samples, but showed no statistically significant differences among samples. Although angiogenic factors showed strong immunohistochemical staining intensity in LMS, anti-angiogenic factors showed minimal immunohistochemical intensity. There was no difference in HIF-1α immunoreactivity compared to LMY, CLM and LMS samples. We suggest that HIF1α protein synthesis could be suppressed by eIF2α and TIA1. Furthermore, VEGF could be activated by pathways such as COX2, Ras, NF-?B or c-myc instead of HIF1α. Angiogenesis could trigger and accelerate tumor development; therefore, anti-angiogenic therapy could be useful for treatment of tumors. 相似文献
Abstract Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of lig-ands such as EGF or transforming growth factor alpha (TGF-α) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10–16, 36–37, 40–47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-α which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor. 相似文献
The thrombospondin type-1 domain containing 7A (THSD7A) protein is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD7A antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this protein contains 21 extracellular domains and with the exception of the first two domains, has a regular repeating pattern of TSP-1-like followed by F-spondin-like domains. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-1-like domains. Of the 21 domains, 18 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like domains have insertions in the canonical TSP fold, most notably the coiled coil region in domain 4, which may be utilized in protein-protein binding interactions, suggesting that this protein functions as a heparan sulfate binding site. 相似文献
AbstractContext: Lipoic acid (LA) and hyperbaric oxygenation therapy (HBOT) improve chronic wound healing.Objective: We compared the effects of LA or its enantiomer R-(+)-lipoic acid (RLA) on wound healing.Materials and methods: Groups LA?+?HBOT (L), RLA?+?HBOT (R) and placebo?+?HBOT (P). Lesion areas measured before treatment and on 20th and 40th day. The biopsies and plasma were harvested before treatment and on 7th and 14th (measurements of VEGF, vascular endothelial growth factor; EGF, epidermal growth factor, TNF-α and IL-6).Results: Ulcers improved more on RLA. In both L and R groups, EGF and VEFG increased in time. RLA decreased IL-6 on T7 and T14, which did not happen with LA. TNF-α levels decreased on T14 in both LA and RLA.Discussion: The improved wound healing is associated with increased EGF and VEGF and reduced plasma TNF-α and IL-6.Conclusion: RLA may be more effective than LA in improving chronic wound healing in patients undergoing HBO therapy. 相似文献
Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that function at cell surfaces, in extracellular matrix (ECM) and as bridging molecules in cell-cell interactions. TSPs are multifunctional and modulate cell behavior during development, wound-healing, immune response, tumor growth and in the homeostasis of adult tissues. TSPs are assembled as oligomers that are composed of homologous polypeptides. In all the TSP polypeptides, the most highly-conserved region is the carboxyl-region, which contains a characteristic set of domains comprising EGF domains, TSP type 3 repeats and a globular carboxy-terminal domain. This large region is termed here the thrombospondin carboxy-terminal cassette (TSP-CTC). The strong conservation of the TSP-CTC suggests that it may mediate ancestral functions that are shared by all TSPs. This review summarizes the current knowledge of the TSP-CTC and areas of future interest. 相似文献
Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression. 相似文献
Fibrillin‐1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs‐like) 6β is one of the fibrillin‐1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6β in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6β and examined their interactions with fibrillin‐1 assembly. Pull‐down assay of the ADAMTSL6β deletion mutants and fibrillin‐1 protein revealed that ADAMTSL6β binds to fibrillin‐1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin‐1 matrix assembly was enhanced in MG63 cells, expressing full‐length ADAMTSL6β, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3‐ADAMTSL6β form showed enhanced assembly formation, Δ TSP2‐ADAMTSL6β form did not enhance that, indicating the difference between Δ TSP2‐Δ TSP3 has a critical role for fibrillin‐1 assembly. As the difference of Δ TSP2‐Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6β influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6β in the process of microfibril formation. 相似文献
Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin, is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood.
Scope of review
This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell–ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs).
Major conclusions
Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively.
General significance
Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell–ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. 相似文献
The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residue aspartate-rich, Ca(2+)-binding repeats; and an approximately 230-residue C-terminal sequence. The Ca(2+)-binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca(2+)-binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca(2+) in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca(2+). Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca(2+)-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern. 相似文献
Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling. 相似文献
AbstractContext: Garcinia hombroniana Pierre, known as manggis hutan in Malaysia is a rich source of xanthones and benzophenones.Objectives: This study was aimed to isolate and characterize potential cholinesterase inhibitors from the extracts of G. hombroniana bark and investigate their interactions with the enzymes.Materials and methods: The dichloromethane extract afforded five triterpenoids which were characterized by NMR and mass spectral techniques. Cholinesterase inhibitory assay and molecular docking were performed to get insight of the inhibitory activity and molecular interactions of the compounds. The compounds were also tested for their antioxidant capacity.Results: The isolated triterpenoids were identified as: 2β-hydroxy-3α-O-caffeoyltaraxar-14-en-28-oic acid (1), taraxerol (2), taraxerone (3), betulin (4) and betulinic acid (5). Compound 1 was the most active dual inhibitor of both AChE and BChE. Compound 1 also showed good antioxidant activities.Conclusion: Compound 1 had dual and moderate inhibitory activity on AChE and BChE worthy for further investigations. 相似文献
The levels of thrombospondin 2 (TSP2) and thrombospondin 3 (TSP3) mRNAs in a variety of human tissues were determined by analysis of multiple-tissue mRNA dot blots. For TSP2 mRNA, aorta and fetal heart had the greatest relative abundance. High levels were also detected for muscle, fetal, endocrine, immune, and nerve tissues. The pattern of expression of TSP3 mRNA was very different: kidney, pituitary gland, trachea, uterus, and fetal kidney had the greatest abundance. In general, TSP3 mRNA was expressed at high levels in endocrine, muscle, and fetal tissues. In addition to the tissue-specific differences, a more even distribution of TSP3 mRNA among tissues was observed. The high relative abundance of the two mRNAs in a variety of tissues and the tissue-specific differences in expression could be significant for understanding the diverse roles implicated for TSP2 and TSP3. 相似文献
Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells
via CD47. Because CD47 binding motifs are conserved in the C-terminal
signature domains of all five thrombospondins and indirect evidence has
implied CD47 interactions with other family members, we compared activities of
recombinant signature domains of thrombospondin-1, -2, and -4 to interact with
CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4
were less active than that of thrombospondin-1 for inhibiting binding of
radiolabeled signature domain of thrombospondin-1 or SIRPα
(signal-regulatory protein) to cells expressing CD47. Consistent with this
binding selectivity, the signature domain of thrombospondin-1 was more potent
than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP
synthesis in vascular smooth muscle cells and downstream effects on cell
adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary
vascular cells from thrombospondin-2-null mice lack enhanced basal and
NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on
NO/cGMP signaling could be detected only in thrombospondin-1-null cells.
Furthermore, tissue survival of ischemic injury and acute recovery of blood
flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore,
thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and
its limiting role in acute ischemic injury responses is not shared by
thrombospondin-2.Nitric oxide (NO) is a major mediator of intracellular and paracellular
signal transduction. NO preserves vascular health by minimizing the adhesion
of inflammatory cells to the vessel wall, limiting platelet activation, and
increasing blood vessel diameter and blood flow by relaxing vascular smooth
muscle cells (VSMC).3
These actions of NO are mediated by activating soluble isoforms of guanylate
cyclase (sGC) to increase cGMP levels, resulting in downstream activation of
cGMP-dependent protein kinases and ion channels
(1).Physiological NO/cGMP signaling is limited by several phosphodiesterases
that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein
that is produced by vascular and inflammatory cells that regulates cellular
behavior by engaging several cell surface receptors. Recently we reported that
TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and
VSMC (2,
3). TSP1 also plays a role in
promoting platelet thrombus formation and hemostasis by antagonizing the
antithrombotic activity of NO
(4). In all of these vascular
cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated
fluxes in cGMP by engaging its receptor CD47
(5). Nanomolar concentrations
of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated
uptake of myristate into vascular cells
(6). In vivo, mice
lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater
increases in regional blood flow in response to a NO challenge than wild type
controls (4). After an ischemic
insult, the absence of TSP1 or CD47 in transgenic mice is associated with
better maintenance of tissue perfusion and enhanced tissue survival.
Similarly, targeting TSP1 or CD47 using function blocking antibodies enhances
ischemic tissue perfusion and survival in wild type mice and pigs
(7,
8).TSP1 belongs to a family of five secreted glycoproteins that share an
evolutionarily conserved C-terminal signature domain
(9). TSP1 and TSP2 form a
distinct subfamily of trimeric proteins that exhibit similar anti-angiogenic
activities for endothelial cells in vitro and activities in
vivo to block tumor growth. Despite their similarities in structure, TSP1
and TSP2 have markedly different expression patterns after tissue injury, with
TSP1 being immediately expressed and maximal at day 3, whereas TSP2 was not
expressed until day 7 and was maximal 10 days after injury
(10). In addition, large
amounts of TSP1 but not TSP2 are stored in platelet α-granules and
released into the wound environment. Polymorphisms in TSP1 and TSP2 have been
linked to altered risk of premature myocardial infarction
(11,
12). A 3′-untranslated
region polymorphism in TSP2 is also associated with type 2 diabetes in men
(13). The molecular basis for
these associations is unclear.Less is known about the roles of the pentameric TSP3–5 in vascular
cells. TSP3 and TSP5 (also known as cartilage oligomeric matrix protein)
appear to serve their primary functions in bone development
(14,
15). However, a polymorphism
in TSP4 is associated with premature myocardial infarcts in certain
populations (11,
16,
17). A proatherogenic activity
for the A387P variant of TSP4 was proposed based on its differential ability
to modulate proliferation of endothelial and VSMC
(18). Cardiovascular functions
of TSP4 may also be linked to the high expression of TSP4 in heart
(19) and its altered
expression in that tissue during hypertensive heart failure
(20).The C-terminal domain of TSP1 is sufficient to mediate CD47-dependent
inhibition of cGMP signaling
(5). Of the two CD47 binding
VVM motifs identified in this domain of TSP1, the first is conserved among all
five TSPs, suggesting that CD47 binding could be a universal attribute of this
family (21). Based on
structural evidence that the VVM motifs may not be accessible
(22,
23), however, conservation of
VVM motifs may not be sufficient to predict CD47 binding. Uncertainty
regarding the location of the CD47 binding site in the G domain of TSP1
therefore limits interpretation of the known sequence homology to predict CD47
binding to other TSP family members.Although CD47 recognition of other TSPs has not been demonstrated
experimentally, a local deficiency of inflammation-associated T cell apoptosis
shared by TSP1-, CD47-, and TSP2-null mice is consistent with this hypothesis
(24). Furthermore, a
21-residue peptide from the C-terminal domain of TSP4 was found to decrease
human umbilical vein endothelial cell proliferation similar to the CD47
binding peptides from TSP1, although it lacks the VVM motif and no interaction
with CD47 was demonstrated
(25).To directly address whether other TSP family members can inhibit NO
responses and signaling in vascular cells, we now compare binding of
recombinant signature domains of TSP1, TSP2, and TSP4 to cell surface CD47 and
inhibition of NO-stimulated cell responses and cGMP signaling by these
domains. We also compared acute tissue blood flow and perfusion responses to
ischemic challenge in TSP1 and TSP2-null mice and cGMP responses in primary
cultures of vascular cells isolated from these mice. These studies clearly
demonstrate that CD47 selectively interacts with TSP1 and that the signature
domains of TSP2 and TSP4 are less potent inhibitors of NO signaling in
vascular cells in vitro. Furthermore, we show that the role of TSP1
to acutely limit recovery from ischemic injury in vivo is not shared
by TSP2. 相似文献
Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE.
OBJECTIVE: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. METHODS: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. RESULTS: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. CONCLUSION: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. 相似文献