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Vol. 19, Issue 2, 563-571, February 2008
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Departments of *Biochemistry and Medicine, University of Washington, Seattle, WA 98195; Millipore, Temecula, CA 92590; and The Center for Vascular and Inflammatory Diseases, and the Departments of Surgery and Physiology, University of Maryland, Rockville, MD 20855
Submitted July 10, 2007;
Revised October 10, 2007;
Accepted November 9, 2007
Monitoring Editor: Josephine Adams
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bornstein, 2001; Kyriakides and Bornstein, 2003; Adams and Lawler, 2004), these proteins are best known for their ability to inhibit angiogenesis (Adams, 2001; Lawler, 2002; Armstrong and Bornstein, 2003). Of particular interest is the capability of TSPs to inhibit tumor growth and metastatic spread (Lawler and Detmar, 2004) and consequently the potential of these proteins to serve as a basis for the development of therapeutic antiangiogenic agents (Zhang and Lawler, 2007).
In view of these properties, it is to be expected that substantial efforts have been made to understand the mechanisms by which the growth of blood vessels can be inhibited by TSPs. The antiangiogenic activity of TSPs was first recognized by Bouck and coworkers, who identified the product of a tumor suppressor gene in hamster cells as a 140-kDa fragment of TSP1 (Rastinejad et al., 1989; Good et al., 1990). Subsequently, an analysis of the phenotype of the TSP2-null mouse and studies of wound healing and the foreign body reaction in these mice confirmed that TSP2 also possessed potent antiangiogenic properties (Kyriakides et al., 1998a; Kyriakides and Bornstein, 2003).
The mechanisms by which TSPs inhibit angiogenesis are complex and comprise both indirect and direct effects on endothelial cells (ECs; Zhang and Lawler, 2007). Indirect effects include direct binding and clearance of a number of growth factors, including vascular endothelial growth factor (VEGF)-A (Greenaway et al., 2007), as well as inhibition of mobilization of growth factors from matrix stores, and clearance of TSP/metalloproteinase complexes from the pericellular environment by the endocytic receptor, low-density lipoprotein–related protein-1 (LRP1). TSP1 is also capable of activating latent transforming growth factor (TGF) β1, which can yield both positive and negative effects on angiogenesis (see review by Zhang and Lawler, 2007, for additional references regarding the indirect effects of TSPs on angiogenesis).
The direct effects of TSPs on ECs are also complex. Angiogenesis can be stimulated by ligation of the N-terminal domain of TSP1 to β1 integrins, whereas interaction of TSPs with either CD36 or CD47 is capable of inhibiting NO-stimulated EC proliferation (Isenberg et al., 2006 and references therein). Migration of both human microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs) is also inhibited by interaction of the type I repeats of TSPs with β1 integrins (Short et al., 2005). However the ultimate fate of such inhibitory effects on ECs was not reported. There is now good evidence that both TSP1 and 2 can cause apoptosis of HMVECs by a pathway that includes ligation of the CD36 receptor and subsequent activation of p58fyn, mitogen-activated protein kinases (MAPKs), and FAS ligand. FAS ligand can then interact with FAS on an adjacent ECs, with the resulting activation of caspases (Guo et al., 1997; Jimenez et al., 2000; Nor et al., 2000; Simantov et al., 2005).
However, it seems apparent to us that cell death will not always represent the most appropriate physiological consequence of the inhibition of angiogenesis, in view of the need for a homeostatic inhibition of EC proliferation in a normal adult animal. Indeed, studies of the rate of replication of aortic ECs in healthy adult rats have revealed life spans ranging from 2 to 10 mo, depending on their anatomical location (Schwartz and Benditt, 1973), and longer life spans might be expected in humans. This quiescence exists despite the fact that the endothelial layer of the vasculature is constantly exposed to plasma, which although it lacks the potent mitogenic activity of serum, does have sufficient VEGF to stimulate EC growth. Thus, in a carefully performed study, the levels of VEGF in normal adult plasma varied from 76 to 108 pg/ml, depending on the method of assay, whereas the level in serum was 249 pg/ml (Webb et al., 1998). Presumably, the VEGF in plasma originates in large part from platelets, the 
granules of which are known to contain the growth factor in substantial amounts (Salgado et al., 2001; Kellouche et al., 2007). It seems plausible that VEGF could be released from platelets in the absence of overt trauma. Furthermore, in the event of trauma, it would seem physiologically preferable for ECs in the periphery of the wound to become quiescent rather than to apoptose in response to the TSP1 released from platelets because, in the latter case, the exposure of additional subendothelial matrix could lead to more extensive and undesirable thrombosis.
Although the very low density lipoprotein receptor (VLDLR) is known to be expressed in capillaries and arterioles of a number of organs including skeletal muscle, heart, liver, ovary, and brain (Wyne et al., 1996), the primary role of this receptor was considered to be nutritional, i.e., the delivery of lipoproteins to cells by virtue of its endocytic function. Presumed binding and endocytosis of TSP1 by the VLDLR, and subsequent degradation of TSP1, were also shown in murine fibroblasts that were genetically deficient in LRP1, but had been transduced with an adenoviral vector expressing the VLDLR (Mikhailenko et al., 1997). However, a signaling function for the VLDLR has also been recognized, as demonstrated by the ability of reelin to modulate neuronal migration, neurodevelopment, and other physiological processes in the CNS (Stolt and Bock, 2006). Binding of reelin to the VLDLR induces phosphorylation of dab1 in neurons. However, to our knowledge, dab1 expression is limited to neurons, and there is no evidence that either dab1 or dab2 is expressed in ECs.
This report establishes another important signaling function for the VLDLR, i.e., the regulation of cell cycle progression in microvascular EC. Moreover, it identifies TSPs as ligands that are capable of eliciting this function, and documents some of the steps in the resulting signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Purification of Recombinant TSP2 from Chinese Hamster Ovary Cells
Chinese Hamster Ovary (CHO) cells constitutively expressing full-length mouse TSP2 and 925 culture medium were kindly provided by Genzyme (Framingham, MA). The CHO cells were grown to confluence in 925 medium with 10% fetal bovine serum (FBS), washed twice with serum-free medium, and then cultured in this medium for 24 or 48 h. The medium was collected, and TSP2 was purified using HiTrap Heparin HP resins (GE Healthcare, Piscataway, NJ). The purity of the protein was determined by silver staining with a Silver Stain Plus kit (Bio-Rad, Hercules, CA).
Endothelial Cell Culture
Human neonatal dermal microvascular ECs were obtained from Cambrex (Walkersville, MD) at passage 3, and experiments were performed between passages 5 and 7. The cells were suspended in EGM2-MV medium, and 40,000 cells per well were plated in 24-well plates and allowed to adhere for several h. The cells were then synchronized in 0.5% FBS for 24 h, followed by overnight stimulation with VEGF and/or TSP2. The cells were then cultured for an additional 3 h in the presence of [3H]thymidine. Additional details are provided in Armstrong et al. (2002).
Overexpression of CD36 by Adenoviral Infection of ECs
HUVEC and HMVEC were suspended in MCDB131 medium (Sigma, St. Louis, MO) with 5% FBS and were plated at 30,000 cells/well in 48-well plates for 4 h. The medium was removed and cells were incubated overnight with an MOI of 50 of adenovirus-expressing CD36, prepared as described by de Villiers et al. (2001). Overexpression was confirmed by immunostaining of ECs with antisera against rat CD36 (de Villiers et al., 2001).
Determination of DNA Synthesis
HMVEC were plated at 40,000 cells per well in 24-well plates in EGM2-MV culture medium (Cambrex). The cells were synchronized in EBM2 basal medium containing 0.5% FBS for 24 h and were stimulated with 10 ng/ml VEGFA165 (Endogen, Woburn, MA) in the absence or presence of 10 µg/ml TSP2 or TSP1 (Hematologic Technologies, Essex Junction, VT), or 1 µM RAP, or a cocktail of VLDLR-specific monoclonal antibodies (1H5, 5F3, and 1H10, 10 µg/ml each (Strickland et al., 1990; Ruiz et al., 2005). The cells were cultured overnight and then labeled with [3H]thymidine for 3 h. The labeled cells were washed twice with cold 5% trichloroacetic acid and twice with cold PBS. DNA was solubilized with 0.25 M NaOH and mixed with EcoLite (+) liquid scintillation cocktail (MP Biomedicals, Irvine, CA) for scintillation counting. In some experiments (see Figures 3 and 4) HMVEC were cultured in EGM-MV medium with 5% serum and were stimulated with a cocktail of growth factors (VEGF, 10 ng/ml; bFGF, 20 ng/ml; insulin-like growth factor, 20 ng/ml, and hepatocyte growth factor, 5 ng/ml; Armstrong et al., 2002).
Apoptosis and Cell Cycle Assays
HMVEC were plated in EGM2-MV culture medium and were cultured for 24 h in EBM2 basal medium containing 0.5–5% FBS. In some experiments, cells cultured in 0.5% FBS were supplemented with 10 ng/ml VEGF in the absence or presence of 10 µg/ml TSP2. Cells were labeled with Annexin V-PE (phycoerythrin) for apoptosis assays according to the manufacturer's recommendations (BD Biosciences, Palo Alto, CA). Briefly, cells were washed twice with cold PBS, trypsinized, and washed with PBS, and 1 x 105 cells were resuspended in 100 µl of 1x binding buffer. Five microliters of Annexin V-PE was added, and the cells were incubated in the dark for 15 min, before analysis by flow cytometry.
For cell cycle analyses, HMVEC were plated in EGM2-MV culture medium. This medium was replaced with EBM2 basal medium containing 0.5% FBS, and the cells were cultured for 24 h with 10 ng/ml VEGF, in the absence or presence of 10 µg/ml TSP2. Cells were washed with cold PBS, trypsinized, and stained with DAPI (4,6-diamidino-2-phenylindole). Cell cycle analysis was performed by flow cytometry.
Coimmunoprecipitation of TSP2 with the VLDLR
HMVEC were incubated at 37°C with 10 µg/ml TSP2 for 4 h or overnight. The cells were washed twice with cold PBS and lysed in buffer containing 1% Triton X-100. Monoclonal antibodies against soluble human VLDLR (5F3, 1H5, and 1H10, 10 µg/ml each; Ruiz et al., 2005) or control purified monoclonal normal mouse IgG (Santa Cruz Biotechnologies, Inc, Santa Cruz, CA) was added to cell lysates, and the solutions were incubated overnight at 4°C. Thirty microliters of protein A/G Sepharose beads was added, and the incubation was continued for an additional 3 h at 4°C. The beads were pelleted and washed four times with lysis buffer and once with 10 mM Tris, pH 6.8. The beads were then boiled in SDS-sample buffer, and the supernatant was resolved by electrophoresis in SDS polyacrylamide gels, electroblotted onto nitrocellulose membrane, and probed with affinity-purified rabbit anti-mouse TSP2 polyclonal antibodies (Kyriakides et al., 1998b) to detect TSP2 that had interacted with the VLDLR.
Surface Plasmon Resonance
Binding of TSP1 and 2 to purified mouse VLDLR was measured with a BIA 3000 optical biosensor (Biacore AB, Uppsala, Sweden) as described previously (Loukinova et al., 2002). For these studies, the BIAcore sensor chip (type CM5; Biacore) was activated with a 1:1 mixture of 0.2 M N-ethyl-N'-(3-dimethylaminopropyl) carbodi-imide and 0.05 M N-hydroxysuccinimide in water. Soluble VLDLR and purified LRP1 were immobilized in a working solution of 10 µg/ml in 10 mM sodium acetate, pH 4.0, at a flow rate of 5 µl/min. The remaining binding sites were blocked by 1 M ethanolamine, pH 8.5, and unbound protein was washed out with 0.5% SDS. An additional flow cell, similarly activated and blocked without immobilization of protein, served as a negative control. A flow cell with immobilized ovalbumin was used as a control for nonspecific protein binding. All binding reactions were performed in 10 mM HEPES, 0.15 M NaCl, 0.005% Tween 20, pH 7.4. Binding of TSP1 and TSP2 were measured at 25°C at a flow rate of 30 µl/min for 4 min, followed by 4 min of dissociation. The bulk shift due to changes in refractive index measured on blank surfaces was subtracted from the binding signal for each condition to correct for nonspecific signals. Chip surfaces were regenerated with subsequent 1-min pulses of 10 mM sodium acetate, pH 4.0, containing 1 M NaCl, and 10 mM NaOH containing 1 M NaCl, followed by a 2-min wash with running buffer to remove this high-salt solution. All injections were performed using Application Wizard in the automated method. Data were analyzed with BIA evaluation 3.0 software (Biacore).
Determination of p-Akt Levels in TSP2-treated HMVECs
HMVEC were cultured in tissue culture plates in EGM2-MV medium with 5% FBS and were used at 50–60% confluence. Growth medium was changed to medium containing 0.5% FBS, and the cells were treated for 4 h with 10 µg/ml TSP2. Cells were washed with PBS and were stimulated with 10 ng/ml recombinant VEGF (Endogen) for 15 min. The cells were lysed on ice for 20 min in a lysis buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, protease inhibitor cocktail (Roche, Indianapolis, IN), 20 mM NaF, 1 mM Na3VO4, and 10 mM β-glycerolphosphate. Cell lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4°C, and protein concentrations were determined by BCA assay (Pierce, Rockford. IL). Equal amounts of total protein in cell lysates (30 µg) were resolved on a 12% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. The Western blot analyses were performed with phospho-Akt or pan-Akt antibodies (Cell Signaling Technology, Beverly, MA), followed by incubation with the appropriate secondary antibody. The protein bands were then visualized by chemiluminescence (Pierce).
Determination of Phosphorylated (Activated) MAPK
HMVEC were cultured in EGM-2 MV medium and used at 60% confluency. Growth medium was changed to medium containing 0.5% FBS, and cells were treated for 4 h with 10 µg/ml TSP2, or 1 µM RAP, or with 10 µg/ml VLDLR antibodies. Cells were washed with PBS and stimulated for 10 min with 10 ng/ml recombinant VEGF in medium containing 0.5% FBS. Cells were lysed and equal amounts of total protein in cell lysates (30 µg) were separated on a 12% SDS-polyacrylamide gel and were blotted onto a nitrocellulose membrane. The blocked membranes were then incubated with a phospho-specific MAPK antibody that detects endogenous levels of p42 MAPK and p44 MAPK (Erk1 and Erk2) dually phosphorylated at threonine 202 and tyrosine 204 (Cell Signaling Technology). The membrane was stripped in β-mercaptoethanol stripping buffer at 50°C for 30 min and was reprobed with an anti-p44/p42 MAPK antibody, which detects total MAPK protein (Cell Signaling Technology).
Quantitative analyses of phospho-Akt and phospho-MAPK levels were performed with Adobe Photoshop 7.0.1 (San Jose, CA). Each bar in Figure 9 represents a relative protein level as determined by densitometry of phospho-Akt or phospho-MAPK bands, divided by the density of the corresponding loading control bands. The results are graphed as the mean ± SD (n = 3). Statistical analysis was performed using a two-tailed Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Conversely, when HUVECs, which lack CD36, were infected with an adenovirus containing a cDNA encoding CD36, the cells remained resistant to the inhibitory effects of TSP2 on DNA synthesis (Figure 3B). Although adenovirus-mediated overexpression of CD36 diminished growth factor–stimulated DNA synthesis in both HMVECs and HUVECs, HMVECs but not HUVECs remained sensitive to inhibition by TSP2, a result indicating that adenoviral infection alone does not interfere with the antiproliferative effects of TSP2 (Figure 3B).
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), and by homology to the same repeats in TSP2 (Simantov et al., 2005). Neither full-length TSP2 nor any of its fragments were effective in inhibiting DNA synthesis by HUVECs (Figure 4). These results suggest that the many integrins that have been identified on HUVECs, including vβ3, vβ5, 2β1, 4β1, 5β1, 6β1, and 6β4, do not play a role in mediating the antiproliferative effect of TSP2. However, we cannot exclude the possibilities that there might be a difference in downstream signaling from integrins in HUVECs compared with HMVECs or that a required coreceptor is lacking in HUVECs.
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; Bu, 2001), to counteract the inhibitory effect of TSP2 on VEGF-stimulated thymidine uptake by HMVECs. To our surprise, RAP completely inhibited the stimulation of thymidine incorporation by VEGF, both in the presence or absence of TSP2 or TSP1 (Figures 5, A and B). We initially considered the possibility that LRP1 was the target of RAP, because this member of the LDL receptor family is known to bind both TSP2 and TSP1, functions as a clearance receptor for TSP-MMP2 complexes and participates in the focal adhesion disassembly that results from the interaction of TSPs with calreticulin (Mikhailenko et al., 1997; Yang et al., 2001; Elzie and Murphy-Ullrich, 2004). However, mouse microvascular endothelial cells (MVECs) have been reported to express little or no LRP1 (Lillis et al., 2005), but rather express the VLDLR, which had been presumed to perform primarily a nutritional function by endocytosis of lipoproteins (Wyne et al., 1996). Indeed, monoclonal antibodies against the VLDLR also inhibited the stimulation of thymidine incorporation by VEGF in HMVECs (Figure 5). Because the effects of TSP2 or TSP1, RAP, and anti-VLDLR antibodies were not additive (Figures 5, A and B), it seems likely that these proteins all target the VLDLR and its signaling pathway.
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; Dardik et al., 2003), whereas mouse MVECs synthesize little if any TSP2 (Armstrong et al., 2002). It is possible that endogenous synthesis of TSP1 confers some resistance to the effects of added exogenous protein, although one would expect a comparable resistance to TSP2 if, as we believe, the intracellular pathways leading to inhibition of cell cycle progression are the same for the two proteins. Perhaps a more likely explanation is a difference in quality or posttranslational modifications of the TSP1, purified from platelets, and recombinant TSP2, synthesized in CHO cells.
Coimmunoprecipitation of VLDLR and TSP2 in Lysates of TSP2-treated HMVECs
To determine whether an interaction occurs between TSP2 and VLDLR, we added recombinant TSP2 to the medium of cultured HMVECs for 4 h or overnight. The cells were then lysed and a cocktail of VLDLR-specific monoclonal antibodies or purified monoclonal normal mouse IgG was used to immunoprecipitate a VLDLR-TSP2 complex. Western blot analysis with a TSP2-specific antibody revealed the presence of TSP2 in the immunoprecipitate (Figure 6, lane 2). TSP2 protein was absent in the lysates of control cells incubated in the absence of exogenous TSP2 (Figure 6, lane 1) and in the lysates of cells treated with the control mouse IgG and then with recombinant TSP2 (not shown). These data suggest, but do not prove, a direct interaction of TSP2 with VLDLR. However, evidence for a direct interaction between the two proteins is provided by the surface plasmon resonance data presented in Figure 7. For reasons that we have yet to explain, several attempts to coimmunoprecipitate the VLDLR with TSP1 from lysates of HMVECs or of rat smooth muscle cells expressing high levels of VLDLR have not been successful.
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In parallel experiments, performed on the same chip, similar analyses of the interaction of TSP1 with the VLDLR and LRP1 were carried out. The results were very similar to those for TSP2: a KD of 11 nM, with an association rate, ka, 8.4 x 10–4 M–1s–1 and a dissociation rate, kd, of 1.01 x 10–3 M–1s–1. For the positive control, the constants for the binding of TSP1 to LRP1 were: a KD of 2.3 nM, an association rate, ka, of 1.12 x 10–5 M–1s–1, and a dissociation rate, kd, of 2.54 x 10–3 M–1s–1.
TSP2 Blocks VEGF-induced Cell Cycle Progression without Triggering Apoptosis
HMVECs were cultured in 0.5% serum for 24 h before addition of VEGF to maximize the effect of the growth factor on cell proliferation. The cells were first cultured for 24 h in the presence of increasing concentrations of serum, ranging from 0 to 5%, to determine whether this low serum level caused apoptosis. HMVECs were subsequently labeled with annexin V-PE, a marker for apoptosis (see Materials and Methods) and examined by flow cytometry. The results of these analyses are presented in Figure 8A. The data indicate that cell culture in 0.5% serum did not cause a significant increase in apoptosis during this time period, compared with cells cultured in 2 or 5% serum. Thus the variability in the low levels of apoptosis determined in cells cultured in 0.5, 2, or 5% serum (Figure 8A) is within the error of the method.
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TSP2 Inhibits VEGF-induced Phosphorylation of Akt and MAPK
It has been shown that both phosphatidyl inositol-3 kinase (PI3K) and the PI3K-dependent serine-threonine kinase, Akt, can be activated in ECs by VEGF (Olsson et al., 2006). In preliminary experiments, we found that maximal phosphorylation of Akt occurs in HMVECs after 15 min of stimulation with 10 ng/ml VEGF. To determine whether TSP2 could interfere with the activation of Akt, HMVECs were plated in growth medium at 60% confluence, the growth medium was replaced with basal medium containing 0.5% FBS, and the cells were cultured for 24 h. Cells were first treated with 10 µg/ml TSP2 for 4 h and were subsequently stimulated for 15 min with 10 ng/ml VEGF; the levels of phospho-Akt (pAkt) were determined by Western blot analysis using a pAkt-specific antibody. As shown in Figure 9A, the level of pAkt was substantially increased in VEGF-stimulated HMVECs, compared with that in control untreated cells (compare lanes 2 and 1). Exposure to TSP2 decreased pAkt levels substantially (lane 3). These data indicate that TSP2 interferes with the PI3K/Akt signaling pathway.
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Figure 9, C and D, shows the results of scanning the gels generated in three independent experiments, similar to those depicted in A and B. The effect of VEGF in increasing the phosphorylation of Akt and MAPKs and that of TSP2 in inhibiting the stimulatory effect of TSP2 are clearly significant. As a control, we show that TSP2 alone has no effect on thymidine incorporation by HMVECs (Supplementary Figure 1) and therefore would not be expected to influence the phosphorylation of either Akt or MAPKs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and is thought to be the basis for the regression of tumors in experimental systems (Jimenez et al., 2000; Lawler and Detmar, 2004; Zhang et al., 2005), other mechanisms of action of these proteins in inhibition of endothelial proliferation have not been thoroughly explored. Indeed, to our knowledge, there is still no direct evidence that TSPs are capable of causing apoptosis of ECs in the normal healthy tissue surrounding tumors in experimental animals or in normal tissues elsewhere.
In an earlier publication, Armstrong et al. (2002) showed that both TSP1 and TSP2 inhibited the proliferation of HMVECs in response to a number of growth factors, including VEGF-A, without causing apoptosis, and that this proliferative response was also not inhibited by a broad-spectrum caspase inhibitor. However, the receptor and the signaling pathway responsible for this effect were not identified at the time. We now report the surprising finding that both TSP1 and 2 are capable of interacting with the VLDLR and propose that these interactions lead to the coordinate ligation of a VEGF-bound VEGFR, although such coordinate ligation is not essential to our conclusions. As a result of these interactions, the PI3K and MAPK pathways that normally stimulate cell cycle progression are inhibited (Figure 10). We propose that these interactions form the basis for an important homeostatic function of TSPs.
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). We do not have a good explanation for this discrepancy. It is possible that HUVECs express substantially lower levels of the VLDLR than other members of the LDL receptor family, such that TSP-induced changes in cell proliferation were not detected in our assays. Alternatively, these cells may express a splice variant of the receptor that does not perform a signaling function or that some of the components of that function (see below) are lacking in these cells. It is of interest in this regard that the closely related ApoER2 receptor exists in several splice forms (May et al., 2005).
The VLDLR was thought to function in most tissues primarily as a means of providing nutrition by endocytosis of lipoproteins (Wyne et al., 1996). However, more recent studies have also established a signaling role for VLDLR and several other members of the LDL family (Stolt and Bock, 2006). One or more NPXY cytoplasmic sequences have been identified in these endocytic receptors and in many other transmembrane proteins (Stolt and Bock, 2006). In the case of the VLDLR, and the closely related LDL family member, ApoER2, this sequence phosphorylates the phosphotyrosine-binding (PTB) domain–containing adapter protein, Dab1, in migrating neurons in the CNS, when these receptors are activated by the ligand, reelin. These interactions are required for cortical and cerebellar lamination during neurodevelopment (Trommsdorff et al., 1999).
The sequence of the VLDLR is highly conserved, with 94% identity in humans and rabbits and 84% in humans and chickens. It is therefore surprising that a preliminary analysis of mice with disruption of both alleles of the VLDLR gene has revealed virtually no consequences, other than a slight reduction in size and in adipose tissue mass (Frykman et al., 1995). This was true even after a number of challenges that included fasting, chilling at 4°C, strenuous exercise, and treatment with triiodothyronine (Frykman et al., 1995). Furthermore, an initial evaluation of VLDLR/LDLR double-null mice revealed no additional abnormalities. It seems likely that compensation by one or more of the 10 members of the LDL receptor family (Bu, 2001), in particular the ApoER2 receptor, could account for these findings.
Our proposed scheme for a partial signaling pathway that enables TSPs to counteract the stimulatory effects of VEGF on cell cycle progression (Figure 10) is based in part on recent studies showing that platelet-derived growth factor (PDGF) binds and activates LRP1, an activity that results in a transient phosphorylation of tyrosine 63 in the second NPXY sequence of the LRP1 cytoplasmic domain (Boucher et al., 2002; Loukinova et al., 2002). This phosphorylation is mediated by Src or Src family members and requires the kinase domain of the PDGF β receptor (Newton et al., 2005). However, the bridging of LRP and the PDGF receptor by a common ligand, PDGF, is apparently not necessary for the phosphorylation of LRP (Newton et al., 2005). PDGF-induced phosphorylation of LRP also produces a docking site for Shc, a group of three homologous adapter proteins that contain a carboxy-terminal Src-homology 2 (SH2), and an amino-terminal PTB domain that is involved in signal transduction by protein tyrosine kinases (Loukinova et al., 2002). Specifically, Shc has been reported to couple activated growth factor receptors to signaling pathways that regulate the proliferation of mammalian cells and plays a role in activation of MAPK (Pelicci et al.,1992; Ravichandran, 2001).
It is of interest that the ability of TSP1 to stimulate activation of the nonreceptor tyrosine kinase, p59fyn, is also inhibited by RAP in a macrophage-like line, J774. This finding has been attributed to a signaling function of LRP1 in these cells (Boucher et al., 2002). Activation of p59fyn had previously been identified as a step in the proapopototic pathway initiated by the engagement of the CD36 receptor by TSP1 in ECs (Jimenez et al., 2000). Although macrophages do express CD36, these findings are not necessarily contradictory, because microvascular EC do not express LRP1 (Lillis et al., 2005). Because RAP is unlikely to inhibit the CD36 receptor, the activation of p59fyn in J774 cells could result from a TSP1-induced association of CD36 and LRP1 or from direct activation by phosphorylated LRP1.
In summary, we have established that the proliferation of HMVECs, and therefore the process of angiogenesis, can be inhibited by the interaction of TSP1 or 2 with the VLDLR, a receptor not previously implicated in the function of TSPs. This process is not mediated by the CD36 receptor and does not require the type I repeats in TSPs. A scheme that depicts our findings in the context of the recent literature is shown in Figure 10. The role of TSPs in bridging the VLDLR and VEGFR is conjectural, but is supported by the known ability of TSP1 to bind VEGFA (Gupta et al., 1999; Greenaway et al., 2007). Alternatively, TSPs could be endocytosed by the VLDLR as previously shown for LRP1-bound TSP1 (Mikhailenko et al., 1995) and TSP2 (Yang et al., 2001), and signaling could be initiated from endocytic vesicles. Indeed, signaling from intracellular compartments has been shown for the VEGFR, a process that is controlled by vascular endothelial cadherin (Lampugnani et al., 2006). In analogy with the activation of LRP1 by PDGF-BB, which leads to activation of Src kinases and Shc, with the participation of PDGF receptor β (see above and Loukinova et al., 2002), we suggest that TSPs may function similarly to elicit a coordinated response from the VLDL and VEGFA receptors. Because PDGF functions as an important mitogen for mesenchymal cells (Heldin and Westermark, 1999) and activated Shc clearly plays a role in the activation of MAPK (Ravichandran, 2001), similarities are likely to exist between the downstream signaling pathways that are engaged by the interactions of PDGF-BB, LRP1, and PDGF receptor β on the one hand and TSPs, VLDLR, and VEGFR on the other.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Paul Bornstein (bornsten{at}u.washington.edu)
Abbreviations used: EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; HMVEC, human microvascular endothelial cell; LDL, low-density lipoprotein; LRP1, low-density lipoprotein–related protein-1; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PTB, phosphotyrosine-binding; RAP, receptor-associated protein; TSP, thrombospondin; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor 2; VLDL, very low density lipoprotein; VLDLR, VLDL receptor.
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REFERENCES |
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Adams, J. C., and Lawler, J. (2004). The thrombospondins. Int. J. Biochem. Cell Biol 36, 961–968.[CrossRef][Medline]
Argraves, K. M., Battey, F. D., MacCalman, C. D., McCrae, K. R., Gafvels, M., Kozarsky, K. F., Chappell, D. A., Strauss, J. F, 3rd, and Strickland, D. K. (1995). The very low density lipoprotein receptor mediates the cellular catabolism of lipoprotein lipase and urokinase-plasminogen activator inhibitor type I complexes. J. Biol. Chem 270, 26550–26557.
Armstrong, L. C., Björkblom, B., Hankenson, K. D., Siadak, A. W., Stiles, C. E., and Bornstein, P. (2002). Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism. Mol. Biol. Cell 13, 1893–1905.
Armstrong, L. C., and Bornstein, P. (2003). Thrombospondins 1 and 2 function as inhibitors of angiogenesis. Matrix Biol 22, 63–71.[CrossRef][Medline]
Bornstein, P. (2001). Thrombospondins as matricellular modulators of cell function. J. Clin. Invest 107, 929–934.[CrossRef][Medline]
Boucher, P., Liu, P., Gotthardt, M., Hiesberger, T., Anderson, R. G., and Herz, J. (2002). Platelet-derived growth factor mediates tyrosine phosphorylation of the cytoplasmic domain of the low density lipoprotein receptor-related protein in caveolae. J. Biol. Chem 277, 15507–15513.
Bu, G. (2001). The roles of receptor-associated protein (RAP) as a molecular chaperone for members of the LDL receptor family. Int. Rev. Cytol 209, 79–116.[Medline]
Dardik, R., Solomon, A., Loscalzo, J., Eskaraev, R., Bialik, A., Goldberg, I., Schiby, G., and Inbal, A. (2003). Novel proangiogenic effect of factor XIII associated with suppression of thrombospondin 1 expression. Arterioscler. Thromb. Vasc. Biol 23, 1472–14777.
Dawson, D. W., Pearce, S.F.A., Zhong, R., Silverstein, R. L., Frazier, W. A., and Bouck, N. P. (1997). CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J. Cell Biol 138, 707–717.
de Villiers, W. J. S., Cai, L., Webb, N. R., de Beer, M. C., van der Westhuyzen, D. R., and de Beer, F. C. (2001). CD36 does not play a direct role in HDL or LDL metabolism. J. Lipid Res 42, 1231–1238.
Elzie, C. A., and Murphy-Ullrich, J. E. (2004). The N-terminus of thrombospondin: the domain stands apart. Int. J. Biochem. Cell Biol 36, 1090–1101.[CrossRef][Medline]
Fischer, J. W., Stoll, M., Hahn, A. W., and Unger, T. (2001). Differential regulation of thrombospondin-1 and fibronectin by angiotensin II receptor subtypes in cultured endothelial cells. Cardiovasc. Res 51, 784–791.
Frykman, P. K., Brown, M. S., Yamamoto, T., Goldstein, J. L., and Herz, J. (1995). Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor. Proc. Natl. Acad. Sci. USA 92, 8453–8457.
Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M., Lemons, R. S., Frazier, W. A., and Bouck, N. P. (1990). A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA 87, 6624–6628.
Greenaway, J., Lawler, J., Moorehead, R., Bornstein, P., Lamarre, J., and Petrik, J. (2007). Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1). J. Cell Physiol 210, 807–818.[CrossRef][Medline]
Guo, N., Krutzsch, H. C., Inman, J. K., and Roberts, D. D. (1997). Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res 57, 1735–1742.
Gupta, K., Gupta, P., Wild, R., Ramakrishnan, S., and Hebbel, R. P. (1999). Binding and displacement of vascular endothelial growth factor (VEGF) by thrombospondin: effect on human microvascular endothelial cell proliferation and angiogenesis. Angiogenesis 3, 147–158.[CrossRef][Medline]
Heldin, C.-H., and Westermark, B. (1999). Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev 79, 1283–1316.
Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991). 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor. J. Biol. Chem 266, 21232–21238.
Isenberg, J. S., Ridnour, L. A., Dimitry, J., Frazier, W. A., Wink, D. A., and Roberts, D. D. (2006). CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1. J. Biol. Chem 281, 26069–26080.
Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L., and Bouck, N. (2000). Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nature Med 6, 41–48.[CrossRef][Medline]
Kellouche, S., Mourah, S., Bonnefoy, A., Schoevaert, D., Podgorniak, M. P., Calvo, F., Hoylaerts, M. F., Legrand, C., and Dosquet, C. (2007). Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation. Exp. Cell Res 313, 486–499.[CrossRef][Medline]
Kyriakides, T. R., and Bornstein, P. (2003). Matricellular proteins as modulators of wound healing and the foreign body response. Thromb. Haemost 90, 986–992.[Medline]
Kyriakides, T. R. et al. (1998a). Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J. Cell Biol 140, 419–430.
Kyriakides, T. R., Zhu, Y. H., Yang, Z., and Bornstein, P. (1998b). The distribution of the matricellular protein thrombospondin 2 in tissues of embryonic and adult mice. J. Histochem. Cytochem 46, 1007–1015.
Lampugnani, M. G., Orsenigo, F., Gagliani, M. C., Tacchetti, C., and Dejana, E. (2006). Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J. Cell Biol 174, 593–604.
Lawler, J. (2002). Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J. Cell Mol. Med 6, 1–12.[Medline]
Lawler, J., and Detmar, M. (2004). Tumor progression: the effects of thrombospondin-1 and -2. Int. J. Biochem. Cell Biol 36, 1038–1045.[CrossRef][Medline]
Lillis, A. P., Mikhailenko, I., and Strickland, D. K. (2005). ). Beyond endocytosis: LRP function in cell migration, proliferation and vascular permeability. J. Thromb. Haemost 3, 1884–1893.[CrossRef][Medline]
Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M. M., Loukinov, D., Ulery, P. G., Mikhailenko, I., Lawrence, D. A., and Strickland, D. K. (2002). Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein receptor-related protein (LRP). Evidence for integrated co-receptor function between LRP and the PDGF. J. Biol. Chem 277, 15499–15506.
May, P., Herz, J., and Bock, H. H. (2005). Molecular mechanisms of lipoprotein receptor signalling. Cell Mol. Life Sci 62, 2325–2338.[CrossRef][Medline]
Mikhailenko, I., Kounnas, M. Z., and Strickland, D. K. (1995). Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. A process facilitated by cell-surface proteoglycans. J. Biol. Chem 270, 9543–9549.
Mikhailenko, I., Krylov, D., Argraves, K. M., Roberts, D. D., Liau, G., and Strickland, D. K. (1997). Cellular internalization and degradation of thrombospondin-1 is mediated by the amino-terminal heparin binding domain (HBD). High affinity interaction of dimeric HBD with the low density lipoprotein receptor-related protein. J. Biol. Chem 272, 6784–6791.
Murphy-Ullrich, J. E., and Poczatek, M. (2000). Activation of latent TGF-beta by thromospondin-1, mechanisms and physiology. Cytokine Growth Factor Rev 11, 59–69.[CrossRef][Medline]
Newton, C. S., Loukinova, E., Mikhailenko, I., Ranganathan, S., Gao, Y., Haudenschild, C., and Strickland, D. K. (2005). Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptor-related protein (LRP). Evidence for co-receptor function. J. Biol. Chem 280, 27872–27878.
Nor, J. E., Mitra, R. S., Sutorik, M. M., Mooney, D. J., Castle, V. P., and Polverini, P. J. (2000). Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J. Vasc. Res 37, 209–218.[CrossRef][Medline]
Olsson, A.-K., Dimberg, A., Kreuger, J., and Claesson-Welsh, L. (2006). VEGF receptor signalling - in control of vascular function. Nat. Rev. Mol. Cell Biol 5, 359–371.
Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992). A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 70, 93–104.[CrossRef][Medline]
Rastinejad, F., Polverini, P. J., and Bouck, N. P. (1989). Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345–355.[CrossRef][Medline]
Ravichandran, K. S. (2001). Signaling via Shc family adapter proteins. Oncogene 20, 6322–6330.[CrossRef][Medline]
Ruiz, J. et al. (2005). The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor. J. Lipid Res 46, 1721–1731.
Salgado, R., Benoy, I., Bogers, J., Weytjens, R., Vermeulen, P., Dirix, L., and Van Marck, E. (2001). Platelets and vascular endothelial growth factor (VEGF): a morphological and functional study. Angiogenesis 4, 37–43.[CrossRef][Medline]
Schwartz, S. M., and Benditt, E. P. (1973). Cell replication in the aortic endothelium: a new method for study of the problem. Lab. Invest 28, 699–707.[Medline]
Short, S. M., Derrien, A., Narsimhan, R. P., Lawler, J., Ingber, D. E., and Zetter, B. R. (2005). Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by beta1 integrins. J. Cell Biol 168, 643–653.
Simantov, R., Febbraio, M., and Silverstein, R. L. (2005). The antiangiogenic effect of thrombospondin-2 is mediated by CD36 and modulated by histidine-rich glycoprotein. Matrix Biol 24, 27–34.[Medline]
Stolt, P. C., and Bock, H. H. (2006). Modulation of lipoprotein receptor functions by intracellular adaptor proteins. Cell Signal 18, 1560–1571.[CrossRef][Medline]
Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990). Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor. J. Biol. Chem 265, 17401–17404.
Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999). Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell 97, 689–701.[CrossRef][Medline]
Webb, N. J., Bottomley, M. J., Watson, C. J., and Brenchley, P. E. (1998). Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin. Sci 94, 395–404.[Medline]
Wyne, K. L., Pathak, K., Seabra, M. C., and Hobbs, H. H. (1996). Expression of the VLDL receptor in endothelial cells. Arterioscler. Thromb. Vasc. Biol 16, 407–415.
Yang, Z., Strickland, D. K., and Bornstein, P. (2001). Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2. J. Biol. Chem 276, 8403–8408.
Zhang, X., Galardi, E., Duquette, M., Lawler, J., and Parangi, S. (2005). Antiangiogenic treatment with three thrombospondin-1 type 1 repeats versus gemcitabine in an orthotopic human pancreatic cancer model. Clin. Cancer Res 15, 5622–5630.
Zhang, X., and Lawler, J. (2007). Thrombospondin-based antiangiogeneic therapy. Microvasc. Res Epub ahead of print.
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