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Jack Lawler

Publications and source records attributed to Jack Lawler.

36 records · Page 2Linked to original sources
PubMed2004

Inhibition of Tie-2 signaling induces endothelial cell apoptosis, decreases Akt signaling, and induces endothelial cell expression of the endogenous anti-angiogenic molecule, thrombospondin-1.

Small molecule inhibitors of endothelial cell specific tyrosine kinases are currently under investigation as potential means to block tumor angiogenesis. We have investigated the utility of blocking Tie-2 signaling in endothelial cells as a potential anti-angiogenic strategy. We have found that interruption of Tie-2 signaling either via RNAi or overexpression of a kinase-dead Tie-2 led to loss of endothelial cell viability, even in the presence of serum. Mechanistically, this is linked to a block in Akt signaling and increased thrombospondin expression. Thrombospondins are endogenous anti-angiogenic matricellular proteins known to regulate tumor growth and angiogenesis. We observed that both Tie-2 and subsequent PI3Kinase signaling regulates thrombospondin-1 expression. These data have lead to the model that Angiopoietin signaling through Tie-2 activates PI3Kinase/Akt, which represses thrombospondin expression. Thus, targeting Tie-2 with small molecules maybe efficacious as an anti-angiogenic therapy.

Angiogenesis Inhibitors↗
PubMed2004

Internalization but not binding of thrombospondin-1 to low density lipoprotein receptor-related protein-1 requires heparan sulfate proteoglycans.

The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

Animals↗
PubMed2004

Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression.

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen alpha3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.

Angiogenesis Inhibitors↗
PubMed2004

Role of thrombospondin-1 in control of von Willebrand factor multimer size in mice.

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr(842)-Met(843) peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet alpha-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets.

Animals↗
PubMed2004

The thrombospondins.

Thrombospondin-1 (TSP-1) was studied in the 1980s as a major component of platelet alpha-granules released upon platelet activation and also as a cell adhesion molecule. In 1993, we published a short review that discussed the exciting identification by molecular cloning of four additional vertebrate gene products related to TSP-1 [Current Biology 3 (1993) 188]. We put forward a structurally based classification for the newly identified proteins and discussed the functional and evolutionary implications of the new gene family. Since that time, the depth and breadth of knowledge on vertebrate TSPs and their functions in cells and tissues in health and disease has expanded into important new areas. Of particular interest is the new knowledge on the complex, domain and cell-type specific effects of TSPs on cell-signaling and cell-adhesion behaviour, the roles of TSP-1 and TSP-2 as anti-angiogenic agents, the roles of TSP-1 and TSP-2 in wound-healing, and associations of point mutations and polymorphisms in TSP-1, TSP-4 and TSP-5/COMP with human genetic diseases. The TSP family also now includes invertebrate members. In this article, we give the 2004 view on TSPs and our perspectives on the significant challenges that remain. Other articles in this issue discuss the functions of vertebrate TSPs in depth.

Animals↗
PubMed2004

Tumor progression: the effects of thrombospondin-1 and -2.

The thrombospondins (TSPs) are a family of proteins that regulate tissue genesis and remodeling. In many tumors, down-regulation of TSPs accompanies activation of oncogenes or inactivation of tumor suppresser genes and appears to be a prerequisite for the aquisition of a pro-angiogenic phenotype. The normal suppression of angiogenesis by TSP-1 and -2 involves multiple mechanisms including direct interaction with vascular endothelial cell growth factor (VEGF), inhibition of matrix metalloproteinase 9 (MMP9) activation, inhibition of endothelial cell migration and induction of endothelial cell apoptosis. The importance of down-regulation of TSPs for tumor progression is further established by the fact that several different approaches that are designed to increase the levels of TSP-1 or -2 in tumor tissue inhibit tumor growth. These approaches include cell-based gene therapy, low dose chemotherapeutics and systemic delivery of recombinant proteins or synthetic peptides that include type 1 repeat (TSR) sequences. Initial studies indicate that these reagents, in combination with established approaches for the treatment of cancer, will offer more efficacious therapies.

Down-Regulation↗
PubMed2004

Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation of transforming growth factor-beta.

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.

Angiogenesis Inhibitors↗
PubMed2004

Role of thrombospondin-1 in the autologous phase of an accelerated model of anti-glomerular basement membrane glomerulonephritis.

BACKGROUND: Thrombospondin-1 (TSP1), a multifunctional, extracellular matrix protein, regulates cellular attachment, proliferation, migration, and differentiation in vitro, and is expressed de novo in many inflammatory diseases, including glomerulonephritis (GN). METHODS: We investigated the role of TSP1 in the autologous phase of an accelerated model of anti-glomerular basement membrane (GBM) GN in mice deficient in TSP1. The model, induced by the injection of rabbit anti-mouse GBM antibody, is characterized by the development of proteinuria and glomerular damage over a 21-day observation period in wild-type mice. RESULTS: Mice deficient in TSP1 developed significantly less proteinuria than their wild-type controls 21 days after induction of disease (5,793 +/- 5,456 vs. 24,293 +/- 15,336 microg albumin/mg creatinine; p = 0.002). Serum creatinine levels were significantly higher in the wild-type mice than in the TSP1 deficient animals (29.03 +/- 2.34 vs. 16.39 +/- 2.87 micromol/l; p = 0.005). Other disease indices as crescent formation, fibrin deposition and macrophage influx, were also diminished in the TSP1 knockout animals. The numbers of interstitial CD4+ and CD8+ T cells were generally less in TSP1-deficient mice and reached statistical significance in CD4+ (p = 0.01) and CD8+ T cells (p = 0.02). The difference in outcome of the disease was not due to the difference in deposition/production of heterologous and autologous antibodies in the two groups of animals. CONCLUSION: This study suggests a proinflammatory role of TSP1 in an experimental model of GN.

Animals↗
PubMed2004

Roles of thrombospondin-1 and -2 in regulating corneal and iris angiogenesis.

PURPOSE: Thrombospondin (TSP)-1 and -2 are important antiangiogenic factors thought to be involved in maintaining corneal avascularity (angiogenic privilege). This study was undertaken to investigate whether deficiencies of these factors altered developmental and inflammation-induced angiogenesis in the cornea and developmental angiogenesis of the iris of mice. METHODS: Expression of TSP-1 and -2 mRNA and protein was assayed in cornea and iris stroma by RT-PCR and Western blot. Corneas and irides of TSP-1(-/-), TSP-2(-/-), and TSP-1,2(-/-) mice aged 2, 3, and 6 months, and wild-type control mice, were analyzed for spontaneous angiogenesis biomicroscopically, histologically, and with CD31 immunohistochemistry. The mouse model of suture-induced, inflammatory corneal neovascularization was used to evaluate the lack of TSP-1,2 and both TSPs on induced-corneal angiogenesis. Seven days after intrastromal placement of three 11-0 sutures, vascularized areas were analyzed morphometrically on CD31-stained corneal flatmounts. RESULTS: Corneas and irises from normal mouse eyes constitutively expressed TSP-1 and -2 mRNAs and proteins. Corneas of TSP-1(-/-), -2(-/-), and -1,2(-/-) mice displayed no evidence of spontaneous developmental-postnatal angiogenesis, although irises of these mice contained significantly increased iris vessel density compared with wild-type animals (P < 0.01). One week after suturing, corneas of all TSP(-/-) mice had significantly greater corneal angiogenesis than those of control mice (P < 0.05). TSP-1(-/-) had a significantly greater effect on induced corneal neovascularization than did TSP-2(-/-), with the opposite being the case in developmental iris angiogenesis (P < 0.01). CONCLUSIONS: Corneal avascularity during development is redundantly regulated, shown by the fact that lack of the antiangiogenic factors TSP-1 and/or -2 resulted in no spontaneous corneal angiogenesis. By contrast, TSP-1, more than TSP-2, helps to suppress inflammation-induced corneal angiogenesis postnatally, implying that angiogenic privilege in the cornea is actively maintained.

Animals↗
PubMed2003

Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy.

Chemotherapeutic drugs chronically administered to tumor-bearing mice, using a frequent schedule at doses substantially lower than the maximum tolerated dose (MTD) (i.e., metronomic dosing), can cause sustained and potent antiangiogenic effects by targeting the endothelial cells of newly growing tumor blood vessels. These effects appear to occur in the absence of an increase in the severity of side effects caused by destruction of other cell types normally sensitive to MTD chemotherapy, suggesting a marked and selective sensitivity of activated endothelial cells, the basis of which is unknown. Here we report that protracted exposure of endothelial cells in vitro to low concentrations of several different anticancer agents, including microtubule inhibitors and an alkylating agent, caused marked induction of gene and protein expression of TSP-1, a potent and endothelial-specific inhibitor of angiogenesis. Increases in circulating TSP-1 were also detected in the plasma of human tumor-bearing severe combined immunodeficient mice treated with metronomic low-dose cyclophosphamide. Most importantly, the antiangiogenic and antitumor effects of low-dose continuous cyclophosphamide were lost in TSP-1-null C57BL/6 mice, whereas, in contrast, these effects were retained by using a MTD schedule of the same drug. Taken together, the results implicate TSP-1 as a secondary mediator of the antiangiogenic effects of at least some low-dose metronomic chemotherapy regimens.

Administration, Oral↗
PubMed2003

Thrombospondin-1-induced vascular smooth muscle cell chemotaxis: the role of the type 3 repeat and carboxyl terminal domains.

Thrombospondin-1 (TSP-1), an acute phase reactant implicated in vascular disease, is a matricellular glycoprotein with six domains that confer different functions. The authors have shown TSP-1 induces vascular smooth muscle cell (VSMC) chemotaxis via extracellular signal-regulated kinases-1 and -2 (ERK) and p38 kinase (p38) and that a fusion protein of the carboxyl terminal (COOH) and type 3 repeat (T3) domains independently induce VSMC chemotaxis. The purpose of this study was to determine whether COOH-, T3-induced VSMC chemotaxis, or both, is dependent upon ERK or p38 activation. To determine if the T3, COOH, or type 2 repeat domain (T2, control domain not associated with chemotaxis) activate ERK, p38, or both, VSMCs were exposed to each fusion protein (20 microg/ml for 15, 30, 60, or 120 min), serum-free media (SFM, negative control), or TSP-1 (20 microg/ml for 30 min, positive control). Western immunoblotting was performed for activation studies. Using a microchemotaxis chamber, VSMCs pre-incubated in SFM, DMSO (vehicle control), PD98059 (10 microM), or SB202190 (10 microM) were exposed to each domain, TSP-1, or SFM. After 4 h (37 degrees C), migrated VSMCs were recorded as cells/five fields (400 x) and analyzed by paired t-test. ERK was activated by T2, T3, and COOH. However, p38 was activated by T3 and COOH, but not T2. T3 and COOH-induced VSMC chemotaxis were inhibited by PD98059 or SB202190, but more completely by SB202190. The T2 domain had no effect on VSMC chemotaxis. These results suggest activation of the p38 pathway may be more specific than ERK for COOH- and T3-induced VSMC chemotaxis.

Animals↗
PubMed2003

Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration.

Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. Its expression is most prominent during the late stages of vascular development and in the adult vasculature. Our previous studies have shown that TSP1 expression promotes a quiescent, differentiated phenotype of vascular endothelial cells. However, the physiological role TSP1 plays during vascular development and neovascularization requires further delineation. Here, we investigated the role of TSP1 during development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy. The retinal vascular density was increased in TSP1-deficient (TSP1-/-) mice compared with wild-type mice. This finding was mainly attributed to increased number of retinal endothelial cells in TSP1-/- mice. During oxygen-induced ischemic retinopathy, the developing retinal vasculature of TSP1-/- mice was less sensitive to vessel obliteration induced by hyperoxia but exhibited a similar level of neovascularization induced by normoxia compared with wild-type mice. This finding is consistent with the similar pattern of VEGF expression detected in wild-type and TSP1-/- mice. Furthermore, the increased expression of TSP1 during development of retinal vasculature was not affected by oxygen-induced ischemic retinopathy. In addition, the regression of ocular embryonic (hyaloid) vessels, as well as the newly formed retinal vessels during oxygen-induced ischemic retinopathy, was delayed in TSP1-/- mice. Therefore, TSP1 is a modulator of vascular homeostasis and its expression is essential for appropriate remodeling and maturation of retinal vasculature.

Animals↗
PubMed2003

Thrombospondin-1 plays a critical role in the induction of hair follicle involution and vascular regression during the catagen phase.

Hair growth is associated with pronounced vascular-endothelial-growth-factor-induced perifollicular angiogenesis, whereas the catagen regression phase is characterized by apoptosis-driven blood vessel regression. The biologic relevance of endogenous inhibitors of angiogenesis in the control of hair cycling, however, has remained unknown. We studied the expression and biologic role of the angiogenesis inhibitor thrombospondin-1 (TSP-1) during the induced adult hair follicle cycle in wild-type, TSP-1 deficient, and TSP-1 overexpressing transgenic mice. TSP-1 expression was absent from hair bulb and dermal papilla cells during early to mid-anagen but was highly upregulated throughout the catagen involution phase. In TSP-1 deficient mice, the follicle growth phase was significantly prolonged, associated with increased perifollicular vascularization and vascular proliferation. Conversely, hair follicle growth was delayed in K14/TSP-1 transgenic mice that expressed high levels of TSP-1 in outer root sheath keratinocytes, associated with reduced perifollicular vascularization. These effects were most probably mediated via its antiangiogenic effects because TSP-1 did not affect the growth of cultured murine vibrissae in the absence of a functional vascular system. These results identify a critical role of TSP-1 in the induction of anagen follicle involution, with potential implications for the therapeutic modulation of hair follicle growth.

Animals↗
PubMed2003

Thrombospondin 1--a regulator of adenoma growth and carcinoma progression in the APC(Min/+) mouse model.

Thrombospondin 1 (TSP-1) is a multifunctional extracellular matrix protein that is an endogenous regulator of tumor angiogenesis. The effects of TSP-1 on adenoma formation and development into cancerous lesions has been evaluated in the Min(/+) (multiple intestinal neoplasia) mouse model. These mice develop multiple adenomas in the small intestine due to a mutation in the homologous APC (adenomatous polyposis coli) gene. As in its human counterpart, these adenomas may progress to carcinomas. Intestines of APC(Min/+) mice were dissected and histologic evaluation of adenomas was then conducted. Significant increases in vascularization and proliferation were observed in adenomatous, as compared with normal, mucosa. TSP-1 immunostaining revealed significant decreases in the number and intensity of positive cells in adenomas, as compared with normal mucosa. TSP-1 scores were inversely correlated with vascularity and proliferation rate. Cross breeding of mice homozygous for a deletion of the TSP-1 gene (TSP-1(-/-)) with mice heterozygous for the APC gene mutation (APC(Min/+)), resulted in animals that showed a significant increase in adenoma number and diameter. Also, histopathological examination of these adenomas showed accelerated dysplasic changes, carcinoma in situ and early invasion, compared with their APC(Min/+) littermates. Moreover, a significant decrease of TUNEL-positive cells was observed in intestinal adenomas of TSP-1(-/-)/APC(Min/+) mice. This study reports the first in vivo impact of TSP-1 during early stages of tumor initiation and development in an intestinal carcinogenesis model and demonstrates that TSP-1 affects both angiogenesis and tumor cell apoptosis.

Adenoma↗
PubMed2002

Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication.

Thrombospondin-1 (TSP-1) contains three type 1 repeats (TSRs), which mediate cell attachment, glycosaminoglycan binding, inhibition of angiogenesis, activation of TGFbeta, and inhibition of matrix metalloproteinases. The crystal structure of the TSRs reported in this article reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end. The front face of the TSR contains a right-handed spiral, positively charged groove that might be the "recognition" face, mediating interactions with various ligands. This is the first high-resolution crystal structure of a TSR domain that provides a prototypic architecture for structural and functional exploration of the diverse members of the TSR superfamily.

Amino Acid Sequence↗
PubMed2002

The lack of thrombospondin-1 (TSP1) dictates the course of wound healing in double-TSP1/TSP2-null mice.

Thrombospondin (TSP) 1 and 2, share the same overall structure and interact with a number of the same cell-surface receptors. In an attempt to elucidate their biological roles more clearly, we generated double-TSP1/TSP2-null animals and compared their phenotype to those of TSP1- and TSP2-null mice. Double-null mice exhibited an apparent phenotype that primarily represented the sum of the abnormalities observed in the single-null mice. However, surprisingly, the wound-healing response in double-null mice resembled that in TSP1-null animals and differed from that in TSP2-nulls. Thus, although the excisional wounds of TSP2-null mice are characterized by increased neovascularization and heal at an accelerated rate, TSP1-null and double-null animals demonstrated delayed healing, as indicated by the prolonged persistence of inflammation and delayed scab loss. Immunohistochemical analysis showed that, similar to TSP1-null mice, the granulation tissue of double-null mice was not excessively vascularized. Furthermore as in TSP1-nulls, decreases in macrophage recruitment and in the levels of monocyte chemoattractant protein-1 indicated that the inflammatory phase of the wound-healing response was impaired in double-null mice. Our data demonstrate that the consequences of a lack of TSP1 predominate in the response of double-null mice, and dictate the course of wound healing. These findings reflect distinct temporal and spatial expressions of TSP1 and TSP2 in the healing wound.

Animals↗
PubMedDate not supplied

Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein that influences cellular phenotype and the structure of the extracellular matrix. These effects are important components of the tissue remodeling that is associated with angiogenesis and neoplasia. The genetic mutations in oncogenes and tumor suppressor genes that occur within tumor cells are frequently associated with decreased expression of TSP-1. However, the TSP-1 that is produced by stromal fibroblasts, endothelial cells and immune cells suppresses tumor progression. TSP-1 inhibits angiogenesis through direct effects on endothelial cell migration and survival and through indirect effects on growth factor mobilization. TSP-1 that is present in the tumor microenvironment also acts to suppress tumor cell growth through activation of transforming growth factor beta in those tumor cells that are responsive to TGF beta. In this review, the molecular basis for the role of TSP-1 in the inhibition of tumor growth and angiogenesis is summarized.

Amino Acid Sequence↗