Monochorionic pseudomonoamniotic twin pregnancy with fetal demise of one twin and development of maternal consumptive coagulopathy.
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Adult↗
Biomedical subjects
J Lawler
Publications and source records attributed to J Lawler.
Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro.
OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.
Animals↗
Patients' and caregivers' expectations and experiences of a physiotherapy intervention 1 year following stroke: a qualitative study.
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Aged↗
Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms.
Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogenesis. The antiangiogenic activity of TSP-1 has been mapped to the procollagen homology region and the type 1 repeats (TSR) using synthetic peptides. To elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the TSRs, we have expressed recombinant versions of these motifs and have assayed their ability to inhibit the growth of experimental B16F10 melanomas and Lewis lung carcinomas. Recombinant proteins that contain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR2) the transforming growth factor-beta (TGFbeta) activating sequence (RFK) have been expressed in Drosophila S2 cells. In addition, recombinant proteins with mutations in either the RFK sequence (TSR2+QFK) or the WSHWSPW sequence [TSR2 (W/T)] of the second TSR have been prepared. Similar to platelet TSP-1, these proteins are potent inhibitors of endothelial cell migration, and 3TSR of human TSP-1 (3TSR/hTSP-1) and TSR2+RFK activate TGFbeta. An 81% inhibition of B16F10 tumor growth is observed at 2.5 mg (135 nmol)/kg/day of the recombinant 3TSR/hTSP-1. A comparable level of inhibition is observed with 2.5 mg (360 nmol)/kg/day of TSR2+RFK. By contrast, 3TSR of mouse TSP-2 (3TSR/mTSP-2), TSR2+QFK, and TSR2 are significantly less effective. TSR2+RFK and TSR2 reduce tumor vessel density, but TSR2+RFK has a greater effect on B16F10 tumor cell apoptosis and proliferation. Concurrent treatment of B16F10 tumor-bearing mice with TSR2+RFK and either a soluble form of the TGFbeta receptor or an antibody to active TGFbeta reduces the inhibition of B16F10 tumor growth to levels that are comparable with those of TSR2 and TSR2+QFK. By contrast, the presence of the TGFbeta-activating sequence does not increase the level of inhibition of Lewis lung carcinoma experimental tumor growth. These data indicate that the TSRs inhibit tumor growth by inhibition of angiogenesis and regulation of tumor cell growth and apoptosis. The regulation of tumor cell growth and apoptosis is TGFbeta dependent, whereas the inhibition of angiogenesis is not.
Animals↗
Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor.
Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.
Animals↗
CD36 associates with CD9 and integrins on human blood platelets.
The membrane glycoprotein CD36 is involved in platelet aggregation, inhibition of angiogenesis, atherosclerosis, and sequestration of malaria-parasitized erythrocytes. In this study, immunoprecipitations with anti-CD36 antibodies were performed to identify proteins that associate with CD36 in the platelet membrane. Platelets were solubilized in 1% Triton X-100, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Brij 96, or Brij 99, and the proteins that coprecipitated with CD36 were identified by peptide mass spectrometry and Western blotting. The tetraspanin protein CD9 and the integrins alphaII(b)beta3 and alpha6beta1 specifically coprecipitated with CD36 from platelets that were solubilized in CHAPS and Brij 99 but not from platelets that were solubilized in Triton X-100. Only CD9 is coprecipitated with CD36 from platelets that were solubilized in Brij 96. Reciprocal immunoprecipitations with antibodies to CD9, alpha6, alphaIIb, or beta3 from Brij 99-solubilized platelets coprecipitated CD36. Coprecipitation of CD36, CD9, and alpha6beta1 was also observed on platelets from a patient with Glanzmann thrombasthenia, indicating that alphaII(b)beta3 is not required for the other proteins to associate. Colocalization of alpha6 and CD36, of CD9 and CD36, and of alpha6 and CD9 was observed on intact platelets prior to solubilization, using double immunofluorescence microscopy. These data indicate that CD36 associates with CD9 and integrins on human blood platelets. These associated proteins may mediate or participate in some of the diverse biological functions of CD36.
Antibodies, Monoclonal↗
Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice.
In vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 +/- 52 days to 149 +/- 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 +/- 103 days in the presence of TSP1 expression, and 426 +/- 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P < or = 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth.
Animals↗
The C-terminal domain of thrombospondin-1 induces vascular smooth muscle cell chemotaxis.
OBJECTIVE: Thrombospondin-1 (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-1 has six domains of repeating homologous amino acid sequences: N-terminal, procollagen homology, type 1 repeat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The purpose of this experiment was to determine which domains of TSP-1 induce VSMC chemotaxis. METHODS: A modified Boyden Chamber chemotaxis assay was used to assess VSMC migration. Serum-free medium, TSP-1, or each of the fusion proteins (10 and 20 microg/mL) synthesized for the different domains were placed in the bottom wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top wells. After 4 hours at 37 degrees C, migrated VSMCs were recorded as cells per five fields (400x) and analyzed with the paired t test. To verify the fusion protein data, we performed chemotaxis assays with antibodies to each of the domains (25 microg/mL) combined with TSP-1 (20 microg/mL) in the bottom wells and VSMCs in the top wells. RESULTS: The COOH domain significantly stimulated VSMC chemotaxis (P = <.001). To a lesser extent, the N-terminal and T3 domains also induced chemotaxis (P <.05). However, only the anti-COOH antibody (C6.7) and the anti-integrin-associated protein portion of COOH antibody (D4.6) significantly inhibited TSP-1-induced VSMC chemotaxis (by 85% and 92%, respectively). CONCLUSIONS: These results implicate the COOH domain as the portion of the TSP-1 molecule primarily responsible for VSMC chemotaxis. This experiment suggests that future strategies in the prevention of VSMC migration, an initial step in the development of vascular lesions, may involve selective inhibition of the COOH domain of TSP-1.
Animals↗
Leadership development in UK companies at the beginning of the twenty-first century: lessons for the NHS?
States that the development of leadership in the NHS is currently high on the agenda of the Department of Health, the government and local health sector organisations. Reports the findings of a study of public and private sector organisations, exploring the development of their in-house leadership skills. Outlines the findings in depth and discusses the implications for health organisations.
Administrative Personnel↗
Cartilage oligomeric matrix protein (thrombospondin-5) is expressed by human vascular smooth muscle cells.
Cartilage oligomeric matrix protein (COMP/thrombospondin [TSP]-5) belongs to the thrombospondin gene family and is an extracellular glycoprotein found predominantly in cartilage and tendon. To date, there is limited evidence of COMP/TSP-5 expression outside of the skeletal system. The aim of the present study was to investigate the expression of COMP/TSP-5 in cultured human vascular smooth muscle cells and human arteries. COMP/TSP-5 mRNA and protein expression was detected in cultured human vascular smooth muscle cells with both Northern blotting and immunoprecipitation. Serum, as well as transforming growth factor (TGF)beta1 and TGF-beta3, stimulated COMP/TSP-5 mRNA expression. COMP/TSP-5 was detected in normal as well as atherosclerotic and restenotic human arteries with immunohistochemistry. The majority of COMP/TSP-5 was expressed in close proximity to vascular smooth muscle cells. In vitro attachment assays demonstrated strong adhesion of smooth muscle cells to COMP/TSP-5-coated surfaces, with the majority of cells spreading and forming stress fibers. In addition, COMP/TSP-5 supported the migration of smooth muscle cells in vitro. The present study shows that COMP/TSP-5 is present in human arteries and may play a role in the adhesion and migration of vascular smooth muscle cells during vasculogenesis and in vascular disease settings such as atherosclerosis.
Cartilage↗
C-mannosylation and O-fucosylation of the thrombospondin type 1 module.
Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of approximately 60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an alpha-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C- and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.
Amino Acid Motifs↗
Cartilage oligomeric matrix protein is a calcium-binding protein, and a mutation in its type 3 repeats causes conformational changes.
Mutations in residues in the type 3 calcium-binding repeats and COOH-terminal globular region of cartilage oligomeric matrix protein (COMP) lead to two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. It has been hypothesized that these mutations cause COMP to misfold and to be retained in the endoplasmic reticulum. However, this hypothesis is not supported by previous reports that COMP, when purified in the presence of EDTA, shows no obvious difference in electron microscopic appearance in the presence or absence of calcium ions. Since this discrepancy may be due to the removal of calcium during purification, we have expressed wild-type COMP and the most common mutant form found in pseudoachondroplasia, MUT3, using a mammalian expression system and have purified both proteins in the presence of calcium. Both proteins are expressed as pentamers. Direct calcium binding experiments demonstrate that wild-type COMP, when purified in the presence of calcium, is a calcium-binding protein. Rotary shadowing electron microscopy and limited trypsin digestion at various calcium concentrations show that there are conformational changes associated with calcium binding to COMP. Whereas COMP exists in a more compact conformation in the presence of calcium, it shows a more extended conformation when calcium is removed. MUT3, with a single aspartic acid deletion in the type 3 repeats, binds less calcium and presents an intermediate conformation between the calcium-replete and calcium-depleted forms of COMP. In conclusion, we show that a single mutation in the type 3 repeats of COMP causes the mutant protein to misfold. Our data demonstrate the importance of calcium binding to the structure of COMP and provide a plausible explanation for the observation that mutations in the type 3 repeats and COOH-terminal globular region lead to pseudoachondroplasia.
Amino Acid Sequence↗
Activation of platelet-transforming growth factor beta-1 in the absence of thrombospondin-1.
Thrombospondin-1 (TSP-1) has been shown to bind and activate transforming growth factor-beta1 (TGF-beta1). This observation raises the possibility that TSP-1 helps to sequester TGF-beta1 in platelet alpha granules and activates TGF-beta1 once both proteins are secreted. Herein, we evaluated the level of active and latent TGF-beta1 in the plasma and in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice on two genetic backgrounds (C57BL/6 and 129Sv). The plasminogen activator inhibitor-1/luciferase bioassay and an immunological assay were used to determine active and latent TGF-beta1. No significant differences were observed in the levels of active and latent TGF-beta1 in the supernatant of thrombin-treated platelets from TSP-1 null and wild-type mice. Active and latent TGF-beta1 were significantly increased in the plasma and platelets of C57BL/6 mice as compared with 129Sv mice. In addition, there was an increase of plasma level of latent TGF-beta1 in TSP-1 null mice as compared with wild-type mice on the C57BL/6 background but not on the 129Sv background. No active TGF-beta1 was observed in the plasma of either TSP-1 null and wild-type mice. These data indicate that TSP-1 does not function as a chaperon for TGF-beta1 during platelet production and does not activate significant quantities of secreted TGF-beta1 despite a vast excess in the number of TSP-1 molecules as compared with TGF-beta1 molecules. Because platelet releasates from TSP-1 null mice contain active TGF-beta1, we suggest that other important mechanisms of physiological activation of TGF-beta1 probably exist in platelets.
Animals↗
Vasopressin pressor effects in critically ill children during evaluation for brain death and organ recovery.
BACKGROUND: Vasopressin (VP) shows promise as a pressor agent in animals and adult human cardiac arrest and resuscitation, but has not been studied for pressor effect in critically ill or arrested children. VP infusion is routine treatment for diabetes insipidus during brain death evaluation and organ recovery. We hypothesized that low dose VP infusion during organ recovery in critically ill children exerts a pressor effect, without major organ toxicity. METHODS: 34 VP-treated and 29 age-matched critically ill controls (C) < or =18 years were retrospectively reviewed during brain death evaluation and organ recovery. VP infusion protocol titrated VP dose clinically to urine output, with high variability. Pressor and inotrope management was titrated clinically to BP, cerebral perfusion and central venous pressures (when available) and peripheral perfusion with similar protocol targets for pre-load in VP and C groups. Outcome measures include dose, type and number of pressors and inotropes. Organ function was assessed at recovery and 48 h post-transplant by independent surgeon and transplant program organ function criteria. Analysis by Odds Ratio (OR), and chi-square. RESULTS: VP dose averaged 0.041+/-0.069 U/kg/h. Average baseline mean arterial pressure (MAP) before VP infusion was 79+/-17 mmHg VP and 76+/-14 mm Hg C (P=0.6). Subsequent average MAP were: 82+/-21 mmHgVP after VP infusion versus 71+/-16 mmHg C (P=0.01) and 80+/-14 mmHg VP versus 68+/-22 mmHg C (P=0.01). Ability to wean/stop pressors and inotropes was: dopamine (14/23) 42% VP versus (10/26) 38% C (P=0.75), dobutamine (4/7) 57% VP versus (0/6) 0% C (P=0.026), epinephrine (4/5) 80% VP versus (0/6) 0% C (P=0.006), norepinephrine/phenylephrine (4/4) 100% VP versus (2/5) 40% C (P=0. 057). Alpha agonist pressor dependence was successfully weaned from 7/9 (78%) VP versus 0/9 (0%) C: odds ratio=7.3, (P<0.01). There was no VP induced dysrhythmia, hypertension, anuria or toxicity reported. Good organ recovery function was not significantly different at recovery or 48 h post-transplant for kidney (79% VP versus 69% C, P=0.068), liver (87% VP versus 95% C, P=0.533), or heart (90% VP versus 71% C, P=0.11). CONCLUSIONS: Low dose vasopressin infusion exerts a pressor effect in critically ill children treated for diabetes insipidus during brain death and organ recovery. VP treated patients were 7.3 times more likely to wean from alpha agonists than comparably managed age matched controls, without adverse affect on transplant organ function. We speculate that further prospective assessment of VP safety and efficacy as a pressor adjunct for resuscitation of critically ill children is warranted.
Adolescent↗
The cell biology of thrombospondin-1.
Thrombospondin-1 (TSP-1) is a matricellular protein that regulates cellular phenotype during tissue genesis and repair. It acts as a molecular facilitator by bringing together cytokines, growth factors, matrix components, membrane receptors and extracellular proteases. TSP-1 binds to a wide variety of integrin and non-integrin cell surface receptors. The binding sites for these receptors on TSP-1 are dispersed throughout the molecule, with most domains binding multiple receptors. In some cases, TSP-1 binds to multiple receptors concurrently, and recent data indicate that there is cross-talk between the receptor systems. Thus, TSP-1 may function to direct the clustering of receptors to specialized domains for adhesion and signal transduction.
Amino Acid Sequence↗
The functions of thrombospondin-1 and-2.
Considerable progress has been made towards understanding the function of thrombospondin-1 and-2. The description of the phenotype of mice with thrombospondin-1 and-2 knocked-out supports in vitro biochemical and cell-biological data and has opened new avenues of research. Recently, our understanding of the roles of thrombospondins in the activation of TGFbeta, inhibition of angiogenesis and the initiation of signal transduction has advanced.
Amino Acid Sequence↗
Investigating recovery from stroke: a qualitative study.
A recent randomized controlled trial evaluated the effects of specialist nurses providing information, advice and support to caregivers and patients at home during the first year after a stroke. Reported here are the results of a complementary study which used qualitative methods to examine the experience of patients and caregivers during the year of recovery after a stroke. We used semi-structured interviews with a purposively selected sample of 30 patients and 15 caregivers at the end of a randomized controlled trial (13-16 months post-stroke). Patients and caregivers provided vivid descriptions of the recovery process. Recovery was perceived in terms of the degree of congruence patients identified between their lives before, and after, stroke. Patients therefore had individual and personal yardsticks for measuring their recovery. In conclusion, further research and interventions must consider the diverse, complex, dynamic and highly personal character of stroke recovery. Traditional outcome measures are too simplistic to capture patients' and caregivers' experiences. There do not appear to be single or simple solutions to the problems of facilitating psycho-social adjustment.
Aged↗
Residues F16-G33 and A784-N823 within platelet thrombospondin-1 play a major role in binding human neutrophils: evaluation by two novel binding assays.
The thrombospondin-1 (TSP1) structural requirements within its heparin-binding domain (HBD)(30 kd) or within the other domains of the molecule (450 kd) that interact with neutrophils (PMNs) have not been delineated. Synthetic peptides based on the HBD, a TSP1 proteolytic fragment lacking the HBD, a large C-terminal domain of TSP1 (210 kd), a TSP1 recombinant fragment (rTSP1(784-932)), and a monoclonal antibody directed against the TSP1 type 3 repeats (mAb D4.6) were utilized to map such structural requirements on TSP1. Synthetic peptides containing a heparin-binding motif and encompassing residues F16-G33 or A74-S95 of TSP1 competed quantitatively with iodine 125-labeled TSP1 for binding to heparinagarose beads. However, only F16-G33 was a competitor of TSP1 binding to PMNs, suggesting that the sequence F16-G33 within the HBD plays a role in PMN binding. The interaction site within the 450-kd fragment was further narrowed. A TSP1 -derived proteolytic fragment (210 kd), a recombinant TSP1 fragment (rTSP1(784-932)), and a type 3 repeat anti-TSP1 monoclonal antibody (mAb D4.6) competed for the binding of 125I-labeled TSP1 to PMNs. The N-terminal of rTSP1(784-932) and C-terminal sequence analysis of TSP1-210 kd delineated the structural requirements for the second binding region for PMNs-namely, residues A784-N823.
Amino Acid Sequence↗