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PubMed2002

Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis.

Hemangiomas are benign tumors of the vascular endothelium and are the most common tumors of infancy. These tumors are characterized by an initial phase of rapid proliferation, which is followed, in most cases, by spontaneous involution. Although most lesions resolve without complication, there are some cases in which hemangiomas can be life threatening when occurring near a vital structure. Treatment for these aggressive tumors represents an unmet clinical need. In addition, this characteristic progression of hemangiomas through distinct phases provides a unique opportunity for studying endothelial cell biology and angiogenesis. Using DNA microarrays representing approximately 10,000 human genes, we identified insulin-like growth factor 2 (IGF-2) as a potentially important regulator of hemangioma growth. IGF-2 was highly expressed during the proliferative phase and substantially decreased during involution. This finding was confirmed at the message level by quantitative reverse transcription-PCR and at the protein level by immunohistochemistry. IGF-2 protein was localized primarily to tumor vessels or vascular channels. Using a human hemangioma explant model, we show that IGF-2 promotes sprouting from intact hemangioma tissue. In addition, several angiogenesis-related factors, including integrins alpha(v)beta3 and alpha5beta1, are present in proliferating hemangiomas. During the involuting phase, an increase in several IFN-induced genes was observed. These studies identify potential regulators of hemangioma growth and involution and provide a foundation on which to build further mechanistic investigations into angiogenesis, using hemangiomas as a model.

Cell Division↗
PubMed2006

Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn).

We describe here the functional implications of an increase in IgG binding to the neonatal Fc receptor. We have defined in a systematic fashion the relationship between enhanced FcRn binding of a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody (MEDI-524) and the corresponding biological consequences in cynomolgus monkeys. The triple mutation M252Y/S254T/T256E (YTE) was introduced into the Fc portion of MEDI-524. Whereas these substitutions did not affect the ability of MEDI-524 to bind to its cognate antigen and inhibit RSV replication, they resulted in a 10-fold increase in its binding to both cynomolgus monkey and human FcRn at pH 6.0. MEDI-524-YTE was efficiently released from FcRn at pH 7.4 in both cases. We show that MEDI-524-YTE consistently exhibited a nearly 4-fold increase in serum half-life in cynomolgus monkeys when compared with MEDI-524. This constituted the largest half-life improvement described to date for an IgG in a primate. For the first time, we demonstrate that these sustained serum levels resulted in an up to 4-fold increase in lung bioavailability. Importantly, we also establish that our non-human primate model is relevant to human. Finally, we report that the YTE triple substitution provided a means to modulate the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of a humanized IgG1 directed against the human integrin alpha(v)beta3. Therefore, the YTE substitutions allow the simultaneous modulation of serum half-life, tissue distribution and activity of a given human IgG1.

Animals↗
PubMed2002

Spontaneous haemothorax: an uncommon presentation of Glanzmann thrombasthenia.

UNLABELLED: Glanzmann thrombasthenia is a rare hereditary qualitative platelet disorder characterized by a lifelong bleeding tendency due to quantitative and qualitative abnormalities of the platelet integrin alpha(IIb)beta3. Common clinical manifestations include purpuric type skin bleeding, prolonged bleeding from minor cuts, epistaxis, gingival bleeding and menorrhagia. Less frequently, gastrointestinal system bleeding may occur. Haemarthrosis, haematuria, intracranial and visceral haemorrhage are very rare symptoms. This study reports a 3-y-old girl with Glanzmann thrombasthenia who presented with life-threatening haemothorax. There was no history of recent trauma or drug usage and no vascular or parenchymal abnormalities to explain the development of haemothorax. CONCLUSION: To the authors' knowledge this is the first case of Glanzmann thrombasthenia complicated by spontaneous haemothorax.

Child, Preschool↗
PubMed2004

Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis.

BACKGROUND: Tumor invasion and metastasis cause most deaths in cancer patients. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells, but few mechanistic details are known. We investigated the roles of CTGF and collapsin response mediator protein 1 (CRMP-1) in metastasis and invasion of human lung adenocarcinoma. METHODS: We compared vector control-transfected cells with corresponding CTGF gene-transfected cells. Invasive activity was measured with a modified Boyden chamber assay, and metastatic activity was measured in an animal model. We used CTGF deletion mutants, CTGF and CRMP-1 antisense oligonucleotides, and anti-integrin and anti-CRMP-1 antibodies to investigate the functional relationship between CTGF and CRMP-1. Expression of CTGF protein in 78 lung adenocarcinoma specimens was investigated immunohistochemically. All statistical tests were two-sided. RESULTS: Invasive (both P<.001) and metastatic (P<.001 and P=.003, respectively) activities were lower in cells that overexpress CTGF than in vector control cells. Expression of CRMP-1 was higher in CTGF-transfected clones than in vector control cells, and its level decreased after cells were treated with anti-integrin alpha(v)beta3 and alpha(v)beta5 antibodies. Reduced levels of CRMP-1 protein after the transfection of CRMP-1-specific antisense oligonucleotides, but not sense oligonucleotides, increased the invasiveness of CTGF-transfected cells (mean numbers of invasive CTGF-transfected cells treated with 20 microM CRMP-1-specific sense and antisense oligonucleotides were 327 and 516 cells, respectively [difference = 189 cells, 95% confidence interval [CI] = 156 to 221 cells; P<.001]). The CT module of CTGF was the region primarily responsible for the increased expression of CRMP-1 and the inhibition of invasion (mean numbers of invasive cells expressing full-length CTGF and CT module-deleted mutant were 148 and 385 cells, respectively [difference = 237 cells, 95% CI = 208 to 266 cells; P<.001]). Reduced expression of CTGF in lung cancer specimens was statistically significantly associated with the risk of more advanced-stage disease (stages III and IV versus stages I and II; P=.001), lymph node metastasis (P =.014), and shorter survival (median survival with high levels of CTGF = 66.7 months and median survival for low levels = 18.2 months; difference = 48.5 months, 95% CI = 33.5 to 63.5 months; P =.02). CONCLUSION: CTGF inhibits metastasis and invasion of human lung adenocarcinoma by a CRMP-1-dependent mechanism.

Adenocarcinoma↗
PubMed2002

Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration.

Collagen-I, which predominates in the neomatrix of fibrotic liver, regulates hepatocyte and hepatic stellate cell (HSC) phenotypes. Recovery from liver fibrosis is accompanied by hepatocyte regeneration, matrix degradation, and HSC apoptosis. Using mice bearing a mutated collagen-I gene (r/r mice), which confers resistance to collagenase degradation, we have investigated the hypothesis that collagen-I degradation is critical to HSC apoptosis and hepatocyte regeneration during recovery from liver fibrosis. During a 28-day recovery period after 8 wk of CCl4 treatment, wild-type (WT) livers had significantly (43%) decreased hydroxyproline (OHP) content. In r/r livers, however, OHP content remained elevated at peak fibrosis levels. Expressed markers of activated HSC (alpha-smooth muscle actin, collagen-I), elevated at peak fibrosis, dropped to control levels in WT livers after 28 days but remained raised in the r/r livers. Moreover, relative to WT livers, r/r livers had significantly reduced stellate cell apoptosis and hepatocyte regeneration during the recovery period. Using extracted collagen-I from each genotype as culture substrata, relative to r/r, we show that WT collagen-I promotes hepatocyte proliferation via stimulation of integrin alpha(v)beta3. Failure to degrade collagen-I critically impairs HSC apoptosis and may prevent the effective restoration of hepatocyte mass in liver fibrosis.

Animals↗
PubMed1998

Growth factors in infant germinal matrix: relationship to extracellular matrix and cell adhesion molecules.

We examined the expression of selected growth factors, growth factor receptors, elements of extracellular matrix and cell adhesion molecules in the germinal matrix layer (GML) utilizing immunohistochemistry and reverse transcriptase polymerase chain reaction. At autopsy brain samples from 10 neonatal infants were used. Epidermal growth factor receptor (EGFR) was significantly expressed in the matrix cells. While transforming growth factor alpha and heparin-binding epidermal growth factor-like growth factor were found in the matrix cells or vascular wall as ligands, epidermal growth factor was not expressed. EGFR and its ligands are thought to be important factors for the maintenance of the matrix cells and cell-to-cell interactions. Insulin like growth factor I, its receptor Ibeta and tenascin were found in the stroma of the GML and periventricular region. Vascular endothelial growth factor and receptor Flk-1, laminin A and B2, fibronectin, collagen type IV and integrins such as beta3, alpha5beta1 and alphaVbeta3 were found mainly in or around the vascular wall indicating their important roles for vascularization. Transforming growth factor beta2 and its receptor II were expressed in the matrix cells and/or vascular wall suggesting a role in proliferation and/or regression of the vasculature. CD44 and Thy-1 were also expressed in the matrix cells.

Autopsy↗
PubMedDate not supplied

FAK signaling in anaplastic astrocytoma and glioblastoma tumors.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic-tyrosine kinase that is activated by several different cell surface receptors shown to be upregulated on glioblastoma cells (integrins alpha(v)beta3 and alpha(v)beta5, and the epidermal growth factor receptor). Activated FAK can signal through several different signaling pathways, which are reviewed here. Published data are summarized that have demonstrated 1) elevated FAK expression in anaplastic astrocytoma and glioblastoma tumor biopsy samples, 2) a role for FAK in the promotion of glioblastoma cell proliferation, survival and migration in vitro, and 3) a role for FAK in the promotion of glioblastoma cell proliferation in vivo in an animal model. The available data suggests that increased levels of FAK protein and activity may contribute to an increased ERK activity and cell proliferation in vivo in these tumors.

Astrocytoma↗
PubMed2004

Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy.

OBJECTIVE: Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? BACKGROUND: Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing "tissue engineering" strategies for the regeneration of the nucleus pulposus. METHODS: Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-beta1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. RESULTS: Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-beta receptor). Likewise, expression of beta3 and alpha2 integrin was upregulated. Transforming growth factor-beta treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-beta. In hypoxia, transforming growth factor-beta sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. CONCLUSIONS: Our results indicate that hypoxia and transforming growth factor-beta drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.

Animals↗
PubMed2005

Three novel beta-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-alphaIIb, but variably impaired progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.

Adult↗
PubMed2006

Von Willebrand factor activates endothelial nitric oxide synthase in blood platelets by a glycoprotein Ib-dependent mechanism.

BACKGROUND: The molecular regulation of endothelial nitric oxide synthase (eNOS) in blood platelets and the signalling events induced by platelet-derived NO are poorly defined. In particular, the ability of von Willebrand factor (VWF) to stimulate cyclic guanosine monophosphate (cGMP) formation in platelets has produced conflicting data. OBJECTIVES: To determine the mechanisms leading to eNOS activation and clarify the downstream signaling pathways activated by platelet-derived NO in response to VWF. METHODS: We used three independent markers of NO signaling, [3H] l-citrulline production, cGMP accrual and immunoblotting of vasodilator-stimulated phosphoprotein (VASP) to examine the NO signaling cascade in response to VWF. RESULTS: VWF increased NO synthesis and bioavailability, as evidenced by increased [3H] l-citrulline production and cGMP accrual, respectively. VWF-induced eNOS activation was GPIb-IX-dependent and independent of integrin alpha(IIb)beta3. cGMP formation in response to VWF required Ca2+ mobilization, Src family kinases, phosphatidylinositol 3-kinase and phospholipase C, but not protein kinase C. This suggests that a cross-talk between the signaling mechanisms regulates platelet activation and NO synthesis. VWF-induced cGMP accrual was completely blocked by apyrase and indomethacin, demonstrating an essential role for platelet-derived ADP and thromboxane A2 (TxA2). Elevated cGMP levels led to increased VASP phosphorylation at serine239 that was both protein kinase G (PKG)- and protein kinase A (PKA)-dependent. CONCLUSIONS: We demonstrate that VWF activates eNOS through a specific Ca2+-dependent GPIb receptor-signaling cascade that relies on the generation of platelet-derived ADP and TxA2. Furthermore, we provide the first evidence to suggest that platelet derived-NO/cGMP activates PKA in addition to PKG.

Adenosine Diphosphate↗
PubMed2006

Inhibition of adhesive and signaling functions of the platelet GPIb-V-IX complex by a cell penetrating GPIbalpha peptide.

BACKGROUND: Interaction between the platelet glycoprotein (GP)Ib-V-IX complex and von Willebrand factor (VWF) is critical for initiating platelet-vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin alpha(IIb)beta3 activation. OBJECTIVE: A cell-penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbalpha subunit in VWF/GPIb-V-IX-dependent adhesion and activation. METHODS: Peptides of 11-13 amino acids, covering the 557-610 region, were coupled to a nine-arginine permeating tag (R9) and the effects of their cell entry on VWF-dependent responses were analyzed. RESULTS: The R9alpha557 peptide corresponding to the 557-569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9alpha557 on GPIb VWF-dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb-IX complex were incubated with R9alpha557. CONCLUSION: This study performed in intact platelets suggests a functional role of the 557-569 domain of GPIbalpha in controlling VWF-dependent adhesion and signaling.

Adenosine Diphosphate↗
PubMed2003

Antithrombotic effects of FK419, a novel nonpeptide platelet GPIIb/IIIa antagonist, in a guinea pig photochemically induced middle cerebral artery thrombosis model: comparison with ozagrel and argatroban.

Platelet activation and subsequent aggregation play a key role in the pathogenesis of ischemic brain damage. Recent studies revealed that enhanced platelet activation is also observed after ischemia, suggesting that secondary thrombus formation might participate in the development of cerebral infarction. The binding of platelet glycoprotein GPIIb/IIIa (integrin alpha(IIb)beta3) to fibrinogen is the final common pathway in platelet aggregation. Therefore, GPIIb/IIIa antagonists might be useful in acute ischemic stroke as well as in the secondary prevention of ischemic stroke. In the present study, we evaluated the effect of three compounds, FK419 ((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl] piperidin-3-ylcarbonyl] amino] propionic acid trihydrate), a novel nonpeptide GPIIb/IIIa antagonist, ozagrel, a selective thromboxane A(2) synthase inhibitor, and argatroban, a thrombin inhibitor, on middle cerebral artery (MCA) patency and ischemic brain damage using photochemically induced MCA thrombosis model in guinea pigs. FK419, ozagrel, or argatroban was administered 5 min after the termination of photoirradiation. FK419 dose-dependently improved MCA patency by decreasing the total occlusion time, time to continuous reperfusion, and the number of cyclic flow reductions, at doses that inhibited ADP-induced platelet aggregation ex vivo. In contrast, ozagrel only improved total occlusion time, and argatroban showed no improvement in MCA patency. FK419 also reduced ischemic brain damage in a dose-dependent fashion, whereas ozagrel and argatroban did not. Finally, FK419 ameliorated neurological deficits, whereas ozagrel and argatroban did not. These results indicate that FK419, a GPIIb/IIIa antagonist, ameliorates ischemic brain damage by improving MCA patency after occlusion and that FK419 is a promising candidate for the treatment of acute ischemic stroke.

Animals↗
PubMed2005

Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.

Astrocytoma↗
PubMed2005

The NO/cGMP pathway inhibits Rap 1 activation in human platelets via cGMP-dependent protein kinase I.

The NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined. We have studied NO/cGMP effects on the activity of Rap 1, an abundant guanine-nucleotidebinding protein in platelets. Rap 1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap 1 inhibition correlated with cGKI activity as measured by the phosphorylation state of VASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp-8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation. Thrombin-,ADP- and collagen-induced formation of Rap 1-GTP in platelets as well as turbulence-induced Rap 1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap 1 activation induced by the Galpha(i)-coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap 1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin alpha(IIb)beta3, we propose that Rap 1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.

Adenosine Diphosphate↗
PubMed1998

Differential involvement of tyrosine and serine/threonine kinases in platelet integrin alphaIIbbeta3 exposure.

The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on 125I-fibrinogen binding to alpha(IIb)beta3 and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of alpha(IIb)beta3 exposure by 30 micromol/L (ADP stimulation) and 35 to 40 micromol/L (alpha-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to approximately 80% (for alpha-thrombin). Studies with a submaximal dose of herbimycin A (approximately 50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as alpha(IIb)beta3 exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in alpha(IIb)beta3 exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP- and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and alpha-thrombin.

Blood Platelets↗
PubMed1996

Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

Adult↗
PubMed2002

Neoplastic cells and proliferating endothelial cells express connective tissue growth factor (CTGF) in glioblastoma.

Connective tissue growth factor (CTGF) was recently described as a cysteine-rich mitogenic peptide that is the prototypic member of the CCN family of protein including CTGF/fisp12, cef/10/cyr61 and nov. Recently CTGF was reported to have a function of mediating endothelial cell adhesion and migration through integrin alpha(v)beta3, promoting endothelial cell survival, and inducing angiogenesis in vivo. To clarify involvement of CTGF in tumor angiogenesis, using brain tissues with glioblastoma, we examined CTGF expression and localization by immunohistochemical staining and reverse transcription-polymerase chain reaction. The result showed that both glioblastoma tumor cells and proliferating endothelial cells are positive for CTGF by immunohistochemical staining, and CTGF mRNA expression was evaluated by RT-PCR in the brain tissues of glioblastoma. These findings suggest that CTGF may have the potential to stimulate angiogenesis in glioblastoma and function as an angiogenic factor in the process of tumor growth.

Adolescent↗
PubMed2004

Nongenomic effects of 17beta-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor beta and Src kinase.

The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.

Adult↗