The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis

The frequency of heterozygous protein C and protein S deficiency, detected by measuring total plasma antigen, in a group (n = 141) of young unrelated patients (less than 45 years old) with venous thrombotic disease was studied and compared to that of antithrombin III, fibrinogen, and plasminogen deficiencies. Among 91 patients not receiving oral anticoagulants, six had low protein S antigen levels and one had a low protein C antigen level. Among 50 patients receiving oral anticoagulant therapy, abnormally low ratios of protein S or C to other vitamin K-dependent factors were presented by one patient for protein S and five for protein C. Thus, heterozygous Type I protein S deficiency appeared in seven of 141 patients (5%) and heterozygous Type I protein C deficiency in six of 141 patients (4%). Eleven of thirteen deficient patients had recurrent venous thrombosis. In this group of 141 patients, 1% had an identifiable fibrinogen abnormality, 2% a plasminogen abnormality, and 3% an antithrombin III deficiency. Thus, among the known plasma protein deficiencies associated with venous thrombosis, protein S and protein C deficiencies (9%) emerge as the leading identifiable associated abnormalities.     

Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or −2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.Angiogenesis is the process through which new blood vessels are formed from pre-existing capillaries. In adulthood the vasculature is normally quiescent, a homeostatic state that is maintained by a precise balance of pro-angiogenic inducers such as vascular endothelial growth factor and anti-angiogenic inhibitors such as thrombospondin (TSP)−1 and −2. This angiostatic balance can be physiologically regulated in favor of new blood vessel formation during processes such as menstruation and wound healing allowing normal tissue regeneration; while pathological disruption of this balance is associated with many disease states, including diabetic retinopathy, atherosclerosis-induced tissue ischemia, chronic inflammation, tumor growth and metastasis, obesity, asthma, and several autoimmune diseases.¹ Therefore, it is extremely important to identify and dissect the pathways involved in vessel growth in both normal and aberrant conditions.Neovascularization is a major component of malignant tumor growth and many therapeutic strategies have been developed to inhibit tumor angiogenesis, including antibodies or decoys that bind and neutralize vascular endothelial growth factor,^2,3 small molecule inhibitors of growth factor signaling pathways,⁴ and peptides based on the anti-angiogenic type I repeat domains (TSR) of TSP-1.^5,6,7 Studies on the mechanisms of TSP-mediated anti-angiogenesis revealed that the TSR domains play an essential role⁸ and that the type B scavenger receptor CD36 functions as the critical endothelial cell surface receptor.^9,10 Using mouse corneal pocket angiogenesis assays we recently demonstrated that CD36 also functions as the receptor for a 120 kDa anti-angiogenic fragment derived from an unrelated TSR-containing protein, Brain Angiogenesis Inhibitor 1 (BAI1). This fragment, known as vasculostatin or Vstat120, suppressed neovessel formation in corneas from wild-type mice yet no effect was observed in CD36 null animals, showing for the first time that a TSR-containing protein distinct from TSP-1 and −2 mediates its anti-angiogenic functions through interactions with CD36.¹¹BAI1 is a 1584-aa brain-specific protein predicted to have seven transmembrane segments and a large extracellular domain. The extracellular domain contains an RGD integrin recognition motif, a putative hormone receptor (HomR) domain, and five TSR domains. Its expression is down-regulated in glioblastomas¹² and inversely correlated with vascularity and metastasis in colorectal cancer,¹³ consistent with an anti-angiogenic role. Kaur et al¹⁴ have shown that the TSR-containing fragment, Vstat120, was released from the cell membrane via proteolytic cleavage at a G protein–coupled receptor cleavage site and that this fragment inhibited microvascular endothelial cell (MVEC) proliferation, migration, and tube formation equivalent to that seen with full-length BAI1. Strikingly, restoration of Vstat120 expression in human glioma cells suppressed tumorigenicity and vascularity and enhanced animal survival in subcutaneous and orthotopic tumor implantation models in nude mice.¹¹The binding site on CD36 for the TSR domains of TSP-1 and −2 and Vstat120 has been localized to amino acids 93 to 120^11,15,16 within a highly conserved region termed the CLESH domain (CD36, LIMP2, EMP structural homology – see Figure 1A). CLESH domains are also found in proteins genetically distinct from the CD36 family,¹⁷ including some, such as HIV gp120 and histidine-rich glycoprotein (HRGP) which are known to bind TSP-1. HRGP, a 75-kDa glycoprotein synthesized in the liver, circulates in the blood at moderately high concentrations (approximately 100 to 150 μg/ml), and is secreted from activated platelets. In addition to TSP-1 and −2, it binds a wide range of ligands including divalent metal cations and several components of the provisional matrix laid down by healing wounds and tumors where angiogenesis is prevalent (for review see¹⁸). It has been implicated in the coagulation and fibrinolytic systems, and high levels have been associated with thrombotic disorders.^19,20 Work in our lab has shown that HRGP acts as a CD36 decoy to promote angiogenesis by binding TSP-1 and −2 and thereby preventing them from binding to CD36 on the endothelial cell surface.^21,22 Because both HRGP and CD36 compete for the identical binding region in TSP-1 and −2, we hypothesized that the relative concentrations of ligands, receptor, and decoy within a given microenvironment would ultimately determine the degree of angiogenesis. Within tumors and other areas of chronic inflammation, HRGP can be released from activated platelets as well as deposited from plasma that gained access via poorly organized hyperpermeable vessels. The angiogenic balance would then be tipped to favor angiogenesis by HRGP binding to TSPs as well as potentially other TSR containing anti-angiogenic proteins.Open in a separate windowFigure 1HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.In this article we show that the CLESH domain of HRGP binds Vstat120 and suppresses its anti-angiogenic activity by reversing inhibition of endothelial cell migration and tube formation. Furthermore, we show in both subcutaneous and orthotopic brain tumor models that HRGP exacerbates glioblastoma tumor growth and enhances tumor vascularity. We also show that the amount of HRGP present in human brain is increased in patients with primary tumors with the protein localized predominantly within the basement membrane. Finally, we offer some insight into the mechanism of action of Vstat120 by showing caspase-3 activation and endothelial cell apoptosis on Vstat120 addition. Together these results suggest that deposition of HRGP into angiogenic microenvironments, perhaps as the result of the inherent “leakiness” of the neo-vasculature and/or of platelet granule release, can modulate the anti-angiogenic processes mediated by the general family of TSR-containing proteins, and may shed light on the mechanism of angiogenesis regulation in the brain.     

Focal adhesion kinase enhances signaling through the Shc/extracellular signal-regulated kinase pathway in anaplastic astrocytoma tumor biopsy samples

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that on activation generates signals that can modulate crucial cell functions, including cell proliferation, migration, and survival. In vitro, overexpression of FAK has been shown to promote cell proliferation by signaling through the Ras/mitogen-activated protein kinase cascade in several cell types. We have shown previously that overexpression of exogenous FAK lacking alternative splicing in malignant astrocytoma clones injected intracerebrally into SCID mouse brains promotes tumor cell proliferation. Here, we show that in anaplastic astrocytoma biopsy samples, FAK is expressed as an unspliced variant and migrates with a faster mobility similar to that observed in embryonic brain. Compared with nonneoplastic adult brain biopsies, the levels of FAK protein are elevated as are its levels of activation as assessed by autophosphorylation and overall tyrosine phosphorylation. The activity of Src kinase in these tumors is also elevated, as well as the activity of Src kinase associated with FAK; the latter may result in enhanced Src kinase phosphorylation of FAK. Phosphorylated Shc is associated with FAK in the anaplastic astrocytoma biopsy samples and in astrocytoma cells overexpressing FAK in vitro but not in nonneoplastic brain biopsy samples. Elevated extracellular signal-regulated kinase-2 activation and elevated expression of cyclins D and E are also found in anaplastic astrocytoma biopsy samples. These data provide evidence that the increased FAK activity in these tumors contributes to phosphorylation of Shc and likely to the promotion of Ras activity, extracellular signal-regulated kinase-2 activation, and cell proliferation in vivo.     

Elevated expression of TGF-beta1 in head and neck cancer-associated fibroblasts

Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood. We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells. Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFalpha, TGF-beta1, HGF, PDGF-alpha, IGFII, bFGF, aFGF, VEGFC, and VEGF. Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro. Fibroblast populations were assessed for TGF-beta1 expression by ELISA and luciferase reporter assay to assess protein expression. We identified TGF-beta1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-beta1 was significantly overexpressed (3.4-fold) in tumor-associated stroma. Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-beta1 in CAFs compared to NMF and NDF populations. Elevated levels of TGF-beta1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis. These results suggest that TGF-beta1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo.     

Oncostatin-M induction of vascular endothelial growth factor expression in astroglioma cells

Oncostatin-M (OSM), a hematopoietic cytokine, and vascular endothelial growth factor (VEGF), a quintessential angiogenic signal, are coexpressed in development, cancer and inflammation. Here, we report that OSM treatment of human astroglioma cell lines increases VEGF levels by approximately threefold. Interleukin-1beta (IL-1beta), in combination with OSM, induces up to sevenfold higher VEGF expression, without significantly inducing VEGF on its own. Specifically examining the OSM contribution to VEGF expression, neutralizing antibodies to OSM receptor subunits gp130 and OSMRbeta, but not LIFRbeta, inhibited OSM induction of VEGF, indicating that the OSM-specific receptor OSMRbeta/gp130 transduces the OSM signal for VEGF synthesis. OSM induction of VEGF promoter activity maps to the (-1171, -786) region of the VEGF promoter, which contains a STAT-3-binding site. STAT-3 is indeed essential for this response, since overexpression of a dominant-negative STAT-3 blocks OSM induction of VEGF promoter activity, as well as endogenous VEGF expression. Finally, we demonstrate that OSM is expressed in glioblastoma multiforme tumor biopsies, a particularly malignant form of brain tumor. This novel mechanism of VEGF regulation in astroglioma cells may be active in pathophysiological states where both OSM and IL-1beta are present.     

Expression of extracellular matrix metalloprotease inducer in laryngeal squamous cell carcinoma

OBJECTIVES/HYPOTHESIS: Head and neck cancer tumor cell invasion is responsible for both local destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesize that EMMPRIN is overexpressed in laryngeal cancer. STUDY DESIGN: Retrospective analysis of patients with supraglottic laryngeal cancer. METHODS: Total protein immunoblotting and immunohistochemical analysis of normal and malignant tissue were performed to determine EMMPRIN expression. EMMPRIN immunoreactivity in 33 patients was correlated with clinicopathological features and survival. RESULTS: Whole-tissue lysates of tumors (n = 8) and metastatic lymph nodes (n = 2), but not normal skin (n = 8) or mucosa (n = 6), expressed significant amounts of EMMPRIN by immunoblotting. EMMPRIN membrane immunoreactivity (transmembrane EMMPRIN score) was associated with nodal positivity (P =.07), and it was a borderline significant predictor of survival (Hazards Ratio = 2.4; 95% CI, 0.88-6.55). As a categorical variable, higher transmembrane EMMPRIN score was associated with higher mortality. CONCLUSIONS: The present study helps to establish EMMPRIN as a widely expressed protein in dysplastic mucosa and supraglottic laryngeal cancer, but not in normal epithelial counterparts.     

Safety of therapeutic beta-blockade in patients with coexisting bronchospastic airway disease and coronary artery disease

Atherosclerotic coronary artery disease and bronchospastic airway disease frequently coexist in older patients. There are substantial data suggesting reduced mortality with the use of beta-adrenergic blocking drugs in patients with symptomatic coronary artery disease, especially patients who have postmyocardial infarction and/or severe coronary artery disease associated with left ventricular dysfunction. Conversely, the use of beta-adrenergic blocking drugs (even selective beta(1)-adrenergic blocking drugs) has the potential of exacerbating bronchospasm. This prospective registry evaluates the safety of use of selective beta(1)-adrenergic blocking drugs in patients with symptomatic coronary artery disease and bronchospastic airway disease. A total of 835 consecutive patients with symptomatic coronary artery disease were prospectively evaluated for coexisting coronary and bronchospastic airway disease. Of these, 30 patients (mean age: 61 +/- 14 years) met the qualifying inclusion criteria. All these study patients except 1 (29/30 [96%]) reached therapeutic beta-blockade (resting heart rate <70 beats per minute). The 1 patient who discontinued use of beta-adrenergic blocking drugs as a result of lifestyle-limiting bronchospasm had no serious adverse outcome. No hospitalizations were required because of worsening bronchospasm. Ten percent of patients reported increased requirement of inhaled beta(2)-agonist use. The patients were followed for 15 +/- 9 months. One patient died of stroke at 22 weeks of follow-up. In conclusion, use of selective beta(1)-adrenergic blocking drugs at a therapeutic dose is safe (as long as careful clinical follow-up is available) and should be considered in all patients with coexisting symptomatic coronary artery disease and bronchospastic airway disease.     

Position Paper of the American Council on Science and Health on Risk Factors for Breast Cancer: Established, Speculated, and Unsupported

Abstract: This article provides the position of the American Council on Science and Health regarding how breast cancer is defined and classified; the magnitude of the public health problem of breast cancer among women; the implications of variation in incidence of breast cancer internationally and with migration; access to health care as a factor in slight differences in incidence and mortality rates among African-American and white women; and the evidence concerning various proposed human-breast-cancer risk factors. The article classifies risk factors as either established, speculated, or unsupported on the basis of available evidence. Specific genes have been identified that may explain as much as 5–10% of new breast cancer cases. Inherited predispositions may be characterized by family history of breast or ovarian cancer, young age at diagnosis, breast cancer diagnosed in both breasts, and male breast cancer. Benign breast disease (BBD), particularly the subtypes of BBD involving atypical hyperplasia, and exposure early in life to ionizing radiation is an established risk factor for breast cancer. Several reproductive characteristics are established as risk factors for breast cancer: early age at menarche, first full-term pregnancy after age 35 years of late age, and late age of menopause. Obesity and low physical activity are established as risk factors for breast cancer and are modifiable. Speculated risk factors for breast cancer that are gaining scientific support include nulliparity, oral contraceptive use, and postmenopausal estrogen replacement therapy. Speculated risk factors for which there is conflicting or preliminary support include not breast feeding, postmenopausal estrogen/progestogen replacement therapy, prescribed diethylstilbestrol, low consumption of phytoestrogens, specific dietary practices, alcohol consumption, not using nonsteroidal antinflammatory drugs, abortion, and breast augmentation. Unsupported risk factors include higher than average consumption of phytoestrogens, premenopausal obesity, electromagnetic fields, and low-dose ionizing radiation after 40 years of age. There is only limited support for xenoestrogens and large breast size as risk factors for breast cancer.