An intermediary role of proHB-EGF shedding in growth factor-induced c-Myc gene expression.

Activation of growth factor receptors by ligand binding leads to an increased expression of c-Myc, a transcriptional regulator for cell proliferation. The activation of transcriptional factors via the activated receptors is thought to be the main role of c-Myc gene expression. We demonstrate here that epidermal growth factor receptor (EGFR)- and fibroblast growth factor receptor (FGFR)-mediated c-Myc induction and cell cycle progression in primary cultured mouse embryonic fibroblasts (MEFs) are abrogated by knockout of the heparin-binding EGF-like growth factor (Hb-egf) gene, or by a metalloproteinase inhibitor, although molecules downstream of the receptors are activated. Induction of c-Myc expression by EGF or basic FGF is recovered in Hb-egf-depleted MEFs by overexpression of wild-type proHB-EGF, but no recovery was observed with an uncleavable mutant of proHB-EGF. The uncleavable mutant also inhibited EGF-induced acetylation of histone H3 at the mouse c-Myc first intron region, which could negatively affect transcriptional activation. We conclude that signal transduction initiated by generation of the carboxyl-terminal fragment of proHB-EGF (HB-EGF-CTF) in the shedding event plays an important intermediary role between growth factor receptor activation and c-Myc gene induction.

Heparin binding EGF is necessary for vasospastic response to endothelin.

Endothelin-1 (ET-1), a powerful vasoconstrictor, is involved in vasospastic diseases such as coronary artery disease and subarachnoidal hemorrhage, as well as in renal and cardiovascular fibrotic remodeling. Transactivation of the epidermal growth factor receptor (EGFR) mediates ET-1 signaling in vascular smooth muscle cells (VSMCs) and isolated arteries. Moreover, EGFR is required for a full constrictive response to ET-1. However, the relevant mechanisms mediating EGFR transactivation in response to ET-1 have not been identified. The present study used isolated arteries and VSMCs to investigate the role of the EGFR ligand heparin binding-epidermal growth factor (HB-EGF) in ET-1-induced transactivation of EGFR, intracellular calcium mobilization, and VSMCs contraction. While baseline blood pressures were similar in HB-EGF-deficient and in wild-type littermate mice, the vasoconstrictor actions of ET-1 were attenuated in HB-EGF-/- animals. In isolated mouse carotid artery segments mounted in an arteriograph, ET-1 caused only a weak increase in isovolumetric tone in HB-EGF-deficient vessels, and this effect was mimicked by inhibition of EGFR tyrosine kinase or phosphoinositide 3-kinase (PI3K) in wild-type arteries with or without endothelium, indicating a specific role in VSMCs. EGFR or PI3K inhibitors had no effect on KCl-induced contraction, which was normal in HB-EGF-deficient mice. To confirm that the abnormal responses in HB-EGF-deficient mice were due to impaired EGFR signaling, we studied VSMCs from waved-2 (wa2) mice; these animals have a mutation causing a partial loss of function of EGFR tyrosine kinase activity. The ET-1-induced calcium peak was reduced by 30% in VSMCs from wa2 mice and from HB-EGF-/- mice. This effect was reproduced by preincubation of wild-type VSMCs with EGFR inhibitor AG1478 and PI3K inhibitors LY294002 and wortmannin. ProHB-EGF is bound to the cell membrane and released after cleavage by metalloproteinases; its action may contribute to effects of GPCR agonists on cell growth. Pretreatment of mouse VSMCs with batimastat, a metalloproteinase inhibitor, significantly attenuated ET-1-induced [Ca(2+)](i) response in wild-type cells. Human proHB-EGF has been shown to be the endogenous receptor for Corynebacterium diphteriae toxin (DT). Mutated DT toxin (CRM197) is devoid of toxicity but it neutralizes HB-EGF binding to EGFR. Pretreatment of human VSMCs from internal mammary arteries with CRM197 significantly blunted ET-1-stimulated calcium transients. In conclusion, these findings suggest that the mechanism of ET-1-induced vasoconstriction involves HB-EGF-mediated transactivation of the EGFR. This functional cascade requires modulation of agonist-induced calcium transient by EGFR and PI3K with extremely fast kinetics, suggesting a novel paradigm for GPCR-mediated calcium signaling, which may offer future therapeutic targets.

Inhibition of thrombosis in melanoma allografts in mice by endogenous mast cell heparin.

An unexplained paradox of malignant melanoma is the apparent failure of the blood within the tumor to clot despite the presence of multiple factors that should promote blood clotting. Here we present histochemical evidence that human and murine melanomas are extensively infiltrated by abundant mast cells. Because mast cells contain the natural anticoagulant heparin, the present studies were aimed at defining the role of mast cell heparin in preventing the blood from clotting within B16 melanoma grafts in C57BL/6 J mice. Mice bearing B16 melanoma grafts were treated with non-specific or specific inhibitors of mast cell heparin (protamine or heparinase, respectively). After the drug treatment there was histologic and functional evidence of selective thrombosis of the blood vessels within the protamine and heparinase treated melanoma grafts. A similar, high degree of thrombosis was also observed in B16 tumors grown in transgenic NDST-2 knockout mice bearing a targeted disruption in the gene coding for mast cell heparin synthesis. The tumors grown in the protamine-treated animals were significantly smaller than the tumors from control (untreated mice). By contrast, the tumors treated with heparinase or grown in the NDST-2 knockout mice were significantly larger than the tumors from control (untreated) mice. We conclude that the intrinsic procoagulant properties of malignant melanoma are neutralized in vivo by the anticoagulant properties of endogenous heparin produced by mast cells that naturally infiltrate the tumor. Our results also suggest that thrombosis and hemostasis within melanoma may play a complex role in modulating the growth of the tumor.

Endogenous heparin activity deficiency: the 'missing link' in atherogenesis?

This paper reviews published studies since 1995 dealing with many atherogenic mechanisms where exogenous heparin was beneficial. In these areas endogenous heparin deficiency is likely to be harmful. Mechanisms included inflammatory factors, lower endogenous plasma heparin levels, lipoprotein lipase, chemokines, APOE e4, lipoprotein(a), among others. Demonstrated reduction of heparan sulfate proteoglycans (HSPG) and of endogenous plasma heparin was reviewed.

Regulation of extravascular coagulation and fibrinolysis by heparin-dependent mast cell chymase.

We recently characterized a heparin-deficient mouse strain generated by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2-/- mice show severe defects in their organization of mast cell (MC) secretory granules, with an almost total absence of the various heparin-binding MC proteases. In the present report we have studied the consequences of heparin/MC protease deficiency for extravascular coagulation and fibrinolysis. Addition of prothrombin to peritoneal cells-a mixture of macrophages, lymphocytes, and MCs-resulted in formation of thrombin but the accumulation of thrombin occurred faster in the NDST-2-/-cells than in normal controls. Further, the generated thrombin was subsequently inactivated in the NDST-2+/+ cell cultures but not in the NDST-2-/- cells. Plasminogen was activated to plasmin at an apparently higher rate in peritoneal cells from NDST-2 null mice than in the normal controls. Similar to thrombin, the generated plasmin was inactivated by NDST-2+/+ but not by the NDST-2-/- cells. Subsequent experiments with normal cells showed that cell surface-associated MC chymase, in a strongly heparin-dependent manner, was responsible for both the thrombin-inactivating- and plasmin-inactivating activities. These results show that MC chymase-heparin complexes have the potential to regulate extravascular coagulation processes, as well as the plasminogen activator/plasmin system.

[Insular system of animals in a chronic heparin deficiency]. / Insuliarnaia sistema zhivotnykh pri khronicheskom defitsite geparina.

The chronic heparin deficiency achieved by long-term treatment (for 3 weeks) with protamine-sulfate is accompanied by the development of stable hyperglycemia, decreased glucose tolerance, and the appearance of insulin resistance. Administration of exogenous heparin promotes the restoration of normoglycemia.

Evidence that endogenous heparin activity deficiency may be an important factor in atherogenesis.

Known atherosclerotic risk factors account today for only 50% of atherogenesis. Evidence is presented that a deficiency of endogenous heparin may account for the other half. Sensitive techniques have shown that there are trace quantities of heparin in plasma of humans. An inverse relationship has been found between plasma heparin levels and triglyceride-bearing Sf 12-400 lipoprotein, and a lower plasma heparin level could be an important determinant of atherogenesis fostering lipid abnormalities. Endogenous heparin also protects endothelium from harmful mediators that can impair normal function. Since atherosclerosis is a chronic inflammatory disease process, with monocyte-mediated release of cytokines and activation of integrins and phospholipase A2-mediated generation of platelet activating factor, heparin inhibits many of these events. Endogenous heparin activity thus opposes the effects of inflammatory activators. Heparin is also kown to inhibit complement activation and to suppress endothelin release from endothelial cells. In addition, endogenous heparin may suppress smooth muscle cell proliferation and decrease microthrombi formation on injured endothelial sites. All of these data seem to suggest that a deficiency of endogenous heparin or heparin-like substances predipose to atherosclerosis. It is conceivable that a genetically determined endogenous heparin deficiency is involved in atherosclerosis.

Sensitivity and specificity of the Hemochron, Hemotec and APTT for the detection of low concentrations of heparin.

This study compared two bedside methods recommended for the detection of low concentrations of heparin and the activated partial thromboplastin time (APTT), with reference to a laboratory measure of heparin concentration. Patients undergoing cardiopulmonary bypass had blood drawn at four stages when low levels of heparin could be expected. At each stage four tests were performed: whole blood clotting time using a Hemochron analyser with a Saline-Rinsed test cartridge, whole blood clotting time using a Hemotec analyser with a High Range Heparinase test cartridge, APTT, and heparin concentration by polybrene neutralization. Thirty patients were studied. The sensitivity of the Saline-Rinsed Hemochron, Hemotec High Range Heparinase, and APTT in detecting concentrations of heparin less than 1 U/ml was 38%, 40% and 97%, respectively, while specificities were 87%, 90%, and 30%, respectively. Neither the Saline Rinsed Hemochron, nor the Hemotec Heparinase cartridge reliably detected concentrations of heparin less than 1 U/ml.

Heparan sulfate proteoglycans are primarily responsible for the maintenance of enzyme activity, binding, and degradation of lipoprotein lipase in Chinese hamster ovary cells.

Various aspects of lipoprotein lipase (LPL) metabolism, including cell surface binding, degradation, and enzymatic activity, were compared between Chinese hamster ovary (CHO) cells and two distinct proteoglycan-deficient CHO cell lines. The contribution of low density lipoprotein receptor-related protein in binding LPL was also analyzed by the use of a 39-kDa receptor-associated protein expressed as a glutathione S-transferase fusion protein (GST-RAP). Equilibrium binding data with 125I-LPL revealed the presence of a class of high affinity binding sites with a KD of 7.8 nM in CHO cells, whereas no high affinity binding was observed for proteoglycan-deficient cells. The high affinity binding of LPL in CHO cells appeared to be concentrated in cell surface projections and was not effectively inhibited by GST-RAP. Moreover, degradation of endogenous and exogenous LPL was significantly greater in control CHO cells than in proteoglycan-deficient cells. Degradation of LPL in CHO cells was not affected by GST-RAP, suggesting that proteoglycans and not low density lipoprotein receptor-related protein are responsible for the majority of binding and degradation of LPL in these cells. Our data also show that proteoglycan binding is not essential for the assembly of active LPL homodimers, although proteoglycan binding controls the distribution of LPL activity. Furthermore, LPL produced by CHO cells was more stable than LPL produced by proteoglycan-deficient cells.

Mapping of heparin-binding structures on bovine herpesvirus 1 and pseudorabies virus gIII glycoproteins.

The gIII glycoproteins of both bovine herpesvirus 1 (BHV 1) and pseudorabies virus (PrV) mediate the initial and dominant interactions between virus and permissive host cells. By studying virus binding to wild-type and heparin-deficient CHO cells, we demonstrated that the cellular heparin-like moieties play an essential role in BHV 1 and PrV gIII-mediated virus attachment. Subsequent studies were carried out to map the gIII structures that are responsible for heparin binding. First, based on the observation that BHV 1 and PrV are differentially sensitive to heparin inhibition of gIII-mediated attachment to cells, we conducted a gIII domain shuffling experiment. This involved the construction of a set of recombinant BHV 1 expressing BHV 1 and PrV gIII chimeras and then using the sensitivity to heparin inhibition as a means of mapping the potential heparin-binding regions on the gIII molecules. Next, we synthesized panels of partially overlapping BHV 1 and PrV gIII peptides and examined their reactivity to heparin. The results from these experiments demonstrated five heparin-binding sites between amino acid 129 and 310 of BHV 1 gIII and four heparin-binding sites between amino acid 90 and 275 of PrV gIII.

[Current concepts of the possibility of correcting blood coagulation disorders in late pregnancy toxemia]. / Sovremennye poniatiia o vozmozhnostiakh korrektsii narushenii gemokoaguliatsii pri pozdnikh toksikozakh beremennykh.

Based on the reported data and their own experience the authors suggested that immediate effect of anticoagulative drug heparin was necessary for the correction of hemo- and homeostatic disorders in females with late gestosis.

[System of regulating the aggregative properties of blood in closed trauma of the chest]. / Sistema reguliatsii agregatnogo sostoianiia krovi pri zakrytoi travme grudi.

The investigation of the system of control of the aggregation properties of blood was carried on in 50 patients with closed traumas of chest in early terms after trauma. Activation of hemostasis was detected which was developing by the type of subacute syndrome of disseminated intravascular coagulation. The cause of these changes is thought to be a release of activators of the coagulating system and thrombocyte aggregation factors into the blood flow from the injury focus. High hemostatic potential is responsible for the disturbed microcirculation participating in pathogenesis of complications observed in closed traumas of the chest.

The role of megakaryocytes and tissue mast cells in the respiratory distress syndrome of adults.

The pathological role of megakaryocytes has been studied during adult respiratory distress syndrome (ARD) in the formation of intraalveolar oedema, microthrombi and hyaline membranes. The giant cells of the bone marrow are also thought to be involved in the development of peribronchial, perivascular and interalveolar oedemas. Heparin produced by tissue mast cells appears to counteract the clotting disturbances and to promote fibrinolysis in the acute phase of ARD. Vasoactive substances liberated from mast cells enhance oedema formation. Heparin has an influence on the lipid metabolism of type II pneumocytes. During regeneration the stimulating effect of heparin on fibrosis becomes predominant in the connective tissue ground substance. No fundamental morphological difference was found between ARD and the infantile respiratory distress syndrome.

[Osteopetrosis in mice caused by ectomesenchymal lesions]. / Osteopetroz u myshi, obuslovlennyi porazheniem ektomezenkhimy.

Pathogenesis of osteopetrosis in mice homozygotic for microphthalmia (symbol mi) mutant gene has been studied. Heparin concentration in the blood of mi/mi mice was demonstrated to be 10 times as little as the normal. Administration of different doses of heparin (1, 5, 10 per g of body weight) at different intervals (2--20, 10--20 and 20--25 days including) of their postnatal development causes partial or complete normalization in the construction of the central part of diaphysis of tubular bones. The number of heparin-secreting mast cells, derivatives of the neural crest is less than normal. Osteopetrosis in mi/mi mice is an integral part of the complex syndrom produced by the damage of neural crest cells.