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Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest--specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo., Introduction Anemia results from insufficient production, excessive destruction, or loss of rbc (1). By limiting the capacity of the blood to carry oxygen, anemia causes tissue hypoxia and may lead [...]

Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis., Author(s): Annelii Ny [1, 5]; Marta Koch [1, 5]; Martin Schneider [1, 5]; Elke Neven [1, 5]; Ricky T Tong [2, 5]; Sunit Maity [1]; Christian Fischer [1]; Stephane Plaisance [...]

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone., Author(s): Monica Autiero [1, 12]; Johannes Waltenberger [2, 12]; Didier Communi [1, 12]; Andrea Kranz [2]; Lieve Moons [1]; Diether Lambrechts [1]; Jens Kroll [2]; Stephane Plaisance [1]; Maria De [...]

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf[sup.164] isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf[sup.164]-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome., Author(s): Ingeborg Stalmans [1, 12]; Diether Lambrechts [1, 12]; Frederik De smet [1]; Sandra Jansen [1]; Jian Wang [5]; Sunit Maity [1, 6]; Paige Kneer [5]; Maren von der Ohe [...]

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na[sup.+] channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A[sup.[DELTA]/+]) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a[sup.[DELTA]/+] mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a[sup.[DELTA]/+] mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome., Author(s): Dieter Nuyens [1]; Milan Stengl [2]; Saran Dugarmaa [4]; Tom Rossenbacker [1]; Veerle Compernolle [1]; Yoram Rudy [5]; Jos F. Smits [3]; Willem Flameng [6]; Colleen E. Clancy [5]; [...]

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf[sup.-/-]). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf[sup.-/-] mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf[sup.-/-] mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders., Author(s): Peter Carmeliet (corresponding author) [1]; Lieve Moons [1]; Aernout Luttun [1]; Valeria Vincenti [2]; Veerle Compernolle [1]; Maria De Mol [1]; Yan Wu [3]; Francoise Bono [4]; Laetitia Devy [...]

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6[sup.-/-] mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6[sup.-/-] mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis., Author(s): Anne Angelillo-Scherrer [1, 5]; Pablo Garcia de Frutos [2, 5]; Cristina Aparicio [2]; Els Melis [1]; Pierre Savi [3]; Florea Lupu [4]; Jef Arnout [1]; Mieke Dewerchin [1]; Marc [...]

Purification of a novel single polypeptide endopeptidase of 24 kDa has been carried out. Results indicate that interactions of 24k-endopeptidase affects plasmin-mediated cellular proteolytic activity.

In the absence of vascular endothelial growth factor genes, VEGF(sub.164) and VEGF(sub.188), in mice, impaired myocardial angiogenesis is seen as are reduced myocardial perfusion and fatal ischemic cardiomyopathy. The pursuit of gene therapy strategies of ischemic heart disease led researchers to generate the mice using targeted embryonic stem cells.

Hypoxia-inducible factor (HIF)-1-alpha deficiency prevents cells from undergoing hypoxic growth arrest, and, in a mitogenic microenvironment, stimulates proliferation. It has been established that tumour vascularization is largely controlled by HIF-1-alpha, partly as a result of upregulation of vascular endothelial growth factor. The HIF-1-alpha-dependent growth arrest in response to hypoxia has similarities with the p53-mediated control of growth arrest/apoptosis in response to genotoxic or physical stresses.

The role of arginine 719 found in human plasminogen in the formation of the staphylokinase:plasmin activator complex was investigated. Alanine-mutagenesis was applied to phage-displayed micro-plasminogen to determine the plasmin residues required for generating the staphylokinase: plasmin activator interface. Results showed that replacement of arginine 719 with Ala dramatically reduces the binding affinity and plasminogen activation potential of the staphylokinase:micro-plasminogen complex.

Genetic research shows that mice born without factor VII (FVII) binding capabilities develop without deformity but die of fatal perinatal haemorrhaging from normal blood vessels. Evidence suggests that tissue factor-FVII-/-initiated fibrin formation is not critical to embryonic development but is involved in other embryonic development processes.

The role of vascular endothelial growth factor in the development of endothelial cell-lined channel was probed through murine gene-targeting studies. Results have shown that urokinase-mediated plasmin proteolysis influenced the migration of smooth muscle cells promoting arterial neointima formation following vascular injury and in the destruction of the media and aneurysm formation during atherosclerosis. This neovascularization can be inhibited by adenovirus-mediated PAI-1 gene transfer.

Embryos containing a single inactive vascular endothelial growth factor (VEGF) gene show abnormal development of blood vessels. The homozygous VEGF-deficient embryos exhibit more abnormality in the blood vessel development, and more fatality than heterozygous mutants. The haploid-insufficient phenotype is associated with heterozygous VEGF deficiency. VEGF participation in vascular development depends on the ligand dose. The embryonic phenotypes from germline transmission and aggregation of heterozygous mutant embryo stem cells with tetraploid hosts are similar.

A study of the three deletion mutants apo A-I(delta-Leu44-Leu126), apo A-I(delta-Glu139-Leu170) and apo A-I(delta-Ala190-Gln243) and recombinant human apolipoprotein reveals that any mutation causing rupture of geometry in apo A-I decreases the activity of lecithin-cholesterol acyltransferase. When apo A-I(delta-Ala190-Gln243) and dimyristoylphosphatidylcholine are combined, the turbidity rate decreases 10 times as compared to other apo A-I proteins. Removal of carboxy terminal decreases the rate but not the degree of lipid binding while, removal of the amino group has little impact on lipid binding.

Experiments on plasminogen activation in mice with defective t-PA and/or u-PA genes reveal that t-PA and u-PA gene products have distinct functional roles in fibrinolytic and macrophage function. They also act cooperatively to bring about several biological processes, and their absence leads to spontaneous fibrin deposition. The deficiency of these genes does not affect embryonic development but can cause morbidity and death.

Vascular endothelial (VE) cadherin can influence vascular morphogenesis. Research findings reveal that VE-cadherin plays a vital role in controlling vascular endothelial growth factor-A-mediated endothelial survival through a pathway that involves the cytoplasmic domain of VE-cadherin, beta-catenin, vascular endothelial growth factor receptor-2 and P13-kinase. A relationship was also established which links the inhibition of normal vascular development with the loss or truncation of VE-cadherin.

Measuring blood levels of malondialdehyde (MDA)-modified low-density lipoprotein (LDL) and myocardial troponin I may distinguish patients with stable coronary artery disease (CAD) and those with unstable angina or a heart attack. MDA-LDL and troponin I are produced by damage to the heart. In a study of 104 patients with a history of CAD, the production of MDA-LDL and troponin I was 98% accurate in detecting patients with unstable angina and 100% accurate in diagnosing a heart attack. The absence of MDA-LDL and troponin was 99% accurate in detecting patients with stable CAD.

Circulating endothelial progenitor cells (EPCs) contribute to neovascularization of ischemic tissues and repair of injured endothelium. The role of bone marrow-derived progenitor cells in hypoxia-induced pulmonary vascular remodeling and their tissue-engineering potential in pulmonary hypertension (PH) remain largely unknown. We studied endogenous mobilization and homing of EPCs in green fluorescent protein bone marrow chimeric mice exposed to chronic hypoxia, a common hallmark of PH. Despite increased peripheral mobilization, as shown by flow cytometry and EPC culture, bone marrow-derived endothelial cell recruitment in remodeling lung vessels was limited. Moreover, transfer of vascular endothelial growth factor receptor-2+/Sca-1+/CXCR-4+-cultured early-outgrowth EPCs failed to reverse PH, suggesting hypoxia-induced functional impairment of transferred EPCs. Chronic hypoxia decreased migration to stromal cell-derived factor-1α, adhesion to fibronectin, incorporation into a vascular network, and nitric oxide production (-41%, -29%, -30%, and -32%, respectively, vs. normoxic EPCs; p < .05 for all). The dysfunctional phenotype of hypoxic EPCs significantly impaired their neovascularization capacity in chronic hind limb ischemia, contrary to normoxic EPCs cultured in identical conditions. Mechanisms contributing to EPC dysfunction include reduced integrin αv and β1 expression, decreased mitochondrial membrane potential, and enhanced senescence. Novel insights from chronic hypoxia-induced EPC dysfunction may provide important cues for improved future cell repair strategies. [ABSTRACT FROM AUTHOR]