Florea Lupu

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA–/–) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA–/– mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Extracellular histones are major mediators of death in sepsis.

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF 164 and VEGF 188

Loss of HIF-2α and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice

Respiratory distress syndrome (RDS) due to insufficient production of surfactant is a common and severe complication of preterm delivery. Here, we report that loss of the hypoxia-inducible transcription factor-2α (HIF-2α) caused fatal RDS in neonatal mice due to insufficient surfactant production by alveolar type 2 cells. VEGF, a target of HIF-2α, regulates fetal lung maturation: because VEGF levels in alveolar cells were reduced in HIF-2α-deficient fetuses; mice with a deficiency of the VEGF164 and VEGF188 isoforms or of the HIF-binding site in the VEGF promotor died of RDS; intrauterine delivery of anti-VEGF-receptor-2 antibodies caused RDS and VEGF stimulated production of surfactant proteins by cultured type 2 pneumocytes. Intrauterine delivery or postnatal intratracheal instillation of VEGF stimulated conversion of glycogen to surfactant and protected preterm mice against RDS. The pneumotrophic effect of VEGF may have therapeutic potential for lung maturation in preterm infants.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

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−/− 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−/− 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.

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

HIF prolyl hydroxylases (PHD1–3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Inhibitory Role of Plasminogen Activator Inhibitor-1 in Arterial Wound Healing and Neointima Formation A Gene Targeting and Gene Transfer Study in Mice

BACKGROUND Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation. METHODS AND RESULTS Neointima formation was evaluated in PAI-1-deficient (PAI-1(-/-)) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001+/-0.001 mm2 in PAI-1(+/+) and 0.016+/-0.008 mm2 in PAI-1(-/-) mice within 1 week after electric injury (P<.02) and 0.055+/-0.008 mm2 in PAI-1(+/+) and 0.126+/-0.006 mm2 in PAI-1(-/-) mice within 3 weeks after mechanical injury (P<.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1(-/-) smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1(-/-) mice were intravenously injected with replication-defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61+/-8 microg/mL with 2x10(9) plaque-forming units (pfu) virus. Luminal stenosis was 35+/-13% in control AdRR5-treated (2x10(9) pfu) and suppressed to 5+/-5% in AdCMVPAI-1-treated (6x10(8) pfu) PAI-1(-/-) mice (P<.002). CONCLUSIONS By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.

Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries.

High plasma levels of plasminogen activator inhibitor type-1 (PAI-1), the principal inhibitor of the fibrinolytic system, have been associated with thrombotic and arterial disease. To study PAI-1 expression in healthy and atherosclerotic human arteries, a detailed analysis was made by light and electron microscopy immunocytochemistry and by in situ hybridization. In healthy arteries PAI-1 was found both at the level of endothelial cells and of smooth muscle cells (SMCs) of the arterial media. In early atherosclerotic lesions PAI-1 was also detected in intimal SMCs and in extracellular areas in association with vitronectin. Immunogold analysis by electron microscopy revealed PAI-1 in vesicular structures in endothelial cells and in SMCs with normal or foam cell characteristics. In advanced atheromatous plaques, PAI-1 mRNA expression in SMCs within the fibrous cap was increased compared with SMCs located in the adjacent media or in normal arterial tissue. PAI-1 mRNA was also detected in macrophages located at the periphery of the necrotic core. The increased synthesis of PAI-1 by cellular components of the atherosclerotic plaque and the extracellular accumulation of PAI-1 may contribute to the thrombotic complications associated with plaque rupture and possibly play a role in the accumulation of extracellular matrix deposits.