Mieke Dewerchin

Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis.

As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor(HIF)-1α helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1α+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1α genes (HIF-1α−/−); however, a deficiency of HIF-1α does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1α-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1α-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1α reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1α tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders,, this new role for HIF-1α in hypoxic control of cell growth and death may be of general pathophysiological importance.

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.

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow–derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.

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.

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

Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1

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.

Anti-PlGF Inhibits Growth of VEGF(R)-Inhibitor-Resistant Tumors without Affecting Healthy Vessels

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.

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.

Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms

The murine VEGF gene is alternatively transcribed to yield the VEGF(120), VEGF(164), and VEGF(188) isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF(164/164) mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF(120/120) mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF(188/188) mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF(164), was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)2 vitamin D [1,25(OH)2D3] action on calcium homeostasis. We assessed the rate of serum 45Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D9K expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA1b) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)2D3 injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D9K expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D9K and PMCA1b expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D9K, should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.