Publications

Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell's decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.

Abstract Introduction of imatinib and second-generation BCR/ABL inhibitors has revolutionized treatment of patients with Philadelphia chromosome positive (Ph+) leukemia, but leukemia cells persist even in successfully treated patients, and some patients develop resistance and ultimately relapse. The reasons for these drawbacks are not entirely resolved, but besides BCR/ABL the host stroma potentially plays an important (independent) role. Placental Growth Factor (PlGF), a homologue of VEGF, was already proven to be abundantly secreted by stromal cells in solid tumors. Therefore, we investigated the role of PlGF, and the therapeutic potential of αPlGF, a monoclonal antibody against PlGF, which we recently reported to have a broad anti-tumoral potential in pre-clinical models of solid tumors (Fischer et al., Cell, 2007), in BCR/ABL+ lymphoid and myeloid leukemia. First, we studied expression of PlGF by 5 different human and murine BCR/ABL+ leukemia cell lines (Bv-173, BaF3, 32D, K562, KCL22) in vitro and found that neither of the cell lines secreted PlGF protein, but expressed its target receptor VEGFR-1. In contrast, primary murine adherent bone marrow stromal cells (BMDSC) expressed abundant amounts of PlGF protein (up to 105 pg/ml/105 cells), indicating a potential stroma-related function of PlGF. Second, we analyzed whether PlGF could induce proliferation and thereby revealed dose-dependent induction of proliferation by recombinant PlGF in all analyzed leukemia cell lines. This pro-proliferative effect of PlGF was nearly completely abrogated by both αPlGF and an extracellular anti-VEGFR-1 antibody, thus indicating that it is mediated primarily by VEGFR-1. Third, we studied potential paracrine interactions between BMDSCs and leukemia cells by performing co-culture experiments. Remarkably, coculture of BMDSC with leukemia cells significantly induced proliferation of both cell types. We hypothesized, that this induction of proliferation might be mediated by PlGF and indeed found its nearly complete abrogation upon addition of αPlGF to the co-cultures. Furthermore, BMDSCs significantly upregulated PlGF secretion (2.1 fold; N=3; P=0.005) when cultured in presence of leukemia cells. Thus, we conclude, that stromal derived PlGF promotes proliferation of leukemia cells in a paracrine fashion and at the same time acts as autocrine pro-proliferative signal for stromal cells. To test this hypothesis in vivo, we established 3 different murine models of BCR/ABL+ myeloid and lymphoid leukemia. Subsequently, we analyzed PlGF protein as present in blood and bone marrow of diseased mice in comparison to healthy mice, and detected no PlGF protein in the peripheral blood of healthy mice and low amounts of PlGF protein in their bone marrow. In contrast, leukemic mice showed PlGF protein (76.5 ± 18.4 pg / ml plasma; N=7) in their circulation at levels comparable to mice bearing solid tumors, and, interestingly more than 8.9 fold (N=7; P<0.0001) elevated PlGF levels in their bone marrow, compared to healthy mice, again indicating that PlGF represents a stroma derived, novel pathogenetic factor in Ph+ leukemia. In order to investigate the therapeutic potential of PlGF inhibition in murine BCR/ABL+ leukemia, we treated mice bearing leukemia induced by syngeneic BCR/ABL+ BaF3 cells with αPlGF, and found a significant prolongation of median survival by 18 days (N=9; P=0.015), compared to control antibodies. Encouraged by these positive results, we established models of imatinib-sensitive and imatinib-resistant (T315I mutant) CML by transducing primary bone marrow cells and subsequent transplantation into lethally irradiated recipient mice. Interestingly, also in these aggressive models, we found a significant prolongation of survival of diseased mice induced by blockade of PlGF compared to controls (median survival prolongation in wt BCR/ABL induced leukemia 5 days; N=11; P=0.002; in T315I mutant 4 days; N=12; P=0.039). Bone marrow histology and phenotypic analysis by FACS revealed decreased infiltration of spleen and bone marrow with leukemia cells (reduction in the bone marrow by 38% and in the spleen by 24%). In conclusion, these data indicate that PlGF represents a stromal derived factor promoting the progression of Ph+ leukemia, independent of BCR/ABL mutational status, and might represent a novel target produced by the leukemic stroma, potentially useful adjunct to BCR/ABL kinase inhibitors.

BACKGROUND: Excessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia. METHODS: The ROS levels were analyzed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia. The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes. RESULTS: Exposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD)-dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1). The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes. CONCLUSIONS: We conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use. FUNDING: This work was supported by grants from the Swedish Research Council, Stockholm County Research Council, Stockholm Regional Research Foundation, Bert von Kantzows Foundation, Swedish Society of Medicine, Kung Gustaf V:s och Drottning Victorias Frimurarestifelse, Karolinska Institute's Research Foundations, Strategic Research Programme in Diabetes, and Erling-Persson Family Foundation for S-B.C.; grants from the Swedish Research Council and Swedish Heart and Lung Foundation for T.A.S.; and ERC consolidator grant for M.M.

Introduction: Murine placentation is associated with trophoblast cells invasion of the maternal endometrium and extensive maternal and foetal angiogenesis. Both processes involve proteases‐dependent extracellular matrix remodelling. Among the protease inhibitors, plasminogen activator inhibitor‐1 (PAI‐1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at days 10.5‐11.5 day post‐coitum (dpc). Although accumulating evidence demonstrates the key role of PA‐1 in pathological angiogenesis, its function during placental vascularisation remains to be elucidated. PAI‐1 knockout mice are fertile and the litter sizes are normal. We have therefore analysed the consequence of PAI‐1 deficiency on murine placentation. Material and Methods: We have studied the possible role of PAI‐1 by quantitating the placental vessel density, the relative thickness of the labyrinth, decidua and spongiotrophoblast at day 10.5, 12.5 and 14.5 dpc in mice deficient for PAI‐1 or in control mice. An original method of computer‐assisted image analysis allowed us to quantify alterations of several placental compartments identified with specific monoclonal antibodies (keratin, desmin, fibrinogen and MECA‐32). To investigate the differentially expressed genes, we performed laser capture microdissection (LCM), followed by genome‐wide expression profiling using high‐density oligonucleotides microarray analysis (GeneChip Mouse Genome 430 2.0 Array, Affymetrix). Data were analysed using Ingenuity Pathways Analysis (Ingenuity Systems ® , http://www.ingenuity.com ). Results: At 10.5 and 12.5 dpc, an abnormal placental morphology was observed in both labyrinth and spongiotrophoblast layers in PAI‐1‐/‐ mice. Lack of PAI‐1 resulted in a transient decreased maternal and fetal vascularisation of the placenta that caused (1) an enhancement in the decidua/labyrinth and labyrinth/spongiotrophoblast thickness ratios, (2) a significant increase of trophoblast density. Normalization of placental morphology occurred by day 14.5 dpc in PAI‐1 deficient mice. Statistical analysis of microarrays revealed 706 genes differentially expressed between PAI‐1 deficient and normal mice in the labyrinth zone at 10.5 dpc. At 14.5 dpc, only 205 genes are differentially expressed. Using Ingenuity Pathways Analysis, most of those genes were found to be associated to lipid metabolism, cellular growth and proliferation. Conclusion: Despite a transient PAI‐1 requirement for optimal placental angiogenesis, this gene does not appear to be essential for trophoblast invasion and placentation.

The murine VEGF gene is alternatively transcribed to yield the VEGF120, VEGF164, and VEGF188 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. VEGF164/164 mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF120/120 mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF188/188 mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF164, 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.