Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Persistent organic pollutants (POPs) and adipose tissue hypoxia have been shown to independently affect adipocyte functions. The goals of this study were to (1) determine the effect of PCB-77, PCB-153, and DDE on the differentiation of human preadipocytes, and (2) investigate the cross-talk between PCB-77 and hypoxia in differentiated human adipocytes. First, human preadipocytes were exposed to PCB-77, PCB-153, or DDE during the entire 14-day differentiation period. We found no effect of low POP levels on lipid accumulation. Second, differentiated human adipocytes were exposed to a combination of PCB-77 and hypoxia. We demonstrated gene-specific cross-talk between PCB-77 and hypoxia, showing an additive effect of PCB-77 on VEGF, MCP-1, and adiponectin, as well as an inhibition of PCB-77-induced expression of CYP1A1 by hypoxia. This work has expanded our understanding of the role of POPs and hypoxia in differentiated human adipocytes.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

The diverse outcomes of ubiquitination primarily relate to the flexibility of ubiquitin in forming homo- or heterotypic chains on each of its seven lysine residues which in turn stimulate distinct downstream signaling pathways. These ubiquitin signals must be selectively initiated on the substrate protein and subsequently decoded to facilitate the desired cellular function. These initiation and decoding steps often involve additional post-translational modifications and ubiquitin receptor proteins, but the enzymes and ubiquitin chains involved for many ubiquitinated substrates are not clear. Here, I have explored the initiation and decoding of ubiquitin signals, focusing on lysine-11 (K11) linked polyubiquitin chains and their role in protein degradation. I established in vitro assays to understand how K11-chains are decoded and whether these chains act as a signal for proteasome-mediated degradation. Pure homotypic K11-chains did not bind the proteasome or its associated ubiquitin binding proteins, but did bind to the mitophagy ubiquitin receptors, MyosinVI and TAX1BP1. Heterotypic K11/K48 linkages not only bound the proteasome but also stimulated degradation of the cell cycle substrate, cyclin B1. To further explore the functions of K11-chains I focused on the hypoxia inducible transcription factor (HIF) pathway, as K11-ubiquitination had been implicated in proteasome-independent degradation of the transcription factor. I established an in vitro assay to initiate HIF ubiquitination, via prolyl hydroxylation, and determine the type of ubiquitin chains involved. Recombinant HIF isoforms were rapidly hydroxylated when incubated with cell extracts. Moreover, the levels of iron and small molecule metabolites within the lysates regulated HIF hydroxylation. However, this hydroxylation was insufficient to reproducibly promote HIF ubiquitination or determine the ubiquitin chains involved. While the nature of the polyubiquitin chains formed in the HIF pathway remain elusive, my studies identify distinct roles for homotypic and heterotypic K11-polyubiquitination in proteasome-mediated degradation.

Areas of hypoxia caused by poor perfusion are a common occurrence in breast cancer. Hypoxia-inducible factors-1 and 2 (HIF1/2) drive the cellular response to hypoxia in such areas, resulting in the upregulation of genes which facilitate the survival of cancer cells and promote growth, invasion, metastasis and angiogenesis, generally leading to more aggressive tumour characteristics. Previous research has demonstrated that growth factor signalling, such as the ligand-mediated activation of HER receptors, can promote the action of HIFs in normoxia, and correlation between HER2 expression and HIFα proteins has been demonstrated in clinical samples of breast cancer. Despite this, little research has been conducted on how the growth factor-driven regulation of HIFα subunits might modify the cellular response to hypoxia. In this thesis, the role of HER2 overexpression in HIFα modulation was assessed in breast cancer cell lines and publically available clinical datasets for breast cancer with the aim of further understanding the implications of hypoxia and HIFα expression in the context of HER2-positive breast cancer. The upregulation of HIF1α and HIF2α by hypoxia was observed across breast cancer cell lines, and the role of HER2 in this process was assessed using an isogenic MCF7 cell line model overexpressing HER2. This demonstrated an increased hypoxic upregulation of HIF2α but not HIF1α when HER2 was overexpressed. The increased upregulation was shown to be facilitated by an increase in normoxic HIF2α, which is driven by a higher transcriptional rate of the EPAS1 (HIF2) gene as a direct result of HER2 overexpression. HER2 overexpression also resulted in the increased hypoxic upregulation of known hypoxia response genes in 2D and 3D culture models. This demonstrates a novel mechanism for growth-factor mediated HIFα regulation in the context of HER2 overexpression, with an important role for HIF2α. Microarray analysis of MCF7 and MCF7-HER2 cells was used to compare the global transcriptional response to acute (24 hrs) and chronic (>10 weeks) hypoxia (0.5% O2) and demonstrated a broadly increased upregulation of hypoxic response genes in the HER2 overexpressing cell line when compared to wild-type MCF7. This included an increase in previously described HIF1 and HIF2 target genes. MCF7-HER2 also illustrated an increased expression of hypoxia response genes in normoxia, and an analysis of the genes involved showed the promotion of a number of pathological processes including proliferation, invasion, angiogenesis and epithelial to mesenchymal transition. Large-scale, publically available expression datasets for breast cancer cell lines and clinical patient data were used to investigate the expression of HIF2α and hypoxia response genes in relation to HER2 expression. A set of pathologically important genes which were primed for hypoxia in MCF7-HER2 were also demonstrated to correlate with HER2 across breast cancer cell lines, suggesting that HER2 may more broadly promote a readiness to respond to hypoxia in breast cancer cells. Assessment of HIF2α in clinical samples has shown its increased expression in the HER2-positive subtype, and HIF2α was shown to be associated with worse disease-specific survival in the context of HER2-positive samples only. To investigate whether HIF2α is a potential target in HER2 overexpressing breast cancer, the effect of HIF2α inhibition through siRNA or HIF2-specific chemical inhibitors was assessed in cell lines with high or low HER2 expression, and this demonstrated an increased sensitivity of HER2 overexpressing cell lines to HIF2α inhibition. This work highlighted HER2 as an important modulator of the cellular response to hypoxia in breast cancer, demonstrating a previously overlooked role for HIF2α in this process. HIF2α expression can be directly driven by HER2 and this differs mechanistically from that previously reported for HIF1α. Finally, further work into the potential for HIF2α as a target for anti-cancer therapy is suggested, as an increased sensitivity of HER2-positive cell lines to anti-HIF2α agents was shown, as well as a HER2-specific relationship between HIF2α expression and worse prognosis. More generally, this work has shown an important interplay between growth factor receptor expression and the cellular response to hypoxia, suggesting that HER2 may promote a stronger response to hypoxia in breast cancer, which may contribute to the increased aggressiveness of HER2-positive tumours.

This thesis is concerned with the role of iron in modulating right ventricular (RV) afterload during exercise in healthy people aged between 50 and 80 years. This is predicated on the requirement of the hypoxia-inducible factor (HIF) pathway for ferrous iron. A secondary objective is to examine the reactive oxygen species (ROS) hypothesis in human hypoxic pulmonary vasoconstriction (HPV) using exposure to hyperoxia. Chapters 3 and 4 describe basal relationships that may affect the HIF pathway and exercise capacity during ageing. These were explored in 113 participants using blood tests and exercise tests. Age and inflammatory factors, C-reactive protein, and ferritin were associated with impaired exercise capacity. In addition, ageing did not significantly affect haematological variables or iron status indicators. Chapters 5 and 6 describe the effect of a single intravenous iron infusion on the haematological variables in 32 participants in a randomised, placebo-controlled and double-blinded study. The effects of iron infusion on RV afterload during light exercise, and exercise capacity during heavy exercise, were examined in these participants. With iron infusion, erythropoietin production, and the increase in RV afterload during light exercise were blunted, potentially indicating involvement of the HIF pathway. However, blunting of RV afterload neither influenced the cardiac output during light exercise nor exercise capacity. Chapter 7 describes a study of 11 healthy volunteers, which investigated the ROS hypothesis in HPV using acute isocapnic hypoxia following an 8-hour exposure to hyperoxia. This sustained hyperoxic exposure did not influence the hypoxic behavior of the pulmonary vasculature. This thesis demonstrates the complex relationship between iron status and exercise capacity in older adults. It shows that the decrease in RV afterload during exercise caused by intravenous iron supplementation does not lead to an augmented cardiac output or exercise capacity. Finally, it calls into question the role of ROS in HPV.

Abstract Oxygen deprivation (hypoxia) is related to many disease conditions, such as anemia, but is also a critical regulatory signal during normal development. Cellular responses to hypoxia are largely mediated through alterations in gene regulation brought about by the transcription factor known as hypoxia inducible facor (HIF). One of the most extensively studied systemic consequences of hypoxia is the induction of red blood cell production, erythropoiesis, which occurs through a HIF-dependent increase in erythropoietin (EPO) gene expression. The amount of HIF in cells is regulated by three HIF prolyl 4-hydroxylases (HIF-P4Hs) while a fourth P4H possessing a transmembrane domain (P4H-TM) is able to act on HIF at least in vitro. The putative role of P4H-TM in regulating erythropoiesis is studied here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null and wild-type mice. By comparing the observed effects with those seen in FG-4497 treated hypomorphic Hif-p4h-2 and Hif-p4h-3 null mice, it is demonstrated for the first time that P4H-TM is involved in the regulation of Epo production in the mammalian kidney, but not in the liver. Long bones are formed via endochondral ossification, in which a cartilaginous template, the growth plate, is first laid down and then replaced with bone. The growth plate is rich in extracellular matrix (ECM) and contains a hypoxic central region in which HIF has been shown to regulate chondrocyte function. Importantly, growth plate chondrocytes are highly active in collagen synthesis. Collagen prolyl 4-hydroxylases (C-P4Hs I-III) provide collagen molecules with thermal stability and are thus necessary for the formation of a proper ECM. Through an in vitro approach it is demonstrated that hypoxia increases the amount and activity of C-P4H in primary mouse epiphyseal growth plate chondrocytes in a HIF-1-dependent manner. Lastly, it was set out to characterize mouse lines with complete inactivation of C-P4H-II with or without partial inactivation of C-P4H-I. A significant reduction in the total amount of C-P4H and its activity was found to result in mild chondrodysplasia and altered bone properties. The above mouse models provided new information on the specific in vivo roles of the C-P4H isoenzymes I and II. / Tiivistelmä Kudosten alentunut happipitoisuus (hypoksia) liittyy osana moniin elimistön patologisiin tiloihin, kuten anemiaan. Lisäksi se on tärkeä säätelytekijä normaalin yksilönkehityksen aikana. Jotta solut havaitsisivat hypoksian ja reagoidakseen siihen, on niille kehittynyt säätelyjärjestelmä, jossa hypoksiassa indusoituva transkriptiotekijä, HIF, on tärkeässä asemassa. Yksi merkittävin HIF:n indusoima systeeminen vaikutus elimistössä on punasolujen tuotannon, erytropoieesin, kiihtyminen. Sitä tapahtuu erytropoietiinia koodittavan geenin (EPO) lisääntyneen ilmentymisen kautta. HIF-tekijän määrää soluissa säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4Ht 1-3). Transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) tiedetään myös vaikuttavan HIF-tekijän määrään soluissa in vitro, mutta sen vaikutusta nisäkkään erytropoieesiin ei ole aiemmin tutkittu. Käyttämällä hyväksi kolmea eri transgeenista hiirilinjaa (P4h-tm-/-, Hif-P4h-2gt/gt, Hif-p4h-3-/-) ja HIF-P4H entsyymeitä inhiboivaa lääkeainetta, FG-4497, tässä työssä osoitettiin ensimmäistä kertaa, että P4H-TM osallistuu nisäkkään Epo-hormonin tuoton säätelyyn. Pitkät luut muodostuvat endokondraalisen luutumisen kautta. Siinä ensin muodostuu rustoinen malli, kasvulevy, joka vähitellen korvaantuu luukudoksella. Kasvulevyn sisin kerros on sen soluille, kondrosyyteille, hypoksinen kasvuympäristö. HIF:illä on todettu olevan tärkeä rooli kondrosyyttien toiminnan säätelijänä. Kasvulevyn soluvälitila sisältää runsaasti kollageeneja. Kollageenin prolyyli-4-hydroksylaasit (C-P4Ht I-III) ovat avainasemassa kollageenien biosynteesissä ja siten niiden toiminta on välttämätöntä kestävän soluvälitilan muodostumiselle. Käyttämällä in vitro menetelmiä, tässä työssä osoitettiin, että hiiren epifyseaalisten kasvulevyjen kondrosyyteissä hypoksia lisää C-P4H:n määrää ja aktiivisuutta HIF-tekijästä riippuvalla mekanismilla. Eri C-P4H-isoentsyymeiden toiminnasta ja merkityksestä in vivo tiedetään vain vähän. Tässä työssä karakterisoitiin hiirilinja, jossa C-P4H-II on täysin inaktiivinen, ja hiirilinja, jossa lisäksi C-P4H-I on osittain inaktiivinen. Merkittävästi alentuneen C-P4H:n aktiivisuuden todettiin aiheuttavan hiirimallissa lievän kondrodysplasian sekä heikentyneet luun ominaisuudet.