Max Gassmann

HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia

Adaptation to hypoxia is regulated by hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐regulated α subunit and a constitutively expressed β subunit. Although HIF‐1 is regulated mainly by oxygen tension through the oxygen‐dependent degradation of its α subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF‐1 activation in vivo, we determined the spatial and temporal distribution of HIF‐1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohisto‐chemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF‐1α is present in mice kept under normoxic conditions and is further in‐creased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF‐1 α expression varies among different organs. In liver and kidney, HIF‐1 α reaches maximal levels after1hand gradually decreases to baseline levels after4hof continuous hypoxia. In the brain, however, HIF‐1α is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF‐1 β is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF‐1 α. Overall, HIF‐1 α expression in normoxic mice suggests that HIF‐1 has an important role in tissue homeostasis.—Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A. H., Bauer, C., Gassmann, M., Candinas, D. HIF‐1 is expressed in normoxic tissue and displays an organ specific regulation under systemic hypoxia. FASEB J. 15, 2445–2453 (2001)

Erythropoietin Gene Expression in Human, Monkey and Murine Brain

The haematopoietic growth factor erythropoietin is the primary regulator of mammalian erythropoiesis and is produced by the kidney and the liver in an oxygen‐dependent manner. We and others have recently demonstrated erythropoietin gene expression in the rodent brain. In this work, we show that cerebral erythropoietin gene expression is not restricted to rodents but occurs also in the primate brain. Erythropoietin mRNA was detected in biopsies from the human hippocampus, amygdala and temporal cortex and in various brain areas of the monkey Macaca mulatta. Exposure to a low level of oxygen led to elevated erythropoietin mRNA levels in the monkey brain, as did anaemia in the mouse brain. In addition, erythropoietin receptor mRNA was detected in all brain biopsies tested from man, monkey and mouse. Analysis of primary cerebral cells isolated from newborn mice revealed that astrocytes, but not microglia cells, expressed erythropoietin. When incubated at 1% oxygen, astrocytes showed >l OO‐fold time‐dependent erythropoietin mRNA accumulation, as measured with the quantitative reverse transcription‐polymerase chain reaction. The specificity of hypoxic gene induction in these cells was confirmed by quantitative Northern blot analysis showing hypoxic up‐regulation of mRNA encoding the vascular endothelial growth factor, but not of other genes. These findings demonstrate that erythropoietin and its receptor are expressed in the brain of primates as they are in rodents, and that, at least in mice, primary astrocytes are a source of cerebral erythropoietin expression which can be up‐regulated by reduced oxygenation.

HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1α (HIF-1α) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood–retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

Induction of HIF-1α in response to hypoxia is instantaneous

Despite the pivotal role the hypoxia‐inducible factor‐1α (HIF‐1α) plays in physiological and pathological processes, little is known regarding the timeframe and mechanisms involved in its regulation. We determined the onset, accumulation, and degradation of HIF‐1α and a number of redox‐sensitive nuclear factors over a range of pathophysiological oxygen concentrations. Experiments were carried out on nonadherent human HeLaS3 cells placed in tonometers to achieve rapid equilibration between the cell suspension and the various hypoxic/reoxygenation conditions. Exposure to hypoxia for less than 2 min already revealed nuclear HIF‐1α protein induction on Western blots and HIF‐1 DNA binding in EMSAs. One hour after anoxic/hypoxic exposure, nuclear HIF‐1α proteins reached maximal levels, which were maintained for 4 h. Reoxygenation reduced HIF‐1 DNA binding within 2 min, and nuclear HIF‐1α protein levels within 4 to 8 min, down to a level below the detection limit within 32 min. Western blot analysis of the redox sensitive nuclear factors NF‐κB, c‐Fos, c‐Jun, Ref‐1, and thioredoxin showed no alteration in their nuclear levels in response to anoxia/hypoxia, but reoxygenation rapidly caused a transient increase in nuclear NF‐κB and thioredoxin protein levels. The instant initiation of HIF‐1α accumulation shown here limits the hypoxic signaling pathway to below 2 min.

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1β resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1β mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1β-deficient subline Hepa1C4, as well as antibodies raised against HIF-1α and ARNT/HIF-1β confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5′ HBS was more efficient in binding HIF-1 than the 3′ HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.

Efficient production of chicken egg yolk antibodies against a conserved mammalian protein.

The egg yolk of immunized chicken is a rich and inexpensive source of specific polyclonal antibodies. In this paper we show that 20‐30 μg of a highly conserved mammalian protein, as exemplified by proliferating cell nuclear antigen, are sufficient to induce an immune response. Immunoblot analysis revealed that specific antibodies appeared 20 days after immunization, reached a plateau after 30 days, and remained high until at least day 81. A total amount of 4 g immunoglobulin was extracted from 62 eggs of one immunized hen, yielding approximately 130 mg of specific antibodies.— Gassmann, M.; Thömmes, P.; Weiser, T.; Hübscher, U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 4: 2528‐2532; 1990.

Embryoid bodies: an in vitro model of mouse embryogenesis.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and ectoderm) of the organism, including the germline. To study early stages of development, as well as to investigate the impact of a gene knock‐out in vitro, ES cells are differentiated into three‐dimensional structures called embryoid bodies, because of their ability to mimick post‐implantation embryonic tissues. This review summarises the work on ES cell differentiation into haematopoietic and vascular cells, neuronal and glial cells, myocytes, and adipocytes, using this in vitro model of early embryogenesis. We also present the potential of this method to analyse the impact of genetic alterations in vitro

Quantifying Western blots: Pitfalls of densitometry

Although Western blots are frequently quantified, densitometry is not documented and appears to be based merely on traditions and guesswork. Confirming previous experience, none of 100 randomly selected and systematically scanned most recent papers provided sufficient information on how Western blot results were translated into statistical values. The importance of such information, however, becomes evident from our correlations of plasma erythropoietin values of various mammals determined using RIA and Western blot densitometry. Different common densitometry procedures applied to the identical Western blot revealed p‐values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. At present, the current lack of any definitions in densitometry opens the door to uncontrollable acquisition of any desired p‐value. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots.

Uncontrolled Expression of Vascular Endothelial Growth Factor and Its Receptors Leads to Insufficient Skin Angiogenesis in Patients With Systemic Sclerosis

Systemic sclerosis (SSc) skin lesions are characterized by disturbed vessel morphology with enlarged capillaries and an overall reduction in capillary density, suggesting a deregulated, insufficient angiogenic response. It has been postulated that this phenomenon is due to reduced expression of the potent angiogenic factor vascular endothelial growth factor (VEGF). In contrast to this hypothesis, we demonstrate that the expression of both VEGF and its receptors VEGFR-1 and VEGFR-2 is dramatically upregulated in skin specimens of SSc patients throughout different disease stages. Interestingly, upregulation of VEGF was not mediated by hypoxia-inducible transcription factor-1 (HIF-1) as indicated by only a weak expression of the oxygen-sensitive &agr;-subunit of HIF-1 in the skin of SSc patients. This was unexpected on measuring low PO2 values in the SSc skin by using a polarographic oxygen microelectrode system. Considering our observation that PDGF and IL-1β costimulated VEGF expression, we propose that chronic and uncontrolled VEGF upregulation that is mediated by an orchestrated expression of cytokines rather than VEGF downregulation is the cause of the disturbed vessel morphology in the skin of SSc patients. Consequently, for therapeutic approaches aiming to improve tissue perfusion in these patients, a controlled expression and timely termination of VEGF signaling appears to be crucial for success of proangiogenic therapies.