HIF-1 and Oxygen-Sensing Matters. Charyl Nicole Warren. February 18, Number of Text Pages: 9. Number of Figure Pages: 5

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1 HIF-1 and Oxygen-Sensing Matters Charyl Nicole Warren February 18, 2004 Number of Text Pages: 9 Number of Figure Pages: 5 Number of Bibliography Pages: 1

2 Introduction Studies of how certain populations adapt to high altitudes have been of continuing interest for over a century. The French physician Viault first sparked curiosity while visiting Peru in He extracted blood samples from himself at sea level in Lima and a few weeks later 15,000 feet higher in Morococha. Upon comparison, Viault noticed a substantial increase in the red blood cell count, which led him to theorize that indigenous Andean highlanders had increase in respiratory oxygen of the blood (West, 1981). Since Viault, numerous studies have been performed on Andeans and other high altitude peoples whose elevated hemoglobin concentration, large lung volume, blunted hypoxic ventilatory response, and other features seemed to represent an ideal adjustment to hypobaric hypoxia (Beall, 2003). Recently, new advances have paved the way for a more extensive understanding of these functional differences. Many genes involved in cellular differentiation are directly or indirectly regulated by hypoxia. Some include erythropoietin (EPO), transferrin, transferrin receptor, and vascular endothelial growth factor (VEGF). Most recently the discovery of hypoxia inducible factor 1 (HIF-1) has been a great step forward in our comprehension of hypoxia response. HIF-1 is a transcription factor which is a key regulator of oxygen homeostasis, and in addition to erythropoietin, HIF-1 responsive genes include examples with functions in cellular energy metabolism, iron metabolism, catecholamine metabolism, vasomotor control and angiogenesis, suggesting an important role in the coordination of oxygen supply and cellular metabolism (Ratcliffe 1998: 1153). The explanation of how hypoxiaresponsive mechanisms function together, particularly Epo and HIF-1, will constitute a 1

3 small portion of this research. The more broad intent is to show how the acknowledgement and/or manipulation of these mechanisms (HIF-1, Epo, and VEGF) can help understand diferent populations adaptations to high altitude. Finaly, there wil be a brief discussion of clinical relevance in understanding hypoxia adaptations. Regulation of Erythropoietin (Epo) First acknowledged in human adaptations to high altitudes was an elevated red blood cell count due to increased erythropoietin (Epo) synthesis. Erythropoietin is a glycoprotein hormone manufactured mainly by the kidneys that stimulates the production of RBCs by stem cells in bone marrow (Zhu 2002: 3). Zhu goes on to state that erythropoietin production is governed by a classic physiological feedback loop. It is made first in the kidney, in particular in response to hypoxic stress. After release into the circulation, it binds to Epo receptors and stimulates increased production of red blood cells. This increase in red blood cell mass, in turn, relieves the hypoxia, thereby decreasing erythropoietin production (Zhu 2002: 3). (See Figure 1). Thus, erythropoiesis is a constant cycle with new RBCs being generated both to compensate for routine cell turnover and to allow adaptive increases in hematocrit, such as the response to anemia, chronic renal disease, inflammation, or hypoxia. Activation of the Hypoxia-Inducible Transcription Factor 1 (HIF-1) HIF-1 is a heterodimer, a molecule consisting of two diferent subunits; an α- and a β-subunit in this case. HIF-1β is involved in other functions and is expresed constitutively and independently of surrounding oxygen tensions, whereas HIF-1α can not be detected unless the system is hypoxic (Zhu 2002). At the transcriptional level, 2

4 genes encoding Epo and vascular endothelial growth factor (VEGF) are both under the control of HIF-1. There is an HIF-1 binding site in the enhancer of the Epo gene and in the promoter of the VEGF gene (Imagawa 2001: ). Figure 2 shows exactly where HIF-1 fits into the hypoxia-reaction equation. First, there is a deficiency in the amount of oxygen reaching body tissues, as in the instance of an individual with hypobaric hypoxia. The next step appears to be the most questionable, as there is little concretely known about how oxygen is sensed or what mechanism is responsible for this. It is known, for example, that not only hypoxia but also transition metals (cobalt, nickel, and manganese) and iron chelators will induce HIF-1 activation. The activation by hypoxia can be suppressed by heme ligands such as carbon monoxide and nitric oxide; this may suggest that the oxygen sensor is a heme protein, although evidence to support the claim is circumstantial (Zhu 2002: 5). Next, the deoxygenated form of this protein activates a signal that may then be transduced to an unknown factor X that activates a pre-existing HIF-1α subunit of the transcription factor. The activated α- subunit then forms a heterodimer with a constitutive β-subunit. Finally, DNA binds to a consensus element, activating a series of genes that are crucial for the continued activity of the hypoxic cells (Keidar 1997). The activation of these genes produces the general responses to hypoxia such as anaerobic metabolism, angiogenesis, vasodilation, erythropoiesis, and increased breathing. Because variation in the gene encoding HIF-1α or in the genes under HIF-1 influence could wel alter an organism s physiological response to hypoxia, HIF-1 has recently been given the title of "global regulator of oxygen homeostasis" (Hochachka 2003: 517). 3

5 Three Adaptations to High Altitude Three main populations have been studied for their differing adaptations to high altitude: Andeans, Tibetans, and Ethiopians. First it was discovered that Tibetans did not acclimatize to high altitude like the clasic Andean model of adaptation. This realization changed scientific understanding of high altitude adaptations and led scientists to explore other populations. Recently this has resulted in the discovery of a third pattern of adaptation in Ethiopian peoples. The goal now is to identify what makes these populations different and why they have adapted to the same stressors in different ways. Andeans The Andean population was the first studied, and much has been learned about their adaptive capabilities. Arterial hypoxemia, insufficient oxygenation of the blood cells, is the primary response to high altitudes; until recently, it was a definitive marker of high altitude adaptation. However, new studies have shown that not all populations display this trait when exposed to high altitudes. Included in the Andean response to high altitude is also an increase in the synthesis of erythropoietin (Epo) that stimulates red blood cells. This stimulation, erythrocytosis, is unique in only the Andean peoples. Tibetans The Tibetan highlanders were the next group examined for their adaptive capacities. At first their response seemed to be the similar since they too experienced arterial hypoxemia. However, the mean hemoglobin concentration of Tibetans was not elevated above sea level values despite very low oxygen concentration (Beall, 2002). Therefore, they did not display erythrocytosis like Andeans. This was a difference that led scientists to study a third group, Ethiopian highlander. 4

6 Ethiopians Cynthia Beall, a physical anthropologist, has been a leader in understanding high altitude adaptations. Most recently, she studied villagers in the Semien Mountains of Ethiopia where she found significant distinctions from Andean and Tibetan highlanders. Ethiopians, she found, have both normal sea-level hemoglobin and normal sea-level oxygen saturations. Beall made the discovery from data collected in a field study of 313 native residents at 3,530 meters, roughly the same altitude as Andeans and Tibetans (2002). So, the Ethiopians are like Tibetans in that they exhibit little or no elevation of hemoglobin concentration; yet, they do not possess very low oxygen concentrations. This poses the question as to how exactly Ethiopians adapt to high altitudes and the great decrease in available oxygen. Beall states that in order to understand their adaptive mechanisms two lines of future investigation must be explored. First, there must be a greater understanding of the underlying biological mechanisms and genetics involved. The other path of inquiry must be into the evolutionary processes that constructed these patterns of adaptation in the first place (Beall, 2002). Clinical Relevance Chronic Mountain Sickness (CMS) Chronic mountain sickness or CMS occurs when indigenous people at high altitudes lose their adaptation and develop intolerance. In response to hypoxia, too many red blood cells are produced, usually consistent with cardiac and respiratory disease. CMS is also known as increased polycythemia or excessive erythrocytosis and is marked by the following conditions: blood vessel proliferation (growing at rapid speeds), polycythemia (increased RBCs), profound hypoxia and neurological symptoms (Appenzeller, 2003). Figure 3 displays two common conditions caused by CMS, cyanosis 5

7 of the lips and clubbing of the fingers. Appenzeller and colleagues did extensive research in Cerro de Pasco (CP), Peru to determine gene product levels of altitude dwellers and if they differed in patients with CMS to those living in the same location who showed no evidence on maladaptation. Because CMS is a disease associated with old age and perhaps related to altitude rather than sea level birth, Appenzeller also studied these two variables in their subjects (2003). The subjects were all men, as follows: 12 normal CP natives (CPC), 15 CMS patients from CP (CPCMS), 13 high altitude natives living now at sea level in Lima for more than 5 years (HANL), 5 Lima natives with no exposure to high altitudes, for sea level controls (SLNL). Blood was drawn from the subjects, total RNA was isolated, and gene expression was assessed by reverse transcription polymerase chain reaction. Results for HIF-1 and vascular endothelial factors (-121,-165, and -189) were found (Appenzeller 2003) HIF-1 HIF-1 appeared no different in the controls from CP (CPC) and sea level natives living in Lima (SLNL). Yet the greatest expression was found in subjects of Cerro de Pasco with chronic mountain sickness (CPCMS). All samples from high altitudes versus those as sea level had considerably higher HIF-1 expression. There was also an adjustment made for age. Thus, a higher chronic mountain sickness score (CMS-sc) was directly related to HIF-1 expression independent of age (Appenzeller 2003: 3-4). Appenzeller's study helped to better understand gene expression in response to altitude, but not in all attempts. The higher HIF-1 and VEGF-1 depict the significance of both proteins in adaptation to hypoxia, as well as maladaptation. Further, there were 6

8 small age-related increases in HIF-1 and VEGF-1. Adjusting for the older age of the CMS group resulted in there being no difference between the controls and the CMS patients of Cerro de Pasco. This supports the view that CMS is definitely related to accelerated aging, and perhaps the effects of altitude, while present, are still not completely understood. CMS then is related to differential gene expression, and it may be the result of mutations to the transcription factor or the control regions of the various genes, including the VEGF variants only found in CMS. Sleep Apnea-Hypopnea Syndrome (SAHS) A number of respiratory events defines the sleep apnea-hypopnea syndrome during sleep and excessive fatigue during the day. The respiratory events during sleep are simply a count of apnea episodes (brief cessations of breathing) and hypopnea episodes (abnormally slow breathing). In an effort to better understand responses to hypoxia, Imagawa and colleagues measured levels of hemoglobin (Hb), serum erythropoietin (Epo), and vascular endothelial growth factor (VEGF) in 106 patients with severe SAHS (2001). These were compared with levels in 45 control individuals, and persons with anemia, renal or liver disease, and coronary heart disease were excluded (Imagawa 2001: 1256). The patients were then separated into 5 groups according to apnea-hypopnea index (AHI), with the controls having AHIs of less than 5. The results depicted in Table 1 show striking increases for Hb, Serum VEGF, and Epo levels. While Hb increases from to , VEGF levels showed increases 5 times higher than the controls, and Epo levels were 1.6 times higher (Imagawa 2001:1256) The increase is quite substantial from those individuals with an AHI less than 5 to 7

9 those with an AHI greater than 110. The levels increase respectively as AHI goes up, until reaching AHI 90 and above. Furthermore, there were no notable relationships between Epo and Hb, between VEGF and HB, or between Epo and VEGF (Imagawa 2003: 1257). The only substantial increase in SAHS patients was vascular endothelial growth factor. To account for this increase, the author states that increased VEGF levels were also reported immediately after marathon runs at high altitudes among his SAHS patients. The VEGF levels continued to increase, however, the longer the runners were exposed to high altitudes. They concluded that the increase in VEGF levels was due to both hypoxia and exercise (Imagawa 2003). However, VEGF levels were increased greatly in Imagawa's SAHS patients and they were not running marathons. Furthermore, the marathon runners reported were those at high altitudes only. It cannot be concluded then that exercise is necessary for VEGF levels to increase. It can be concluded that hypoxia from patients with sleep apnea-hypopnea syndrome causes VEGF elevation and that this operates through HIF-1 expression in SAHS. Tumor Angiogenesis Angiogenesis is the formation of new blood vessels in the body. Tumor angiogenesis occurs when these blood vessels create the blood supply for a tumor, allowing it to grow. Since hypoxia most often occurs in the complex setting of ischaemia (insufficient blood supply to a body part), it is relevant to assess HIF-1 to see if other responsive genes might exist (Ratcliffe 1998). Ideal studies would be able to assess the HIF-1 activation that can be applied to heterogenous tissues for whole animals (Ratcliffe 1998:1155). In the meantime, Ratcliffe and colleagues sought to address the issue through 8

10 analysis of tumor xenografts from tissue culture cells in nude mice (1998) To begin, wild-type (WT) hepa-1 cells were chosen from one group that were deficient in HIFβ/ARNT and thus not able to form the HIF-1 complex. These cells (c4) that displayed defective oxygen-regulated gene expression were compared to two other cell types, a revertant which had regained wild-type levels of HIF-1β/ARNT (Rc4) and a line that was functional for HIF-1(c31). Compared were gene expression, vascularization, and growth in tumors from wild-type cells, and Figure 4 depicts these results. In WT tumors, the areas with the most intense expression of the glucose transporter Glut-3 and VEGF mrna were often close to necrotic regions (Ratcliffe 1998: 1156). It was not, however, intense in the c4 cells, only in the Rc4 and c31 cells--those that make HIF-1 normally (Ratcliffe 1998). Similar correlations occurred with vascularity and growth, as HIF deficient cells displayed an obvious reduction in vascularity (Ratcliffe 1998). This experiment suggests that the activation of HIF-1 is largely responsible for this pattern of gene expression within the tumor (Ratcliffe 1998:1156). HIF-1 is activated within solid tumors, and the pathophysiological role of such knowledge is evident and warrants further research. Conclusions Understanding the adaptive mechanisms of high altitude populations is important not only to become aware of those differences but also to employ that knowledge for future endeavors. Since Viault, there have been many steps forward in our comprehension of adapting to high altitude and hypoxia. The recent discovery of hypoxia-inducible factor-1 (HIF-1) was only possible through the understanding of other 9

11 oxygen regulating mechanisms like erythropoietin and vascular endothelial growth factor. These are some of the key players in being able to survive at mountainous altitudes, and analyzing them is crucial for further comprehension. Specifically, there is more to learn about HIF-1 and how it controls the expression of other genes. Further, the oxygensensing mechanism warrants more extensive research as well. While it may very well be a heme protein, there is still no conclusive evidence to fully support this. Determining how different populations adapt differently is essential to understand high altitude adaptations. If the responses are dissimilar then it is necessary to learn more about the underlying biological mechanisms at play. Finally, if there are no obvious distinctions biologically between these populations, then the other obvious path, as Beall suggests, is to investigate the evolutionary history of the populations. To focus altitude adaptive studies in beneficial ways is the ultimate goal, and more clinical applications appear every day. Oxygen regulation and HIF-1 knowledge can be applied in better understanding anemia, tissue ischaemia, chronic mountain sickness, and sleep apnea-hypopnea, just to name a few. Pursuit of the mechanisms that underlie these disorders and others will hopefully lead to a more thorough comprehension, as well as more therapeutic directions. 10

12 References Cited Appenzeller Otto, Minko Tamara, Pozharov Vitaly, Bonfichi Maurizio, Malcovati Luca, Gamboa Jorge, and Luciano Bernardi Gene expression in the Andes; relevance to neurology at sea level. Journal of the Neurological Sciences, 2: Beall Cynthia, Decker Michael, Brittenham Gary, Kushner Irving, Gebremedhin Amha, and Kingman Strohl An Ethiopian pattern of human adaptation to highaltitude hypoxia. PNAS, 26: Hochachka Peter, and Jim Rupert Fine tuning the HIF-1'global' O 2 sensor for hypobaric hypoxia in Andean high-altitude natives. BioEssays, 25: Imagawa S, Yamaguchi Y, Higuchi M, Neichi T, Hasegawa Y, Mukai H, Suzuki N, Yamamoto M, and Toshiro Nagasawa Levels of vascular endothelial growth factor are elevated in patients with obstructive sleep apnea-hypopnea syndrome. Blood, 4:

13 Keidar Danit, Efrati Edna, Elaad Shlomit, Shamovsky Ilya, Weinreb Orly, Lavie Lena, and David Gershon Molecular biology of ageing: age-associated attenuation in the regulation of the expression of stress response genes Haifa, Israel. (February 02, 2004); c0021fig2.gif&imgrefurl= 1.htm&h=357&w=477&sz=5&tbnid=FuLVU7E14QMJ:&tbnh=94&tbnw=125&pr ev=/images%3fq%3dhif- 1%2Bhypoxia%26svnum%3D20%26hl%3Den%26lr%3D%26ie%3DUTF- 8%26oe% 3Dutf-8%26safe%3Doff%26sa%3DN. Ratcliffe Peter, O'Rourke John, Maxwell Patrick, and Christopher Pugh Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. The Journal Of Experimental Biology, 201: West John B Adaptation to extreme altitudes. Environmental Physiology: Aging, Heat and Altitude. Amsterdam: North Holland Biomedical Press. Zhu Hao, Jackson Tim, and H. Franklin Bunn Detecting and responding to hypoxia. Nephrol Dial Transplant, 17:3-7. Figure 1 12

14 back 13

15 Figure 2 back (Keidar 1997) 14

16 Figure 3 back (Drugge 1996) 15

17 Table 1 Table 1. Levels of Hb, serum Epo, and VEGF in patients with severe OSAHS and controls AHI Hb (g/dl) Epo (mu/ml) VEGF (pg/ml) n <5 (Controls) 14.5 ± ± ± ± 1.1 * 17 ± ± 202 * ± 1.2 * 13 ± ± 415 * ± 1.2 * 16 ± ± 517 * ± ± ± 250 * 6 > ± ± ± 182 * 4 Numbers represent mean ± 1 SD. AHI indicates apnea-hypopnea index; Hb, hemoglobin; Epo, erythropoietin; VEGF, vascular endothelial growth factor; OSAHS, obstructive sleep apnea-hypopnea syndrome. * P <.005 compared to AHI < 5. P <.025 compared to AHI < 5. Back (Imagawa, S. 2001) 16

18 Figure 4 Gene expression and vascularity in derivative c4. Immunoperoxidase labelling tumour grafts from wild-type (WT) hepa-1 for vascular endothelium using a and HIF-1 /ARNT-deficient c4 cells (C4). monoclonal antibody to CD31/PECAM. (A) In situ hybridisation for vascular More capillaries are present in the wild-type endothelial growth factor (VEGF) and tumour (left-hand panel) than in the c4- glucose transporter (Glut-3) mrna in wildtype derived tumour (right-hand panel). Hepa-1 (left) and c4 (right) tumours. Back (Ratcliffe 1998) 17