CHAPTER 7 The endothelial genomic response to chronic hypoxia Robert E. Verloop1 Anton J. G. Horrevoets2 Marten A. Engelse1 Oscar L. Volger3 Sophie Nadaud4 Pieter Koolwijk1 Victor W.M. van Hinsbergh1 1Laboratory for Physiology and 2Department of Molecular Cell Biology and Immunolo- gy, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherland 3Department of Biochemistry, Academic Medical Center, Amsterdam, the Netherlands 4UPMC University Paris 06 and INSERM, UMR_S 525, F-75005, Pa- ris, France Abstract: Exposure of endothelial cells to hypoxia shifts their gene expression pattern to me- tabolically adjust to the change in oxygen tension. Little is known of the effects of chronic hypoxia on these cells as experienced during many pathological conditions, because only short-term hypoxia (up to 24 hours) has been investigated extensi- vely. Therefore, we studied human umbilical vein and microvascular endothelial cells under culture conditions that allowed continuous culture, inspection and medium renewal under defined oxygen tensions. Endothelial cells remained healthy and maintained their proliferation properties at 1% and 5% oxygen atmosphere com- parably or better than at 21% oxygen for up to 2 weeks. Microarray analysis of gene expressions revealed a significant regulation of 435 defined genes, grouped into 8 panels after these culture conditions. Pathway analysis showed hypoxia/HIF- dependent genes specifically regulated at 1% but not 5% oxygen, while the glyco- lysis pathway was well represented in the genes that displayed a gradual increase at lowering (5% and 1%) oxygen concentration. Surprisingly, comparison with five previously published microarray studies on short-term (24 hours) hypoxia-exposed endothelial cells showed substantial overlap in identity of the genes. Next, a panel of 55 genes selected from our results and published studies was studied in detail by Chapter 7 real-time (RT)-PCR in six additional cultures that were exposed for either 24 hours or 2 weeks to 1% oxygen. Most genes showed similar regulation by both acute and 196 chronic hypoxia, but tended to increase to higher levels after prolonged hypoxia. In- deed, only 8 out 55 genes, including PDH3, showed a significant difference between 24 hours and 2 weeks exposure. Still, the expression of HIF-1a and HIF-2a mRNA were suppressed after 2 weeks of hypoxia, but their cellular protein levels remained induced, whereas the potentially inhibitory HIF-3a/HIF-3a4 also markedly increased, suggesting additional complexity of HIF-1 regulation. One of the candidates for such additional regulation was identified as KLF2, as we observed a decrease of KLF2 mRNA and its direct targets like thrombomodulin and eNOS. In contrast, proinflam- matory and TGF-β responsive genes normally suppressed by KLF2 were markedly enhanced. These results identify HIF-1 as the most important transcription factor for chronic hypoxia, despite the complex adjustments in its regulatory circuit, and identify a novel interaction between KLF2 and HIF activity and their regulated genes. Endothelial Gene Expression During Chronic Hypoxia 197 Introduction Oxygen is required for proper functioning of tissues. It is delivered by the blood and replenished via the circulatory system that is lined by endothelial cells. When hypox- ia occurs, tissue cells switch to a change in metabolic activity and an altered gene expression pattern, which is largely determined by hypoxia inducible transcription factors (HIF) as well as by additional mechanisms1, 2. This results in reduced energy expenditure, enhanced glycolysis, and the production of factors, such as VEGF and erythropoietin, that help to restore vascularization and oxygen transfer. Being part of the circulation, the vascular endothelium is normally well oxygenated, and can respond to angiogenic factors produced by hypoxic tissues3, 4. However, after occlu- sion of a proximal vessel, by high local oxygen consumption e.g. in inflamed tissues or tumors, and in transplanted tissue, endothelial cells become part of a hypoxic environment. In such cases the endothelium has to maintain its survival and to alter its properties towards a phenotype that restores the circulation and blood supply, i.e. inducing vasodilation and participating in angiogenesis5, 6. This requires a change in gene expression, which may be dependent on the duration and severity of hypoxia. 7 Hypoxia is defined as insufficient delivery of O2 to meet the demands of the tissue . While normal tissues, such as resting skeletal muscles and heart, are exposed to a 4 - 6.5% O2 tension, the oxygen concentration of hypoxic regions is usually reduced 8-14 to 0.5-2% O2 , but can incidentally decrease to anoxic values . The lack of oxygen causes the immediate availability of HIF and induces several HIF-dependent and independent pathways that contribute to hypoxia tolerance 2, 15, 16. HIF are heterodi- meric transcription factors composed of a stable, constitutively expressed, HIF-1β (ARNT) subunit and a HIF-α protein, either HIF-1α or HIF-2α, which are negatively regulated by oxygen17, 18. HIF-1α is ubiquitously expressed, while HIF-2α, also called EPAS1, is encountered only in a limited number of cell types, including endothelial cells19-22. The HIF-α subunits are continuously synthesized. However, when oxygen is present, they are degraded after hydroxylation of specific proline residues by oxy- gen-sensitive prolyl hydroxylases (PHD), after which they bind to the von Hippel- Lindau protein and become degraded in the proteasome15, 23. An additional aspara- gine hydroxylation by factor-inhibiting hypoxia (FIH-1) prevents the formation of an active HIF-containing transcription complex. Conversely, in the absence of sufficient oxygen the HIF-a-subunits escape from hydroxylation and degradation and become able to bind as active HIF complexes to many hypoxia-responsive elements (HRE), thereby inducing a large number of different genes. Because of the central role of endothelial cells (EC) in angiogenesis, a number of groups have studied the genomic responses of EC to hypoxic exposure5, 24, 25. Mana- lo et al.5 selected genes that were both induced/reduced by hypoxia as well by HIF- 1a overexpression. On the other hand, Takeda et al. 26 selected hypoxia-responsive genes by evaluating overexpression of HIF-2a in EC. Other studies utilized chemical agents that induced a hypoxia-like response24, 25, 27. Each of the studies came with a different set of genes that only partially overlapped with one another. In addition a number of studies identified individual genes that were up- or downregulated in hypoxic or anoxic conditions, usually studied within a 24-hour period of time. Mostly due to technical limitations, to date studies have largely ignored the influ- ence of prolonged exposure to hypoxia on hypoxia-regulated gene expression. This is rather contradictory since disease conditions, like cancer, heart ischemia and chronic infection, are usually accompanied by prolonged hypoxia. Chaplin et al.28, was among the first who described the concepts of acute (short-term) and chronic (prolonged) hypoxia with relation to the radio-sensitivity of specific areas of human tumors exposed for various durations of hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, a limited number Chapter 7 of papers have demonstrated adverse effects of a fully active HIF system during prolonged hypoxia29, 30. Ginouvès et al.31 made a detailed analysis of the HIF system 198 and demonstrated that chronic hypoxia induced HIF-1α and -2α desensitization in vitro and in vivo. This was accomplished by increasing the abundance and activity of the PHD. These feedback mechanisms31, 32 aimed to protect cells against necrotic cell death and thus to adapt them to chronic hypoxia. In addition it also prepares the system for a rapid response to an acute more severe hypoxic stimulus33. This may underlie the recent observation that much higher concentrations of HIF-1a, VEGF and SDF-1 were present in tissue sections of human ischemic legs after acute-on- chronic hypoxia than in chronically hypoxic legs34. Because of the lack of data on chronic hypoxic exposure we made a detailed ge- nomic analysis of human endothelial cells subjected to short and prolonged periods of hypoxia (1% O2), normoxia (5% O2), and hyperoxia (21% O2), which is the stan- dard culture condition. We focused on the expression of angiogenesis-regulating genes to determine whether endothelial cells are capable of initiating this essential survival pathway during chronic hypoxia. Endothelial Gene Expression During Chronic Hypoxia 199 Materials and Methods Materials Medium 199 (M199), penicillin/streptomycin, Hank’s balanced salt solution (HBSS) and trypsin were purchased from Lonza (Basel, Switzerland). Tissue culture plastics were from Corning Life Sciences (Lowell, MA, USA) and Greiner Bio-One (Krem- smünster, Austria). Heparin was from Leo Pharmaceutical Products (Weesp, The Netherlands). A crude extract of endothelial cell growth factor (ECGF) was isolated from bovine brain as described by Maciag et al.35. Newborn calf serum (NBCS) was obtained from Invitrogen (Carlsbad, CA, USA). For HUVEC culture, human serum was purchased from the local blood bank (Sanquin Blood Supply Foundation, Am- sterdam, The Netherlands) and was prepared from at least 20 healthy donors (after informed consent). hMVEC were cultured in human serum from PAA Laboratories (Pasching, Austria); both sera were heat-inactivated