Age-dependent Defect in Vascular Endothelial Growth Factor Expression Is Associated with Reduced Hypoxia-inducible Factor 1 Activity

Abstract

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6–8 months) or old rabbits (ages 4–5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in oldversus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1α-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1α protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia. Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6–8 months) or old rabbits (ages 4–5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in oldversus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1α-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1α protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia. vascular endothelial growth factor hypoxia-inducible factor 1 erythropoietin vascular smooth muscle cell enzyme-linked immunosorbent assay electromobility shift assay base pair(s) One of the characteristics of aging is the decline in the ability of the organism to respond to different types of stress. For instance, we have recently demonstrated that advanced age is associated with a defect in compensatory neovascularization in response to tissue ischemia (1Rivard A. Fabre J.E. Silver M. Chen D. Murohara T. Kearney M. Magner M. Asahara T. Isner J.M. Circulation. 1999; 99: 111-120Crossref PubMed Scopus (647) Google Scholar). Such impaired angiogenesis in ischemic tissues of old animals was found to be associated with reduced expression of vascular endothelial growth factor (VEGF),1 an endothelial-specific growth factor that is essential for embryonic (2Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3475) Google Scholar,3Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K. Powell-Braxton L. Hilan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3061) Google Scholar) and postnatal (4Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar) neovascularization. The molecular alterations responsible for this age-dependent decline in VEGF expression, however, have not been elucidated. Among those factors that have been implicated in the regulation of VEGF expression, hypoxia appears to play a major role, both in vitro (5Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4176) Google Scholar) and in vivo (6Banai S. Shweiki D. Pinson A. Chandra M. Lazarovici G. Keshet E. Cardiovasc. Res. 1994; 28: 1176-1179Crossref PubMed Scopus (412) Google Scholar). The transcriptional and post-transcriptional mechanisms involved in the hypoxic regulation of VEGF are similar to those factors responsible for erythropoietin (Epo) expression. Transcriptional regulation of VEGF expression at the 5′-promoter of VEGF is conferred by a sequence that is homologous to the hypoxia-inducible factor 1 (HIF-1) binding site within the Epo gene (7Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar). HIF-1 is a heterodimeric (HIF-1α and HIF-1β) basic helix-loop-helix protein that activates the transcription of the Epo gene in hypoxic cells (8Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5103) Google Scholar). When reporter genes containing VEGF sequences are co-transfected with expression vectors that encode HIF-1α and HIF-1β, the reporter gene transcription is much higher in both hypoxic and normoxic cells than in cells transfected with the reporter gene alone (9Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3232) Google Scholar). Moreover, the results of gene-targeting experiments have shown that HIF-1α deficiency is associated with a complete loss of VEGF gene induction under hypoxic conditions, marked reduction in vascularization of embryos, and early lethality by embryonic day 10.5 (10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2068) Google Scholar). In this study, we used an in vitro system to investigate potential transcriptional and post-transcriptional mechanisms involved in the age-dependent defect of VEGF induction by hypoxia. Our results indicate that the reduction in VEGF expression with aging is primarily attributed to a lower transcriptional activity under hypoxic conditions. Thus, this lower transcriptional activity is related to an age-dependent defect in HIF-1 activity. Cultures of primary vascular smooth muscle cells (VSMCs) were isolated from the aortas of young New Zealand White rabbits (ages 6–8 months) or old New Zealand White rabbits (ages 4–5 years) as described by Mader (11Mader S.L. J. Gerontol. Biol. Sci. 1992; 47: B32-B36Crossref Scopus (26) Google Scholar). The maximum age for New Zealand White rabbits was previously reported to be 7 years (12Weisbroth S. Weisbroth S. Flatt H. Kraus A. Neoplastic Diseases. Academic Press, New York1974: 331Google Scholar). Cells were incubated in Dulbecco's modified of Eagle's medium (Sigma), supplemented with 10% fetal bovine serum, 2 mml-glutamine, 200 units/ml penicillin, and 0.25 mg/ml streptomycin. Cells were cultured at 37 °C in a humidified atmosphere of normoxic conditions (5% CO2, 21% O2, and 74% N2), or placed in a modular incubator chamber (Billups-Rothenberg, New York), flushed with hypoxic gas (95% N2 and 5% CO2), and incubated at 37 °C in a humidified atmosphere. All experiments were performed with early passage (P1-P3) rabbit VSMCs at 70% confluence. VSMCs from young and old rabbits were incubated under normoxic or hypoxic conditions for a 24-h period. The supernatant was collected and VEGF levels were determined by using an immunoassay according to the manufacturer's instructions (Quantikine, R&D Systems). Results were compared with a standard curve of human VEGF with a lower detection limit of 5 pg/ml. Samples were checked by serial dilution and were performed at least in duplicate. VEGF levels were normalized relative to the protein concentration of VSMC cellular extracts (Bio-Rad protein assay). VSMCs (P1) from young and old rabbits were incubated under normoxic or hypoxic conditions for a 24-h period, washed with phosphate-buffered saline, and lysed in lysis buffer. Total protein extracts were quantified with the Bio-Rad protein assay. The protein extract (100 μg per sample) was separated on a 4–12% polyacrylamide gel (Bio-Rad) and electroblotted on a nitrocellulose membrane. The membrane was blocked with 10% nonfat dry milk in 0.2% Tween phosphate-buffered saline and then probed with VEGF rabbit polyclonal antibody (1:200 dilution, Santa Cruz Biotechnology, Inc.), an HIF-1α mouse monoclonal antibody (1:400 dilution, Novus Biologicals), or an actin goat polyclonal antibody (1:200 dilution, Santa Cruz) for 2 h at room temperature. The membrane was washed three times in Tween phosphate-buffered saline and then incubated with anti-rabbit (1:7000), anti-mouse (1:5000), or anti-goat (1:2000) horseradish peroxidase IgG for 1 h. Antigen-antibody complexes were visualized after incubation for 1 min with enhanced luminescence reagent (Amersham Pharmacia Biotech) at room temperature, followed by exposure to hyperfilms (Amersham Pharmacia Biotech). Each experiment was repeated at least twice with different cellular extracts. Representative results are shown below. Total cellular RNA was isolated from VSMCs by acid guanidinium phenol/chloroform extraction (13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63231) Google Scholar). The total denatured RNA (15 μg) was subjected to electrophoresis in a 1.0% agarose/formaldehyde gel and then transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech). A 675-bpEcoRI/BglII fragment of pSVI.VEGF.21 (14Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (537) Google Scholar), or a 345-bp fragment of human HIF-1α cDNA (pBluescript/HIF-1α 3.2–3T7) (8Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5103) Google Scholar) were used as specific probes. All probes were radio-labeled by random-primed labeling (Rediprime kit, Amersham Pharmacia Biotech). Filters were hybridized overnight with 106 cpm/ml of the probe and then washed in 0.5 × SSC,0.1% SDS for 10 min at room temperature, twice for 30 min at 65 °C, and subsequently exposed to Kodak XAR-5 films. For VEGF mRNA stability studies, VSMCs were exposed to hypoxic conditions for 24 h before the addition of 5 μg/ml actinomycin D to block gene transcription. RNA isolation was performed at different time-points (0, 1, 2, and 3 h). Subsequently, VEGF mRNA was analyzed by Northern blot and by densitometric analysis of the autoradiograms that were exposed in the linear range of film density using a Hewlett-Packard scanner with National Institutes of Health Image Software. Least squares regression analysis of the resulting lines was performed, and half-life values were determined from the regression curves. Each experiment was repeated at least twice with different cell extracts. Representative results are shown below. For transfection experiments, we used a reporter construct containing the luciferase gene under the transcriptional control of the VEGF promoter (a 2.3 kilobase pair KpnI/NheI fragment of VEGF 5′-promoter region). From this initial full-length reporter gene, a deletion construct lacking the HIF-1 binding site was generated by restriction endonuclease digestion, PstI, as described previously (9,15). VSMCs were transiently transfected with 10 μg of reporter construct and with 30 μg of LipofectAMINE reagent (Life Technologies, Inc.). After transfection, VSMCs were allowed to recover for 3 h in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum before exposure to normoxic or hypoxic conditions. To correct for differences in transfection efficiency, luciferase activity was normalized relative to the level of alkaline phosphatase activity that was produced from co-transfected pSVAPAP plasmid (0.5 μg), which contained the reporter gene under the control of the simian virus 40 enhancer-promoter. The cells were incubated with the transfection mixture for 3 h and then exposed to normoxic or hypoxic conditions. After 24 h, luciferase and alkaline phosphatase activities were measured in the VSMCs in old and young rabbits. Results are expressed as the ratio of luciferase to alkaline phosphatase activities. Cells were plated on four-well chamber slides before being exposed to either normoxic or hypoxic culture conditions for 6 h. Cells were fixed in 10% neutral buffered formalin for 10 min before staining. They were then incubated in 3% hydrogen peroxide to block endogenous peroxidase and 10% horse serum as a protein block before overnight incubation with the monoclonal HIF-1α antibody (1:5000 dilution). Signal amplification and detection were then carried out using the peroxidase-Catalyzed Signal Amplification System (Dako, Carpinteria, CA) according to the manufacturer's instructions (16Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google Scholar). Finally, the reaction product was visualized by incubation with 0.05% (w/v) 3,3′-diaminobenzidine tetrahydrochloride dihydrate, and a counterstain of 10% Gill was before was performed in a containing 10 1 0.5 0.5 and mg/ml After 10 min of in μg of cell extracts were incubated for 30 min with 1 × of probe the VEGF HIF-1 binding site HIF-1 site (9Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3232) Google Scholar). was to the mixture for were performed with a of μg of cell separated at °C in in 0.5 × buffer. Each was performed in duplicate. The gel was and exposed to Kodak XAR-5 films. To the of aging on VEGF expression in VSMCs that were isolated from young or old rabbits were exposed to normoxic or hypoxic conditions for 24 h, and the VEGF expression was at mRNA and protein shown in 1 VEGF mRNA levels were and similar young and old VSMCs under normoxic conditions. VEGF levels were significantly by hypoxia in both However, the of VEGF induction by hypoxia in old VSMCs increase versus was significantly lower than that of young VSMCs increase were at the protein VEGF levels in the supernatant were and similar young and old VSMCs in normoxic conditions 1 VEGF values were significantly higher in both young and old VSMCs under hypoxic conditions, the of VEGF induction was significantly reduced in old versus young VSMCs (100 induction by was by blot experiments on cellular extracts from which were exposed to hypoxic conditions. seen in 1 VEGF levels were significantly reduced in old versus young hypoxic results indicate that aging VEGF induction under hypoxic conditions. VEGF expression is regulated not at the transcriptional level as E. Breier G. Risau W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), we VEGF mRNA half-life under hypoxic conditions was reduced by VSMCs were exposed to hypoxic conditions for 24 h before the addition of 5 μg/ml actinomycin D to block gene transcription. RNA isolation was performed at different and the mRNA half-life was determined after Northern shown in 2, significant difference in mRNA half-life was found young and old VSMCs versus h, p not that the age-dependent impairment of VEGF induction under hypoxic conditions is not because of a in mRNA To investigate the transcriptional regulation of VEGF is by we transfected VSMCs that were isolated from young or old rabbits with a reporter construct containing the luciferase gene under the transcriptional control of the VEGF The cells were then exposed to hypoxic conditions for a 24-h period, and promoter activities were measured in young and old VSMCs VEGF promoter activity under hypoxia in old VSMCs was of that in young VSMCs versus p < However, when a promoter construct lacking the HIF-1 binding site was transfected of the full-length reporter gene, VEGF promoter activity was not from hypoxia and significant difference was found the young and old HIF-1α in the age-dependent defect of VEGF induction by hypoxia. we HIF-1α expression at the mRNA and protein levels in young and old VSMCs HIF-1α mRNA was not by and similar levels were found in young and old VSMCs under normoxic or hypoxic conditions the protein HIF-1α expression was under normoxic conditions. hypoxic conditions, HIF-1α protein expression was in young was reduced in old VSMCs HIF-1α reduced expression of HIF-1α protein in oldversus young VSMCs that were exposed to 6 h of hypoxia To the of aging on HIF-1α DNA binding activity, we used a probe to the VEGF HIF-1 binding and we performed electromobility shift on protein extracts from young or old VSMCs specific HIF-1 DNA binding activity be under normoxic conditions in either young or old hypoxic conditions, HIF-1 activity was in young VSMCs was in old In this study, we have HIF-1 as a major factor involved in the age-dependent impairment of VEGF expression. We in vitro the previously described reduction of VEGF expression in ischemic tissues of old animals (1Rivard A. Fabre J.E. Silver M. Chen D. Murohara T. Kearney M. Magner M. Asahara T. Isner J.M. Circulation. 1999; 99: 111-120Crossref PubMed Scopus (647) Google Scholar). VEGF expression in VSMCs that were isolated from old animals was seen in hypoxic versus normoxic conditions at both the mRNA and protein that aging is associated with a defect in VEGF up-regulation under hypoxic conditions. VEGF expression been attributed to in both transcriptional and post-transcriptional mechanisms E. Breier G. Risau W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Cancer Res. 1999; 59: 5830-5835PubMed Google A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google U. 1995; PubMed Scopus Google M. Shweiki D. Itin A. Keshet E. Mol. Cell. Biol. 1995; PubMed Scopus Google G. D. Levy A.P. 1999; PubMed Scopus Google J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Previous have demonstrated the of hypoxia in transcriptional up-regulation of VEGF expression (14Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (537) Google J. J. 1998; PubMed Scopus Google Scholar). In this study, transfection experiments with a reporter gene under the control of the VEGF promoter that aging is associated with a reduction in hypoxia-induced VEGF transcriptional activity. the age-dependent reduction in transcriptional activity was not seen when VSMCs were transfected with a reporter construct lacking the HIF-1 binding HIF-1 is a transcription factor that is to be involved in to hypoxia. are by specific genes that are transcriptionally by as the genes for G.L. Jiang B.H. Leung S.W. P. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), erythropoietin M.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google J. L.E. J.W. H. J. Biol. 1998; Google Scholar), and VEGF HIF-1 been shown to be essential for VEGF transcriptional both in vitro (9Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3232) Google Scholar) and in vivo (10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2068) Google Scholar). sequences transcriptional of reporter gene expression, for (9Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3232) Google Scholar) that a sequence at to to the VEGF transcription site was for VEGF induction in hypoxic cells. A HIF-1 binding site was demonstrated in the response and a the ability of the to HIF-1 and transcription in response to hypoxia. Moreover, the results of gene-targeting experiments have shown that HIF-1 to VEGF expression under hypoxic conditions, to a marked reduction in vascularization of the and early lethality by embryonic day 10.5 (10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2068) Google Scholar). the essential of HIF-1 in VEGF induction under hypoxic conditions. In this study, we found that HIF-1α mRNA levels were not by hypoxia and were similar young and old is with a in which HIF-1α and are shown to be expressed in and cells with significant induction by hypoxia. In the of HIF-1 by hypoxia is shown to be primarily determined by the of HIF-1α protein L.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our analysis of HIF-1α protein expression under hypoxic conditions that the of HIF-1α was impaired with The mechanisms involved in this age-dependent reduction of HIF-1α protein expression are HIF-1α protein been shown to be by the system under normoxic conditions and by hypoxia S. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google L.E. J. M. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google S. L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). mechanisms are by aging to be in mRNA stability a control for the hypoxic induction of VEGF in different cell lines E. Breier G. Risau W. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google U. 1995; PubMed Scopus Google Scholar). of VEGF mRNA by hypoxia is to be by the binding of G. D. Levy A.P. 1999; PubMed Scopus Google J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) to the sequences in both the and of VEGF mRNA A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Our indicated that VEGF mRNA half-life under hypoxic conditions was similar in VSMCs that were isolated from young and old that the mechanisms involved in VEGF mRNA by hypoxia are not with The results of in the experiments indicated that DNA binding activity of HIF-1 under hypoxic conditions was significantly reduced in VSMCs that were isolated from old this was because of a reduction of HIF-1α protein expression is is that aging to a reduction in the ability of HIF-1 to to the hypoxia response within the VEGF or that the ability of HIF-1α to is Such loss of previously been described with aging for U. D. J. Biochem. PubMed Scopus Google H. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and transcription factors K. J. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Mol. 1997; PubMed Google Scholar). The age-dependent in HIF-1 activity as being primarily responsible for reduced expression of VEGF and impaired angiogenesis associated with aging (1Rivard A. Fabre J.E. Silver M. Chen D. Murohara T. Kearney M. Magner M. Asahara T. Isner J.M. Circulation. 1999; 99: 111-120Crossref PubMed Scopus (647) Google Scholar). HIF-1 is involved in the induction of Epo and expression of and genes been reported in with advanced aging 1999; PubMed Scopus Google Scholar). lower of Epo been in old animals Lu Chen S.W. J. Gerontol. A Biol. Sci. Sci. 1996; PubMed Scopus Google Scholar) and in J. K. L. N. J. PubMed Scopus Google D. M. A. P. L. F. 1999; PubMed Google Scholar). The of this that an age-dependent defect in HIF-1 in a reduction in the expression of genes that are involved in the to hypoxia.