Acute Hypobaric Hypoxia (5486 m) Induces ... - Mary Ann Liebert, Inc.

HIGH ALTITUDE MEDICINE & BIOLOGY Volume 8, Number 4, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/ham.2007.1031

Acute Hypobaric Hypoxia (5486 m) Induces Greater Pulmonary HIF-1 Activation in Hilltop Compared to Madison Rats BARBARA J. ENGEBRETSEN,1,3 DAVID IRWIN,2 MARIA E. VALDEZ,3 MARY K. O’DONOVAN,3 ALAN TUCKER,3,† and MARTHA TISSOT VAN PATOT2,3

ABSTRACT Engebretsen, Barbara J., David Irwin, Maria E. Valdez, Mary K. Donovan, Alan Tucker, and Martha Tissot van Patot. Acute hypobaric hypoxia (5486 m) induces greater pulmonary HIF-1 activation in hilltop compared to Madison rats. High Alt. Med. Biol. 8:312–321, 2007.—Compared to Madison strain Sprague–Dawley rats, the Hilltop strain is resistant to acute hypoxic pulmonary vasoconstriction and pulmonary leak, a pathology resembling high altitude pulmonary edema (HAPE) in humans. Hypoxia inducible transcription factor-1 (HIF-1) mediates transcription of proteins that can “rescue” tissue from hypoxia, including vasoactive and angiogenic proteins such as inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF). Because these proteins have theoretical relevance to the etiology of HAPE, we hypothesized that hypoxia-resistant Hilltop rats acutely exposed to high altitude would have greater HIF-1 activity and expression of iNOS and VEGF as compared to hypoxia-sensitive Madison rats. Animals were exposed to normobaric normoxia or hypobaric hypoxia (18 h at 5486 m). The presence of nuclear HIF-1 heterodimer subunits, HIF-1–DNA binding, and iNOS and VEGF protein expression were determined in lung tissue. Hypoxic HIF-1 expression, HIF-1–DNA binding, and iNOS and VEGF expression were greater in Hilltop than in Madison rats. After 18-h hypobaric hypoxia, HIF-1 activity and HIF-mediated protein expression were elevated in Hilltop rats, but not in Madison rats. To our knowledge, this is the first report of differing HIF-1 activation between two strains of animals with clearly divergent physiological responses to identical hypoxic conditions. Key Words: high-altitude pulmonary edema; hypoxic sensitivity; HIF-1-DNA binding; inducible nitric oxide synthase (iNOS); vascular endothelial growth factor (VEGF) INTRODUCTION

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N THIS ARTICLE, we show that two rat strains with well-characterized divergent physio-

logical responses to hypoxia experience differing HIF-1 activity in response to identical acute hypoxic conditions. Compared to Madison strain Sprague–Dawley rats, the Hilltop strain

1Departments 2Department

of Biology and Health, Human Performance and Sport, Wayne State College, 68787 of Anesthesiology, Cardiovascular Pulmonary Research, University of Colorado Health Sciences Cen-

ter, 80206 3Department of Biomedical Sciences, Colorado State University, 80525. †Deceased.

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is resistant to acute hypoxic pulmonary vasoconstriction and pulmonary leak, resembling high altitude pulmonary edema (HAPE) in humans (Colice et al., 1995; West et al., 1995). Comparisons of the physiological responses to hypoxia between the Hilltop and Madison strains have been extensively described since the early 1980s (Ou et al., 1984; Langleben et al., 1987; Ou et al., 1992; Colice et al., 1995; Salameh et al., 1999). In contrast to their acute hypoxic resistance, Hilltop rats are sensitive to chronic hypoxic exposure, developing exaggerated polycythemia, pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy, resembling chronic mountain sickness (CMS) in humans. Divergent responses to hypoxia between the strains do not appear to be related to ventilatory, endocrine, fluid handling capabilities, or initial blood gas differences (Ou et al., 1992; Ou et al., 1994; Colice et al., 1995). Several studies suggest that blunted pulmonary vasoconstriction during acute hypoxia in Hilltop rats may be mediated by differences in nitric oxide-mediated vascular relaxation (Salameh et al., 1999; Karamsetty et al., 2001). Hypoxic sensitivity is quite variable in mammalian species. A key player in the integrated cellular response to hypoxia is hypoxia inducible factor (HIF), a ubiquitous heterodimeric nuclear transcription factor tightly regulated by physiologically relevant hypoxia (Semenza, 2001). During normoxia, the heterodimeric subunit HIF-1 is ubiquitinated by oxygen-dependent hydroxylation of prolyl residues and proteosomically degraded in the cytosol; however, during hypoxia, HIF-1 is stabilized and translocated to the nucleus, where it binds the HIF-1 subunit. HIF-1 is the principal regulator of HIF-1 dimerization (Gassmann and Wenger, 1997; Semenza, 1998). HIF-1 was the first and to date most extensively characterized transcription factor responding to hypoxia; however, three HIF species have currently been identified, underscoring the complex molecular integration of hypoxic signaling and response. HIF-1 and HIF-2 are ubiquitously expressed with slightly different target genes but some functional redundancy (Maxwell, 2005), whereas HIF-3 may be a negative regulator of angiogenesis in some tissues

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during hypoxia (Makino et al., 2002). A number of additional cofactors may modulate the intensity of the HIF–DNA binding in response to hypoxia, including HIF-1 hydroxylation and HIF-1 phosphorylation (Semenza, 1998; Ivan et al., 2001; Jaakkola et al., 2001). HIF-1 induces transcription of genes encoding proteins that may in turn mediate many of the pathophysiological processes involved in the contrasting responses of Hilltop rats to acute and chronic hypoxia, such as inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and erythropoietin (EPO) (Yu et al., 1999; Semenza, 2001). In studies using heterozygous Hif1a/ knockout mice, a blunted HIF-1 response to chronic hypoxia resulted in reduced polycythemia, pulmonary vasoconstriction, right ventricular hypertrophy and pulmonary angiogenesis (Hampl and Herget, 2000; Hampl et al., 2006). Because the chronic hypoxic phenotype of the Hif1a/ knockout mice contrasts with the Hilltop phenotype, we were led to examine the role of HIF-1 activation in the contrasting responses to acute hypoxia of Hilltop as compared to Madison rats. It is possible that HIF-1-mediated iNOS and VEGF, contributors to the development of chronic hypoxic pulmonary hypertension and remodeling, are associated with the chronic hypoxic Hilltop phenotype (Maggiorini et al., 2004). Therefore, we speculated that acutely increased HIF-1-induced iNOS would increase NO potential, attenuating hypoxic vasoconstriction and subsequently pulmonary leak (Colice et al., 1995). Our approach was to measure nuclear HIF-1 heterodimers, HIF1–DNA binding, expression of iNOS and VEGF, and vascular leak in the lungs of Hilltop and Madison strains exposed to hypobaric hypoxia for 18 h, because that is a time point at which Madison, but not Hilltop, rats develop pulmonary vascular leak (Colice et al., 1995).

METHODS Animals All protocols were conducted in accordance with the Colorado State University Animal Care and Use Committee guidelines for the use

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of research animals. Adult, male, high-altitudesensitive Hilltop rats (n 12 unless otherwise indicated, Hilltop Laboratory, Scottsdale, PA, USA) were compared with sex- and agematched Madison rats (n 12 unless otherwise indicated; Harlan Laboratories, Madison, WI, USA, Barrier 205). All animals were allowed free access to food and water and were subjected to a 12-h day–night light cycle. All animals were allowed to acclimate (minimum of 7 days) to ambient pressure (4920 ft; PB 640 mmHg) before experiments. Experimental design To determine the effect of hypoxia on these two strains, one group of animals was studied at ambient altitude (n 28; n 14 Hilltop and n 14 Madison rats) and the other group of animals was exposed to a simulated altitude of 5486 m (18,000 ft; PB 380 mmHg) for 18 h in a hypobaric chamber (n 26; n 13 Hilltop and n 13 Madison rats). Lung wet weight/dry weight ratios were determined using the left hemilung from all treated animals. The right hemilung was obtained from a smaller subgroup of treated animals for the nuclear and total lung preparations for molecular analysis (n 12; normoxic, n 3 Hilltop and n 3 Madison rats; hypoxic, n 3 Hilltop and n 3 Madison rats). Between 12 and 24 h of hypobaric hypoxia, Madison but not Hilltop rats develop pulmonary leak; Therefore we chose to conduct our studies at 18 h so that HIF activity could be determined in association with a hypoxia-induced pathology (Hagenbuchle and Wellauer, 1992). All terminal hypobaric hypoxic procedures were conducted in a main chamber after gaining access through an independently controlled antechamber. In this manner, the hypoxic animals were never exposed to ambient PB during hypobaric hypoxic exposure. Investigators collected all hypobaric hypoxic data at a simulated altitude of 4572 m (15,000 ft; PB 483 mmHg) for investigator safety. Tissue and blood collection Rats were anesthetized with intraperitoneal ketamine (90 mg/kg1), xylazine (10 mg/kg1), and acepromazine (1 mg/kg1).

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Midline thorocotomies were performed and the proximal pulmonary arteries were clamped. Following exsanguination, the lungs were removed en bloc; right lungs were frozen in liquid N2 for molecular analyses, while the left lungs were dissected free of connective tissue for determination of blood-free wet weight/dry weight ratio as an indication of pulmonary vascular leak. Protein extraction and analyses Total lung protein was isolated from 300 mg of frozen lung tissue per animal and was pulverized, suspended in 2.5-mL Laemmli buffer supplemented with 7 mol/L urea, 1% (v/v) pepstatin, 0.5% dithiothreitol, 0.05% phenylmethylsulfonyl fluoride, and 1 mini-tab protease inhibitor tablet/10 mL buffer (Complete Mini Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland, #1 836 153). Suspended tissues were homogenized for 1 min and centrifuged at 13,400 g at 4°C for 15 min. The supernatant was stored at 80°C until Western blot analysis. Nuclear protein was extracted by modification of procedures described by Hagenbuchle and Wellauer (Shen et al., 2002). Briefly, 500 mg of lung tissue from each animal was pulverized and suspended in 2.5-mL sample buffer, homogenized for 1 min, and centrifuged for 10 min at 800g at 4°C. The pellet representing the nuclear fraction was resuspended in 1-mL lysis buffer, extracted for 30 min at 4°C on a rocker, and centrifuged at 13,000g for 60 min. The supernatant was transferred to dialysis tubing and slowly stirred overnight on ice in dialysis buffer for equilibration of the salts. Each sample, total lung or nuclear protein, was analyzed for protein concentrations by a modified method of Lowry assay (Bio-Rad DC Protein Assay Kit #500-0116, Bio-Rad Laboratories, Hercules, CA) and stored at 80°C. Western blot analysis Primary monoclonal antibodies were obtained for HIF-1, HIF-1 (Novus Biologicals, Littleton, CO, USA, NB100-105, NB100-124), iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-7271), VEGF (Santa Cruz Biotechnology, sc-7267), or polyclonal eNOS (Santa


Cruz Biotechnology, sc-654) and -actin (Sigma, St. Louis, MO, USA, A-5441). -actin was used as a control for protein loading. Relative quantification by densitometry was performed using LabWorks 4.0 software (UVP, Inc. Upland, CA, USA). DNA binding activity of HIF-1 An ELISA kit was used to assess HIF-1 activity on all samples in a single experiment (K2077-1, BD Biosciences, Clontech, Palo Alto, CA, USA). Briefly, nuclear extract (20 g) was added to a 96-well plate coated with the DNA consensus binding sequence for HIF-1. Bound HIF-1 was detected by the addition of mouse monoclonal primary antibody to HIF-1, followed by HRP-conjugated secondary antibody. A microtiter plate reader (Multiskan Ascent; ThermoLab Systems, Helsinki, Finland) was used to measure the enzymatic product. An HIF-1 wild-type competitor oligonucleotide control was used to demonstrate DNA–HIF-1 binding specificity. The assay is able to detect DNA binding by HIF-1 with greater sensitivity than EMSA and is described by Shen and colleagues (Pearce et al., 1965; Parker and Ivey, 1997). All samples were run in triplicate and results were accepted when the coefficient of variance was less than 10%. We validated the ELISA by testing HIF-1–DNA binding using electromobility shift assays on a subset of tissue (data not shown). HIF-1–DNA binding was elevated in the same samples using both methods. Lung wet weight to dry weight ratios Pulmonary vascular leak was assessed by calculating the lung-wet weight/dry weight ratios corrected for estimated residual lung blood

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volume using a modification of the methods of Parker and colleagues and Pearce and colleagues (Ou et al., 1986; Colice et al., 1995). Statistical analysis Gravimetric data are reported as mean standard deviation (SD) and were analyzed by a two-factor ANOVA. Differences among treatment, strain, and interaction variability were determined to be significant if p 0.05. Immunoblots normalized for -actin and ELISA data were analyzed for treatment or strain differences by a Student’s t-test. Differences were considered to be significant if p 0.05. RESULTS Because Hilltop rats have an attenuated acute hypoxic pulmonary vasoconstrictive response and are less prone to high altitude-induced pulmonary vascular leak after 12 to 24 h of hypobaric hypoxia in comparison to Madison rats, and HIF-mediated proteins are integral to these processes (Semenza, 1999; Wenger, 2000), we assessed HIF-1 activity after 18-h hypobaric hypoxia in Hilltop and Madison rat strains. We did not detect HIF-1 stabilization at 18-h hypobaric hypoxia in either rat strain (Fig. 1); however, nuclear HIF1 (Fig. 2) and HIF-1 DNA binding (Fig. 3) were greater than normobaric normoxia levels in Hilltop but not Madison rats. Because iNOS is a HIF-1-mediated protein and a potent generator of nitric oxide (NO), which can attenuate hypoxic pulmonary vasoconstriction, we determined iNOS expression in the lungs of Madison and Hilltop rats exposed to normobaric normoxia and hypobaric

FIG. 1. Nuclear proteins were extracted from the lungs of Hilltop and Madison rats following normoxia and 18-h hypobaric hypoxia and analyzed for HIF-1 and -actin by Western blot analyses (top). -actin was used to control for protein loading in each lane. There was no strain or treatment difference in HIF-1 levels.

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DISCUSSION

FIG. 2. Nuclear proteins were extracted from the lungs of Hilltop and Madison rats following normoxia and 18h hypobaric hypoxia and analyzed for HIF-1 and -actin by Western blot analyses (top). -actin was used to control for protein loading in each lane. Data are expressed as the ratio of relative densitometry units of HIF-1 to actin (mean standard deviation) (bottom). *p 0.05 when compared to Madison hypoxic and Hilltop normoxic control values.

hypoxia. In accordance with greater HIF1–DNA binding, 18-h hypobaric hypoxia elevated the expression of iNOS in Hilltop but not Madison rats (Fig. 4). HIF-1 also mediates hypoxic expression of VEGF (Uchida et al., 2004); therefore, VEGF expression was determined as an indication of HIF-1 activation. Similar to iNOS, Hilltop but not Madison rats expressed greater VEGF at 18h hypobaric hypoxia as compared to normobaric normoxia (Fig. 4). To correlate HIF activity with divergent hypoxia-induced physiologic events in Hilltop as compared to Madison rats, we determined pulmonary vascular leak in Hilltop and Madison rats; extravascular lung water was calculated from the blood-free wet weight/dry weight ratios. After 18-h hypobaric hypoxia, pulmonary extravascular lung water was elevated in Madison but not in Hilltop rats (Fig. 5). To summarize, at 18 h of hypobaric hypoxia HIF-1–DNA binding activity and HIF-1-mediated protein expression were elevated in Hilltop but not Madison rats, and only Madison rats experienced significant pulmonary vascular leak.

In this study, we have described distinctive molecular divergence associated with physiologic differences between Hilltop and Madison rats in response to 18 h of hypobaric hypoxia. As hypothesized, hypoxia-induced HIF-1 activity to a greater extent in Hilltop compared to Madison rats, and this was associated with greater expression of downstream the HIF-1mediated proteins iNOS and VEGF. Moreover, greater HIF activity was associated with resistance to hypoxia-induced pulmonary vascular leak. Two issues are presented by these data. (1) HIF-1–DNA interaction is elevated despite the absence of HIF-1a elevation in the Hilltop rats, and (2) HIF-1 is elevated in the Hilltop rats at 18 h of hypoxic exposure. One potential explanation for the lack of HIF-1 stabilization is that HIF-1 was likely upregulated in the first 0 to 12 h of exposure and was downregulated by 18 h. Differential activity and the time course of HIF-1 and HIF-2 homologues may partially explain and support our observations (Wiesener et al., 2003). Acutely, both HIF regulators appear to be similarly activated by hypoxia. In cell culture (lung epithelial), while HIF-2 remains elevated, HIF-1 levels begin to decline after 4 h, returning to normoxic levels at 12 h.

FIG. 3. Nuclear proteins were extracted from the lungs of Madison and Hilltop rats following normoxia and 18h hypobaric hypoxia and analyzed for HIF–DNA binding activity using an ELISA transcription factor activity assay. *p 0.01 compared to Madison hypoxic and Hilltop normoxic values (mean standard deviation; n 3 per group).


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FIG. 4. Total proteins were extracted from the lungs of Hilltop and Madison rats following normoxia and 18-h hypobaric hypoxia and analyzed by Western blot for iNOS, VEGF, and -actin. -actin was used to control for protein loading in each lane. Data from blot shown are expressed as the ratio of relative densitometry units of the protein of interest to -actin. *p 0.05 when compared to Hilltop normoxia and Madison hypoxia. **p 0.05 when compared to Hilltop and Madison normoxia and Madison hypoxia. Data are presented as mean standard deviation.

HIF-1 mRNA also falls due to increased antisense HIF-1 transcripts (Uchida et al., 2004). Thus, in our model it is possible that at 18 h, HIF-1 had been actively downregulated. However, this 18-h time frame did capture elevated HIF-1–DNA interaction, perhaps simply a reflection of residual binding. Moreover, the data show increased iNOS and VEGF expression in the Hilltop but not Madison rats. Elevated HIF activity was associated with resistance against pulmonary leak, because the Madison rats did not exhibit elevated HIF1–DNA binding and iNOS or VEGF expression, but had significantly greater pulmonary leak. The elevation of HIF-1 is an interesting observation. HIF-1 has been identified as the principal regulator of HIF-1 dimerization and transcriptional activity, and its stabilization is tightly coupled to physiologically relevant hypoxia. Although reviews on HIF often refer to HIF-1 as the “hypoxia insensitive” unit of the HIF-1 dimer, many publications report hypoxia-induced increases of HIF-1 mRNA and protein in various tissue and cell types (Yu et al., 1998; Gassmann et al., 2000; Stroka et al., 2001) and specifically in hypoxic rat lungs (Yu et al., 1998). In addition to these observations

of hypoxic increases in HIF-1 levels, Chilov et al., (1999) has reported that free HIF-1 tends to leak from the nuclear envelope. In normoxia, this is not a problem because of the constitutive presence of HIF-1. However, during hypoxic stress, the authors suggest that there may

FIG. 5. Blood-free lung wet weight/dry weight ratios were measured to determine pulmonary vascular leak in rats after 18-hours hypobaric hypoxia. *p 0.001 when compared to Hilltop hypoxia; p 0.02 when compared to Madison normoxia. Data are presented as mean ± standard deviation.

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be a need for increased HIF-1 availability for HIF-1 dimerization. Indeed, in vivo some tissues of hypoxic mice increased expression of HIF-1 prior to increasing HIF-1 and continued to express HIF-1 after HIF-1 had diminished (Stroka et al., 2001). Further, these data indicate elevated expression of pulmonary iNOS and VEGF after 18-h hypobaric hypoxia. We propose that this is additional evidence of enhanced HIF activity; however, we acknowledge that other factors may be responsible for the increased expression of these proteins. While it is possible that iNOS and VEGF expression is elevated in Hilltop rats independently of HIF-1 signaling, both are clearly recognized as products of hypoxic signaling through HIF pathways. What remains to be determined is how the interaction between HIF-1, HIF-2, and perhaps even HIF3 negative regulation may contribute to the molecular and physiological differences observed between Hilltop and Madison rats. Indeed, both heterozygous Hif-1a/ and Hif-2a/ mice appear to be resistant to chronic hypoxic pulmonary hypertension (Semenza, 2004), indicating that both forms are associated with pulmonary vascular responses to hypoxia and could contribute to the observed Hilltop–Madison differences. Previous studies of Hilltop rats support our premise that Hilltop rats have a HIF-1-mediated elevation of transcriptional response to hypoxia. Ou and colleagues (1992) reported that renal EPO mRNA was greater in Hilltop than in Madison rats following exposure to 5500-m simulated altitude. The acute increase in EPO expression was not attributable to differences in PO2 because arterial and renal venous PO2 did not differ between strains at days 1 and 3 when EPO was initially elevated. Only after 14 days of hypoxia do strain differences in PO2 begin to develop, possibly as a result of exaggerated polycythemia in the Hilltop rats. Taken together with data from the current study, the evidence supports the hypothesis that Hilltop rats respond with greater HIF1–DNA binding and subsequent elevation of HIF-mediated protein expression than do Madison rats when exposed to equivalent hypoxic conditions. The exact role of elevated iNOS and VEGF

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in conferring resistance to acute hypoxic pulmonary edema is speculative and merits further study. The importance of NO in the etiology of hypoxic pulmonary edema is well documented. Excessive hypoxic pulmonary vasoconstriction is an essential predisposing condition for producing pulmonary vascular leak (Maggiorini et al., 2001; Bartsch et al., 2005). Moreover, evidence linking reduced NO availability to HAPE pathogenesis is growing (Duplain et al., 2000; Busch et al., 2001; Berger et al., 2005;). Greater iNOS in hypoxic Hilltop as compared to Madison rats most likely elevates nitric oxide synthesis, which could in turn attenuate acute hypoxic pulmonary vasoconstriction (Hampl and Herget, 2000). While greater iNOS may impart short-term protection from the pathological consequences of acute hypoxia, continued HIF-1 activity and iNOS expression may eventually contribute to the pathogenesis of pulmonary hypertension and right ventricular hypertrophy in response to chronic hypoxia (Kendall et al., 1996; Hastings et al., 2003). This paradigm corresponds with the paradoxical response of Hilltop rats to acute and chronic hypoxia. Interestingly, these data show that increased VEGF expression, considered a permeability factor, is associated with resistance to pulmonary vascular leak in Hilltop rats. Moreover, Madison rats experienced pulmonary edema despite normoxic VEGF levels after 18 h of hypoxic exposure. While VEGF is one of many factors that can increase vascular permeability, its exact role in the development of HAPE is controversial (Hanaoka et al., 2003). Development of hypoxic pulmonary edema requires both a critical threshold in capillary hydrostatic pressure (Maggiorini et al., 2001) and increased permeability (Sartori et al., 2000). It is likely that increased NO-generating potential and decreased hypoxic pulmonary vasoconstriction in Hilltop rats (Salameh et al., 1999) maintain vascular pressures below the critical threshold needed to produce hydrostatic leak. Consequently, any increase in VEGF-induced permeability in the absence of pulmonary hypertension may not be sufficient to create hypoxia-induced pulmonary edema in the Hilltop strain. It is also possible that VEGF bioavailability


is restricted by increased soluble Fms-like tyrosine kinase receptor-1 (sFlt-1), which is known to bind VEGF from the circulation, effectively reducing VEGF-induced changes in permeability (Gerber et al., 1997). Soluble Flt-1 is also a HIF-mediated protein and may have been present in greater concentrations in the plasma of Hilltop than in Madison rats, effectively preventing VEGF-induced pulmonary vascular leak. Supporting this hypothesis is evidence that plasma sFlt-1 concentration is elevated in humans exposed to hypobaric hypoxia, yet attenuated in those developing acute mountain sickness (Tissot van Patot et al., 2005).

CONCLUSION The picture emerging from this study and other research suggests that heightened hypoxic sensitivity at the molecular level may be a valuable short-term defense in individuals exposed to environmental hypoxia. However, this same heightened sensitivity may contribute to long-term pathological consequences. Moreover, the Hilltop–Madison rats have been well established as strains with divergent physiological responses to acute and chronic hypoxia consistent with this emerging picture. In addition to novel data showing an association between HIF activity and acute hypoxic pulmonary leak, this report introduces evidence supporting the potential value of the Hilltop–Madison model as a powerful tool for understanding the integrated molecular response of the HIF cascade in coordinating acute accommodation and chronic adaptation to hypoxia. In contrast to models using genetically modified mice, the Hilltop–Madison model involves the use of physiologically normal animals representing the extreme ends of the normal distribution of hypoxic sensitivity. These data suggest that HIF mediates protection from acute hypoxia-induced pulmonary leak, while HIF depression permits development of leak. This is the first report of strain-dependent differences in HIF activity in response to identical hypoxic challenges; Hilltop rats, which are resistant to acute hypoxic pathologies demonstrated elevated HIF activ-

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ity, and Madison rats, which are susceptible to acute hypoxic pathologies, did not.

ACKNOWLEDGMENTS This research was supported by the Colorado State University College Research Council Grant 1-67985 and the National Institutes of Health IN-BRE NIH Grant-2 P20 RR- 16469-05.

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Address reprint requests to: Barbara J. Engebretsen Wayne State College Wayne, NE 68787 E-mail: [email protected] Received April 17, 2007; accepted in final form July 11, 2007.