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Pulmonary Vascular Responses to Chronic Hypoxia Mediated by Hypoxia-inducible Factor 1

Program in Vascular Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland

Correspondence and requests for reprints should be addressed to Gregg L. Semenza, M.D., Ph.D., Program in Vascular Cell Engineering, Johns Hopkins University School of Medicine, 733 N. Broadway, Suite 671, Baltimore, MD 21205. E-mail: gsemenza{at}jhmi.edu

	ABSTRACT

TOP ABSTRACT ANALYSIS OF HIF-1{alpha}... ANALYSIS OF KNOCKOUT MICE... INVOLVEMENT OF HIF-1 IN... CONCLUSIONS REFERENCES

 
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of oxygen homeostasis that controls transcriptional responses to hypoxia. HIF-1 plays critical roles both during development and in response to physiologic and pathophysiologic stimuli in the adult. Here, the involvement of HIF-1 in lung pathophysiology will be discussed.

Key Words: gene expression • oxygen sensing • pulmonary hypertension

Hypoxia-inducible factor 1 (HIF-1) is a global regulator of oxygen homeostasis. Both the protein half-life and specific activity of the HIF-1 subunit are precisely O₂-regulated via hydroxylation events that provide a direct mechanism for transducing changes in O₂ concentration into changes in gene expression.

HIF-1 is a heterodimer consisting of an O₂-regulated HIF-1 subunit and a constitutively expressed HIF-1ß subunit (1). Proline residues 402 and 564 of HIF-1 are hydroxylated by HIF-1 prolyl hydroxylases (2). The K_m of these enzymes for O₂ is such that any decrease in PO₂ will result in a reduction in the rate of hydroxylation (3). Prolyl hydroxylation of HIF-1 is required for binding of the von Hippel-Lindau tumor suppressor protein (VHL), which is the recognition component of an E3 ubiquitin-protein ligase (4, 5). Under normoxic conditions, VHL-mediated ubiquitination of HIF-1 targets the protein for proteasomal degradation (6). Under hypoxic conditions, the rates of prolyl hydroxylation, ubiquitination, and degradation of HIF-1 are reduced, resulting in rapid accumulation of the protein.

Asparagine residue 803 of HIF-1 is also hydroxylated in an O₂-dependent manner by FIH-1 (factor inhibiting HIF-1) (7, 8). Hydroxylation of asparagine 803 blocks the interaction of HIF-1 with the coactivators p300 and CBP, which is required for HIF-1–mediated transcriptional activation (9).

The HIF-2 protein is structurally similar to HIF-1, dimerizes with HIF-1ß, and contains proline and asparagine residues that are hydroxylated in an O₂-dependent manner (2, 6, 9). Unlike HIF-1, which is active in all hypoxic cells, HIF-2 only appears to be transcriptionally active in a restricted number of cell types, most notably endothelial cells, and may play an important role in responses to oxidative stress (10–12).

	ANALYSIS OF HIF-1 EXPRESSION

TOP ABSTRACT ANALYSIS OF HIF-1{alpha}... ANALYSIS OF KNOCKOUT MICE... INVOLVEMENT OF HIF-1 IN... CONCLUSIONS REFERENCES

 
Immunoblot analysis of isolated ferret lung preparations demonstrated that HIF-1 induction occurred in a time- and O₂ concentration–dependent manner (13). Immunohistochemistry revealed expression of HIF-1 in most cell types within the hypoxic lung including bronchial epithelium and smooth muscle, alveolar epithelium, and vascular endothelium (13).

Exposure of cultured cells to hypoxia also resulted in the induction of HIF-1 expression and HIF-1 DNA-binding activity, including alveolar epithelial and macrophage, bronchial epithelial, and microvascular endothelial cell lines, as well as primary cultures of pulmonary artery endothelial cells and aortic endothelial and smooth muscle cells (13). Pulmonary artery smooth muscle cells were unusual in expressing high levels of HIF-1 under nonhypoxic conditions. The physiologic significance of this observation remains to be established.

Subsequent studies have demonstrated induction of HIF-1 expression in cells exposed to a wide variety of growth factors and cytokines, including epidermal growth factor, fibroblast growth factor 2, hepatocyte growth factor, insulin-like growth factor-1 and -2, interleukin-1ß, insulin, prostaglandin E₂, transforming growth factor- and -ß, thrombin, and tumor necrosis factor-. Whereas induction of HIF-1 in response to hypoxia involves decreased degradation of the protein, HIF-1 induction in response to growth factor/cytokine stimulation of receptor tyrosine kinases and G protein–coupled receptors results from increased synthesis of the protein that is mediated via the phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways (14-16).

	ANALYSIS OF KNOCKOUT MICE DEFICIENT FOR HIF-1 OR HIF-2

TOP ABSTRACT ANALYSIS OF HIF-1{alpha}... ANALYSIS OF KNOCKOUT MICE... INVOLVEMENT OF HIF-1 IN... CONCLUSIONS REFERENCES

 
Gene targeting experiments that generated a null allele at the mouse Hif1a locus revealed that complete HIF-1 deficiency results in embryonic lethality at midgestation due to cardiac and vascular defects (17). Mice that are heterozygous for the knockout allele develop normally but when subjected to chronic hypoxia (10% O₂ for 3 weeks) have impaired pulmonary vascular remodeling (18). The dramatic increase in the muscularization of pulmonary arterioles that is observed in wild-type (WT) mice is significantly reduced in heterozygous-null (HET) littermates. Electrophysiologic studies revealed a dramatic increase in capacitance (a measure of cell volume) in pulmonary artery myocytes from WT mice subjected to chronic hypoxia. This response was completely lost in HET mice (19). Measurements of membrane potential revealed depolarization of myocytes from chronically hypoxic WT mice, whereas the degree of depolarization was significantly reduced in myocytes from HET mice. In WT mice, chronic hypoxia induced a dramatic reduction in voltage-gated K⁺ currents, a response that was completely lost in HET mice (19). Thus, partial HIF-1 deficiency significantly impaired hypertrophy and depolarization, the two critical responses of pulmonary arterial myocytes leading to hypoxia-induced pulmonary hypertension.

Mice that are homozygous for a null allele at the locus encoding HIF-2 have impaired fetal lung maturation that can be rescued by vascular endothelial growth factor (VEGF) treatment (20). Heterozygosity for a null allele at the locus encoding HIF-2 manifest a complete absence of pulmonary vascular remodeling in response to chronic hypoxia that is associated with an absence of hypoxia-induced endothelin 1 expression, consistent with the expression of HIF-2 in vascular endothelial cells (21).

Thus, both HIF-1 and HIF-2 contribute to the process of hypoxia-induced pulmonary vascular remodeling characterized by medial thickening that is characteristic of mild forms of secondary pulmonary hypertension. In contrast, the severe form of primary pulmonary hypertension is characterized by the dysregulated proliferation of pulmonary arterial endothelial cells that results in the formation of plexiform lesions. The pathogenesis of this condition appears to involve the autocrine stimulation of endothelial cells that express both VEGF and VEGF receptors (22). The high levels of HIF-1 in these lesions provide a mechanism for the high levels of VEGF and may be a direct consequence of the dysregulated signaling via the bone morphogenetic protein receptor type II that underlies primary pulmonary hypertension (23).

	INVOLVEMENT OF HIF-1 IN ANGIOGENESIS

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HIF-1 also plays a critical role in angiogenesis by regulating the expression of key angiogenic growth factors including vascular endothelial growth factor, placental growth factor, angiopoietin-1 and -2, and platelet-derived growth factor-B (24). Injection of mice with adenovirus AdCA5 encoding a constitutively active form of HIF-1 is sufficient to induce angiogenesis of nonischemic tissue in vivo (24). Recent studies have demonstrated that exposure of cultured endothelial cells to hypoxia or AdCA5 induces activation as manifested by Matrigel invasion and tube formation assays (25). Hypoxia-induced endothelial cell invasion and tube formation are significantly inhibited when the cells are exposed to cigarette smoke extract for 24 hours (26). Hypoxia-induced expression of HIF-1 and VEGF protein are also inhibited under these conditions. Chronic exposure of mice to cigarette smoke inhibits ischemia-induced angiogenesis that is associated with inhibition of ischemia-induced HIF-1 and VEGF protein expression (26). Given the important role of HIF-1 in regulating VEGF gene expression (17), the finding that VEGF receptor blockade results in emphysema (27) suggests that inhibition of HIF-1 activity in smokers may lead to VEGF production that is insufficient to maintain normal alveolar structure and thus play a major role in the pathogenesis of emphysema.

	CONCLUSIONS

TOP ABSTRACT ANALYSIS OF HIF-1{alpha}... ANALYSIS OF KNOCKOUT MICE... INVOLVEMENT OF HIF-1 IN... CONCLUSIONS REFERENCES

 
Taken together, these studies suggest that HIF-1 may contribute to multiple aspects of pulmonary pathophysiology in patients with chronic lung disease. Further studies in clinically relevant animal models are required to investigate the hypothesis that manipulate of HIF-1 activity, either by genetic or pharmacologic means, may be of therapeutic benefit in patients with COPD.

	FOOTNOTES

 
Conflict of Interest Statement: G.L.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form April 12, 2004; accepted in final form August 6, 2004)

	REFERENCES

TOP ABSTRACT ANALYSIS OF HIF-1{alpha}... ANALYSIS OF KNOCKOUT MICE... INVOLVEMENT OF HIF-1 IN... CONCLUSIONS REFERENCES

 

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