1.Oxygen Homeostasis: Phylogeny, Ontogeny, Physiology, and Pathobiology

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of carbon bonds. In this process of photosynthesis, CO2 and H2O are utilized to generate a compound containing multiple carbon bonds (glucose) and, as a side product, O2. The resulting progressive rise in the atmospheric O2 concentration led to the second major evolutionary event, which was the establishment of a symbiotic relationship between single-celled organisms and internalized primitive cells, which over several billion years became highly specialized (as mitochondria) to perform a series of chemical reactions in which glucose and O2 were utilized to generate ATP through the process of oxidative phosphorylation. The dramatic increase in energy production attendant with the development of oxidative phosphorylation provided the resources required to undertake the daunting and costly task of coordinating individual cells to act in concert as a multicellular organism. The evolution of oxidative phosphorylation may also have been critical for the establishment of a chemical equilibrium on the planet, by regenerating the CO2 and H2O that are consumed during photosynthesis. Thus, the balanced equation for life on Earth as we know it can be written simply as: CO2 + H2O + hν → C6H12O6 + O2 → ATP + CO2 + H2O.

For simple metazoans, exemplified today by the roundworm Caenorhabditis elegans, which consists of approximately 103 cells, O2 can diffuse to all cells at adequate concentrations for efficient mitochondrial respiration. However, as metazoans became increasingly larger and more complex, diffusion was no longer sufficient to insure all cells of adequate oxygenation. In organisms with multiple layers of cells, O2 becomes limiting at a distance of 100–200 µm from the source, which, in the case of simple metazoans, was the exterior of the body. To solve this limitation of O2 diffusion, organisms evolved with cell types that are specialized for O2 delivery. In the fruit fly Drosophila melanogaster, a series of tracheal tubes conduct O2 from the exterior to all cells, including those further removed from the source. The spacing of these tubules is such that the distance between any cell and the nearest tubule is less than that at which O2 becomes diffusion limited.

In the case of mammals, the further dramatic increase in body size and cell number (more than ten orders of magnitude greater than C. elegans) necessitated even greater specialization through the development of the lung, an organ in which surface area for O2 diffusion from the

environment was greatly increased; the erythrocyte, a cell specialized for high affinity O2 capture in the lung and release in peripheral tissues; the blood vessels, an organ for the delivery of O2 and glucose to peripheral tissues and the removal of CO2 and other metabolic wastes from them; and the heart, an organ for pumping erythrocytes through the blood vessels circuiting the lungs and peripheral tissues. During mammalian development, the circulatory system is the first functioning organ system. In mice, which have a gestational period of 20 days, establishment of the circulation must occur by embryonic day 9, when embryo size increases to the point where diffusion from the uterine cavity becomes insufficient to supply all cells with adequate amounts of O2. Major defects in the development of the red blood cells, blood vessels, or heart result in embryonic lethality at this stage. In humans, the functioning circulatory system is established during the first trimester.

Although increased O2 levels on Earth provided opportunities for increased energy production, the utilization of O2 was not without its risks. The goal of oxidative phosphorylation is the orderly transfer of electrons through a series of acceptor cytochromes in order to generate a proton gradient within the inner mitochondrial membrane. The potential energy of this gradient is used to synthesize ATP. O2 is utilized as the ultimate electron acceptor, resulting in the production of H2O. However, this process is not 100% efficient, so that electron transfer to O2 may occur at complex I or complex III, resulting in the generation of superoxide radicals. These reactive oxygen species can oxidize cellular proteins, lipids, and nucleic acids and, by doing so, interfere with their normal structure and function, resulting in cell death or carcinogenesis.

Due to the opposing perils of O2 deprivation (hypoxia) and O2 excess (hyperoxia), cellular O2 concentrations are very tightly regulated. In the context of angiogenesis, this means that new blood vessels form rapidly under conditions of hypoxia but that any excessive vascularization resulting in hyperoxia stimulates vascular regression. Thus, O2 delivery is matched to O2 consumption. The molecular physiological mechanisms by which O2 homeostasis is maintained are discussed in the following section. This ability to maintain O2 homeostasis through angiogenesis is lost in many disease conditions, most notably ischemic cardiovascular disease. Hence, a more complete understanding of these

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mechanisms may lead to novel therapeutic approaches to the most common cause of mortality in Western societies.

2.Hypoxia-Inducible Factor 1: Master Regulator of O2 Homeostasis

Blood O2-carrying capacity is maintained by O2-regulated production of erythropoietin (EPO), which stimulates the proliferation and survival of red blood cell progenitors. Analysis of cis-acting sequences required for increased transcription of the human EPO gene in response to hypoxia led to the identification,1 biochemical purification,2 and molecular cloning3 of hypoxia-inducible factor 1 (HIF-1). EPO is produced primarily by a rare cell type in the kidney. However, HIF-1 is expressed in all cell types and functions as a master regulator of oxygen homeostasis by playing critical roles in embryonic development, postnatal physiology, and disease pathobiology. HIF-1 has been identified in all metazoan species that have been analyzed from Caenorhabditis elegans to Homo sapiens, suggesting that the appearance of HIF-1 represented an adaptation that was essential to metazoan evolution.

HIF-1 is a transcription factor that binds to specific cis-acting sequences, which are designated hypoxia response elements (HREs) and contain one or more copies of the core HIF-1 binding-site sequence 5 -(A/G)CGTG-3 .4 HREs containing a single HIF-1 binding site also contain a second essential sequence which has the consensus 5 -CACAG-3 . In the EPO gene, mutation of either the 5 -ACGTG-3 HIF-1 binding site or the 5 -CACAG-3 sequence (Fig. 1) results in loss of HRE activity.2 Thus, an HIF-1 binding site is necessary but not sufficient for HRE activity, which can only be demonstrated by transcriptional assays in which the insertion of a putative HRE into a reporter gene is shown to result in increased expression in hypoxic cells.4,5

The expression of over 40 genes is known to be activated at the transcriptional level by HIF-1 as determined by the most stringent criteria (Table 1), including the induction of gene expression in response to hypoxia, the presence of a functionally-essential HIF-1 binding site in the gene (Fig. 1), and an effect of HIF-1 gain-of-function or

Fig. 1. Hypoxia response elements. Nucleotide sequences from the human genes encoding erythropoietin (EPO), vascular endothelial growth factor (VEGF), aldolase A (ALDA), ceruloplasmin (CP), enolase-1 (ENO1), and lactate dehydrogenase A (LDHA) are shown. The core HIF-1 binding site (5 -RCGTG-3 ) is overlined with an arrow and the accessory sequence (consensus, 5 -CACAG-3 ) is underscored. Ellipses indicate cases where the 5 or 3 end of the minimal hypoxia response element has not been determined and N19 denotes an additional 19 nucleotides that are not shown. See Table 1 for references.

loss-of-function on expression of the gene. This list of HIF-1 target genes probably underestimates the total number of genes regulated by HIF-1 by more than an order of magnitude.7 The battery of genes regulated by HIF-1 is different in each cell type and, for some genes, expression can be induced or repressed by HIF-1 depending upon the cell type.6 Among critical physiological processes regulated by HIF-1 target genes are angiogenesis, erythropoiesis and glycolysis, which represent examples of both cell-autonomous and non-cell-autonomous (systemic) adaptive responses to hypoxia. Additional candidate HIF-1 target genes have been identified by the analysis of HIF-1-deficient mouse embryonic stem cells8 and fibroblasts,9 tissue culture cells expressing constitutively-active forms of HIF-1α or HIF-1β,10 and VHL-null renal carcinoma cells.11–16

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Table 1. Genes that are directly regulated by HIF-1.

HIF-1 is a heterodimeric protein that is composed of HIF-1α and HIF-1β subunits.2 The amino-terminal half of each subunit consists of basic helix-loop-helix and PAS domains that mediate heterodimerization and DNA binding.3,17 The carboxyl-terminal half of HIF-1α contains two transactivation domains18,19 that mediate interactions with co-activators such as CBP and p300.20−26 Co-activators interact with both sequence-specific DNA binding proteins such as HIF-1 and with the general transcription factors associated with RNA polymerase II (reviewed in Ref. 27). Co-activators also have histone acetyltransferase activity that is required to make the DNA embedded in chromatin accessible to the polymerase complex for transcription into RNA.

The HIF-1β subunit is constitutively expressed, whereas the expression and activity of the HIF-1α subunit are precisely regulated by the cellular O2 concentration.3,28 HIF-1α accumulates instantaneously under hypoxic conditions and upon reoxygenation is rapidly degraded with a half-life of less than five minutes in post-hypoxic tissue culture cells.3,29 This represents an overestimate of the half-life since it includes the time required for O2 to diffuse out of the culture medium. In isolated perfused and ventilated lung preparations subjected to hypoxia and reoxygenation, the half-life of HIF-1α is less one minute.30 No protein has been shown to have a shorter half-life.

In addition to HIF-1α, a structurally and functionally-related protein designated HIF-2αs which is the product of the EPAS1 gene, can also heterodimerize with HIF-1β.31 HIF-1α:HIF-1β and HIF-2α:HIF- 1β heterodimers appear to have overlapping but distinct target gene

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specificities.10,32 Unlike HIF-1α, HIF-2α is not expressed in all cell types and when expressed can be inactive as a result of cytoplasmic sequestration.33 A third protein, designated HIF-3α, has also been identified.34 Its role has not been well defined although a splice variant, designated IPAS, has been shown to bind to HIF-1α and inhibit its activity.35,36

The O2-dependent degradation of HIF-1α involves ubiquitination and degradation by the 26S proteasome.37−39 The von Hippel-Lindau tumor suppressor protein (VHL) is required for this process and renal carcinoma cells lacking functional VHL constitutively express HIF- 1α and HIF-2α as well as mRNAs encoded by HIF-1 target genes, under non-hypoxic conditions.40 VHL forms a complex with elongin B, elongin C, cullin 2, and RBX1 to form an E3 ubiquitin-protein ligase capable of functioning with E1 ubiquitin-activating and E2 ubiquitinconjugating enzymes to mediate the ubiquitination of HIF-1α.41 Proline (Pro) residue 564 is hydroxylated in an O2-dependent manner and this modification is required for VHL binding.42−44 Pro-402 represents a second site of hydroxylation and VHL binding.45 Pro-402 and -564 are each contained within a similar amino acid sequence (LXXLAP; A, alanine; L, leucine; P, Pro; X, any amino acid). HIF-2α and HIF-3α expression are also regulated by prolyl hydroxylation and VHL binding.40,46,47

Three prolyl hydroxylases were identified in mammalian cells and shown to utilize O2 as a substrate to generate 4-hydroxyproline at residue 402 and/or 564 of HIF-1α.48−50 These proteins are homologues of EGL-9, which was identified as the HIF-1α prolyl hydroxylase in C. elegans by genetic studies.49 Alternative designations for the three mammalian homologues include EGLN (EGL Nine homologue), PHD (Prolyl Hydroxylase Domain protein), and HPH (HIF-1α Prolyl Hydroxylase) 1–3. The hydroxylation reaction also requires α-ketoglutarate as a substrate and generates succinate as a side product.49,51,52 Ascorbate is required as a co-factor. The prolyl hydroxylase catalytic site contains an Fe (II) ion that is coordinated by two histidine and one aspartate residue. Unlike heme-containing proteins, the Fe (II) in dioxygenases can be chelated or substituted by Co (II), rendering the enzyme inactive. Most importantly, these prolyl hydroxylases have a relatively high Km for O2 that is slightly above its atmospheric concentration, such that O2 is rate limiting for enzymatic

activity under physiological conditions.46,49 As a result, changes in cellular O2concentration are directly transduced into changes in the rate at which HIF-1α is hydroxylated, ubiquitinated, and degraded.

Remarkably, HIF-1α transactivation domain function is regulated by O2-dependent hydroxylation of asparagine residue 803, which blocks the binding of the co-activators CBP and p300.53 FIH-1 (factor inhibiting HIF-1), which was identified as a protein that interacts with and inhibits the activity of the HIF-1α transactivation domain,54 functions as the asparaginyl hydroxylase.55,56 As in the case of the prolyl hydroxylases, FIH-1 appears to utilize O2 and α-ketoglutarate and contain Fe (II) in its active site, although it has a Km for O2 that is three times lower than the prolyl hydroxylases.57

The more cells that are present in a tissue, the more O2 is consumed. Thus, it is not surprising that major pathways that transduce proliferative and survival signals from growth factor receptors also induce HIF-1α expression in what can be viewed as a pre-emptive strategy for maintaining oxygen homeostasis. The increase in HIF-1α levels in response to growth factor stimulation differs in two important respects from the increase in HIF-1α levels in response to hypoxia. First, whereas hypoxia increases HIF-1α levels in all cell types, growth factor stimulation induces HIF-1α expression in a cell type-restricted manner. Second, whereas hypoxia is associated with decreased degradation of HIF-1α, growth factors, cytokines, and other signaling molecules stimulate HIF-1α synthesis via activation of the PI-3-kinase or MAP kinase pathways.58−61

Pulse-chase studies of MCF-7 breast cancer cells stimulated with heregulin demonstrated increased HIF-1α protein synthesis that was inhibited by treatment with rapamycin, a macrolide antibiotic which inhibits mTOR, a kinase that functions downstream of PI-3-kinase and AKT.59 The effect of heregulin was mediated via the 5 -untranslated region of HIF-1α mRNA.59 The known targets for phosphorylation by mTOR are regulators of protein synthesis. The translation of several dozen different mRNAs are known to be regulated by this pathway and specific sequences in the 5 -untranslated region may determine the degree to which the translation of any particular mRNA is modulated by mTOR signaling. HIF-1α protein expression is particularly sensitive to changes in the rate of synthesis because of its extremely short half-life

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under non-hypoxic conditions. In addition to effects on HIF-1α synthesis, activation of the RAF-MEK-ERK signaling pathway has also been shown to stimulate HIF-1α transactivation domain function.62 This effect is due at least in part to ERK phosphorylation of the coactivator P300 with which the transactivation domains interact.26

As described earlier, O2 delivery to cells of the developing embryo becomes limited by diffusion such that establishment of a functioning circulatory system is required for embryonic survival by embryonic day 9 (E9) in the mouse. In wild-type mouse embryos, HIF-1α expression increases dramatically between E8.5 and E9.5 whereas embryos that lack HIF-1α expression die between E9.5 and E10.5 with cardiac malformations, vascular regression, and massive cell death.63−66 Thus, HIF-1α is essential for embryonic survival and vascularization.

3.Control of Angiogenic Growth Factor and Cytokine Production by HIF-1

Angiogenesis is mediated by a complex network of cell-cell communication, which begins when HIF-1 activity is induced within parenchymal cells in response to either hypoxia (e.g. ischemic tissue) or growth factor stimulation (e.g. tissue undergoing hyperplasia or hypertrophy). HIF-1 then mediates increased transcription of the VEGF gene,5 leading to increased secretion of the VEGF protein. Binding of VEGF to its cognate receptor VEGF-R2 on endothelial cells induces endothelial cell activation, which is characterized by changes in gene expression that resulting in decreased interactions with pericytes/smooth muscle cells and increased cell proliferation, motility, invasiveness, and cell autonomous survival. The combination of VEGF and hypoxia induces the expression by vascular cells of a variety of angiogenic growth factors and cytokines in a temporaland cell-type-specific manner. For example, endothelial cells produce endothelin 1 (EDN1), which binds to receptors on smooth muscle cells, inducing proliferation and hypertrophy.

As compared to growth/survival factors, cytokines primarily function to promote cell recruitment from distant sites and to regulate cell-cell interactions within the vasculature. Examples of cytokines regulated

by HIF-1 are placental growth factor (PLGF) and stromal-derived growth factor 1 (SDF-1), which in concert with VEGF, promote the recruitment to ischemic tissue of bone marrow-derived cells expressing VEGF-R1 (for PLGF and VEGF) or CXCR4 (for SDF-1) on their cell surface.6,67,68

Another group of secreted molecules, including angiopoietin 1 (ANGPT1), angiopoietin 2 (ANGPT2), and platelet-derived growth factor B (PDGFB), regulate short-range interactions between endothelial cells and pericytes/smooth muscle cells, which are inhibited during vascular budding and then re-established once a new blood vessel branch has been formed. Several factors, including VEGF and transforming growth factor β, have effects on both the proliferation/survival and migration of endothelial cells. Both gain-of-function and loss-of- function studies have demonstrated that HIF-1 regulates the expression of the genes encoding ANGPT1, ANGPT2, EDN1, PDGFB, PLGF, SDF1, TGFB3, VEGF, and VEGFC both in primary cultures of vascular cells and in vivo.6,7,66–74 Other secreted factors that are regulated by HIF-1 may promote angiogenesis in certain tissues or in response to specific physiological or pathological states include adrenomedullin, connective tissue growth factor, endocrine gland-derived vascular endothelial growth factor, erythropoietin, insulin-like growth factor 2, and leptin (see Table 1 for references).

4.Cell-Autonomous Effects of HIF-1 in Vascular Endothelial Cells

Gene expression profiles were compared in arterial endothelial cells cultured under non-hypoxic versus hypoxic conditions and in non-hypoxic cells infected with AdLacZ, an adenovirus encoding β-galactosidase, versus AdCA5, which encodes a constitutively-active form of HIF- 1α.7 Two hundred and forty-five gene probes showed at least 1.5-fold increase in expression in response to hypoxia and in response to AdCA5; 325 gene probes showed at least 1.5-fold decrease in expression in response to hypoxia and in response to AdCA5. The largest category of genes downregulated by both hypoxia and AdCA5-encoded proteins involved in cell growth/proliferation. Many genes upregulated by both