HIGH ALTITUDE MEDICINE & BIOLOGY Volume 14, Number 2, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/ham.2013.1016
Cellular Responses to Hypoxia in the Pulmonary Circulation David J. Welsh and Andrew J. Peacock
Abstract
Welsh, David J., and Andrew J. Peacock. Cellular responses to hypoxia in the pulmonary circulation. High Alt Med Biol 14:111–116, 2013.—Hypoxia can be defined as a reduction in available oxygen, whether in a whole organism or in a tissue or cell. It is a real life cause of pulmonary hypertension in humans both in terms of patients with chronic hypoxic lung disease and people living at high altitude. The effect of hypoxia on the pulmonary vasculature can be described in two ways; Hypoxic pulmonary vasoconstriction (HPV) (resulting from smooth muscle cell contraction) and pulmonary vascular remodelling (PVR) (resulting from pulmonary vascular cell proliferation). The pulmonary artery is made up of three resident cell types, the endothelial (intima), smooth muscle (media) and fibroblast (adventitia) cells. This review will examine the effects of hypoxia on the cells of the pulmonary vasculature and give an insight into the possible underlying mechanisms. Key Words: vascular smooth muscle, hypobaric hypoxia, hypoxia inducible factor, hypoxic pulmonary vasoconstriction, pulmonary hypertension
Hypoxia: Relevance in Contemporary Pulmonary Hypertension Research?
pulmonary hypertension; due to a combination of polycythemia and pulmonary vascular remodeling (Meyrick, 1997).
H
Hypoxia: Definition
ypoxia has been used as a model of pulmonary hypertension for many years (Meyrick, 1979) and is a reallife cause of pulmonary hypertension in humans, both in terms of patients with chronic hypoxic lung disease and people living at high altitude. Indeed, > 140 million individuals live at > 2500 m worldwide, including 80 million in Asia and 35 million in South America. An electrocardiographic survey of 741 Kyrgyz highlanders demonstrated signs of cor pulmonale in 14% of subjects, and an independent group of 136 male highlanders revealed established pulmonary hypertension in 20% of subjects (Aldashev et al., 2002). Chronic hypoxia is used as a model (in animals) of pulmonary arterial hypertension, along with other models, such as shunt, monocrotaline, and sugen/hypoxia models (with sugen being an antivascular endothelial growth factor agent), which some believe are better models of human idiopathic disease. Despite these misgivings, chronic hypoxia is still the most widely studied animal model of pulmonary hypertension and has been particularly well studied with regard to the associated structural and cellular changes it causes. Pulmonary hypertension in response to hypoxia develops in most species, including humans. Hypoxia results, within minutes, in an acute increase in pulmonary arterial pressure (hypoxic pulmonary vasoconstriction) followed, if hypoxia persists, by sustained
Hypoxia can be defined as a reduction in available oxygen, whether in a whole organism or in a tissue or cell, and is a relative term because the normal environment of some tissues or cells may be generally lower in available O2 than others. The available oxygen in a ‘‘normal’’ environment depends on altitude. The Po2 of oxygen at sea level is 147 mmHg but the amount of available O2 will be reduced at higher altitudes because of decreasing Patm so that the oxygen availability at Everest base camp (5500 m) is reduced by half and on its summit (8848 m) by 70%. Another relative term with regards to hypoxia is its duration. ‘‘Acute’’ hypoxia can be anything in duration from seconds to days, whereas ‘‘chronic’’ hypoxic, days to weeks. The effects of acute and chronic exposure to hypoxia can be very different in the whole animal or in cells and can have different effects in different organs or circulations. This review will focus on the effects of hypoxia in the cells of the pulmonary vasculature. Hypoxia and the Pulmonary Vasculature The effect of hypoxia on the pulmonary vasculature can be described in two ways; hypoxic pulmonary vasoconstriction
Scottish Pulmonary Vascular Unit, Regional Heart and Lung Center, Glasgow, United Kingdom.
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112 (HPV) (resulting from smooth muscle cell contraction) and pulmonary vascular remodeling (resulting from pulmonary vascular cell proliferation). HPV (or the von Euler-Liljestrand mechanism) is a response of the lung to distribute pulmonary capillary blood flow to areas of high oxygen availability. It is thought that HPV may precede pulmonary vascular remodeling and be the initiation of the pathological process resulting in pulmonary hypertension (Fishman 1976, See review by Swenson in this issue). Under normal conditions, the thickness of the vascular wall is maintained at an optimal level by a fine balance between proliferation and apoptosis of the resident cell types. Pulmonary vascular remodeling can take place if this balance is disturbed in favour of proliferation with the vascular wall thickening and eventually obliterating the vessel lumen, leading to increased resistance (Kato, 1966) (Fig. 1). Cells of the Pulmonary Vasculature The pulmonary artery is made up of three resident cell types, the endothelial (intima), smooth muscle (media), and fibroblast (adventitia) cells. The endothelial cell layer forms a permeable barrier between circulating blood cells and the underlying vascular tissue and as such, it is in a unique position to respond to circulating factors and serves as a signal integrator and transducer to modulate events in the vasculature via paracrine effects. Medial smooth muscle cell thickening is the main determinant of pulmonary vascular resistance. Precapillary segments of the pulmonary vascular bed contribute the majority of pulmonary vascular resistance. It therefore follows that small changes in tone and/or structure in this area can lead to a large elevation of pulmonary arterial pressure. These vessels are normally only partially muscularized, but hypoxic pulmonary vascular remodeling leads to enhanced muscularization (Meyrick 1979) and hence are a key feature of hypoxic pulmonary vascular remodeling. The adventitial fi-
WELSH AND PEACOCK broblast also plays an important role in pulmonary vascular remodeling. The vascular adventitia can act as a biological processing center for the production, storage, and release of key regulators of vessel wall function. In response to stress or injury (e.g., hypoxia), resident adventitial cells can be activated and reprogrammed to exhibit different functional and structural behaviours, which include proliferation, differentiation, upregulation of contractile and extracellular matrix proteins, and release of factors that directly affect medial smooth muscle cell tone and growth. Mechanisms of Hypoxic Smooth Muscle Cell Contraction Of the three resident cell types, the smooth muscle cells are responsible for maintaining vascular tone. The mechanisms of HPV is a very complex topic and one that can only be touched upon in a review of this length but is considered at length by Swenson in this issue of High Altitude Medicine & Biology. The hypoxic contraction of pulmonary artery smooth muscle cells results from an influx of extracellular calcium and a release of intracellular calcium (Weir, 2006). This is a complex system that is still not entirely understood but involves L-type calcium channels, Kv channels, and nonselective cation (NSCC) channels. There is debate in the literature as to whether internal release of calcium (Gelband, 1997) or the influx of extracellular calcium (Weissman et al., 2006) is the ‘‘trigger’’ of smooth muscle cell contraction, but regardless of this, both play an essential role. Internal calcium release results from cell surface receptor mediated events resulting in inositol trisphosphate (IP3) generation from phospholipase C (PLD) and phosphoinositol bis-phosphate (PIP2), and the generation of diacylglycerol (DAG) from PLD and phosphatidyle choline (PC) resulting in protein kinase C (PKC) generation. Rho kinases (Rho/RhoA) also play an important role in intracellular calcium release by inhibiting the dephosphorylation of the myosin light chain kinase (MLCK) by
FIG. 1. Images of transverse sections of (a) a normal intralobar pulmonary artery from the lung of a control rat, and (b) a remodeled intralobar pulmonary artery from the lung of a chronically hypoxic rat (rat exposed to 10% oxygen for 4 weeks). The internal and external E.L. mark the boundaries of the media. Note that in the remodeled artery, there is marked thickening of the media due to an increase in smooth muscle. The adventitia is also thickened, due to increased deposition of collagen. Scale = *50 lm. The vessels were viewed under a light microscope, and images were captured via a video camera linked to a computer. (From Jeffery et al. 2001).
CELLULAR RESPONSES TO HYPOXIA myosin light chain phosphatase (MCLP). Such is the importance of this pathway that new pulmonary hypertension drugs such as Fasudil (a Rho kinase inhibitor) are being marketed. External calcium entry via L-type calcium channel regulation appears to be regulated via reactive oxygen species (ROS) and/or potassium channel (K + ) membrane depolarization (of which many have been described; e.g., Kv9.3, Kv1.5 (Archer et al., 1998, Hulme et al., 1999). The regulation of ROS itself appears to be via mitochondrial nicotinamide adenine dinucleotide (NAD) (Weir, 1995). As the mitochondria consumes oxygen, this organelle may well play a role in O2 sensing.
113 with SB203580, a specific p38 MAPK inhibitor (Mortimer et al., 2007). The relationship between p38 MAPK and HIF-1a is an attractive explanation for the role of p38 MAPK in hypoxia-mediated HPAF proliferation as HIF-1a is known to be responsible for the upregulation of hypoxia-sensitive gene products (Pouyssegur, 2003). The mechanism of this relationship is unclear at present: HIF-1a may be a downstream effector of p38 MAPK or p38 MAPK may contribute towards HIF-1a stability. HIF-1: The Master Regulator
Mechanisms of Hypoxic Pulmonary Vascular Cell Proliferation
HIF-1 is induced in all cells that have a nucleus (HamptonSmith, 2009). Under ‘‘normal’’ conditions, HIF-1 subunits are bound to the von Hippel-Lindau (VHL) protein; however, this binding relies on the hydroxylation of proline-564 which re-
It is well accepted that hypoxia is a cause of pulmonary vascular cell proliferation and vascular remodeling, but the mechanisms remain unclear. In vitro studies have demonstrated that hypoxia has direct effects on cell proliferation in some but not all cell preparations (Welsh et al., 1998; 2001; 2006). Hypoxia is able to increase cell proliferation by inhibition (production and/or release) of antimitogenic factors (e.g., NO, prostacyclin) and/or increasing the production and/or release of different mitogenic stimuli [e.g., serotonin (5-hydroxytryptamine (5-HT)), endothelin-1, PDGF, serotonin, VEGF], and inflammatory mediators (e.g., IL-6, IL-8, monocyte chemoattractant factor-1) from SMC, fibroblasts, EC, and platelets (Kourembanas et al., 1990, 1991; Yan et al., 1995, Mukhopadhyay et al., 1995, Eddahibi et al., 1999, Humar et al., 2012). Furthermore, hypoxic exposure leads to increased production of extracellular matrix components (Stenmark, 1997). Several possible pathways have been implicated in the cellular response to hypoxia. For example, as described earlier, in SMC, hypoxia dramatically increases the level of Ca2 + in the cytoplasm (Ashida, 1987). Increased Ca2 + levels lead to activation of Ca2 + /calmodulin and MAP kinases and expression of the early responsive gene, c-fos (Berridge, 1994; Hardingham et al., 1997,). Elevated Ca2 + level in SMC has been shown to modulate proliferation and growth (Berridge, 1994). These signaling processes may be different for different cell types. We know that acute hypoxic exposure leads to early proliferation of pulmonary artery fibroblasts (PAF) and this proliferation appears to be dependent on p38 mitogenactivated protein kinase (MAPK) (Welsh et al., 1998, 2001, 2006). p38 MAPK is among the key mechanisms that transmit signals from the cell surface to the nucleus. It belongs to the Ras/ERK (Ras/extracellular-signal-regulated kinase) signaling pathway (Sturgill, 1991, Lewis et al., 1998). p38 MAP kinase can directly influence gene transcription—a growing number of transcription factors are known to be direct targets of p38 (ATF-1, ATF-2, and ATF-6, the myocyte factor 2C and A (MEF2 C, A), the signaling lymphocytic activation molecule associated protein 1A (SAP1A), and others (Pouyssegur, 2003). Another important target of p38 MAP kinase is the tumor suppressor protein p53 (Katsoulidis et al., 2005). A link has also been established between p38 MAPK and HIF-1a (hypoxia inducible factor), the key transcription factor in the biochemical response to hypoxia. There was a reduction in HIF-1a expression in human PAF (HPAF) cells grown in conditions of acute hypoxia if the cells were preincubated
FIG. 2. MicroRNA biogenesis and mechanisms of action in hypoxia. Hypoxia can regulate the expression of miRNA through HIF-dependent and HIF-independent mechanisms. Hypoxia-responsive miRNA may undergo several nuclear and cytosolic processing steps prior to expression as mature and biologically active species. It has been reported that hypoxia increases Ago2 hydroxylation to increase miRNA expression and activity; hypoxia also decreases Dicer expression to depress miRNA expression. Mature miRNA are taken up into the RNA induced silencing complex (RISC) in order to recognize complementary sites in the 3’untranslated region (UTR) of target gene messenger RNA (mRNA) via Watson-Crick base pairing. At this point, miRNA negatively regulate gene expression via either translational repression or mRNA degradation. Proven to be critically important in the regulation of numerous other cellular processes and cardiovascular diseases, the functions of hypoxamirs in pulmonary hypertension remain mostly undefined. (From Hale et al., 2012).
114 quires O2. The hydroxylation of proline-564 takes place via prolyl-4-hydroxylase domain proteins (PHD) that contain Fe(II) in their catalytic center (Epstein et al., 2001). In hypoxic conditions, this hydroxylation of proline-564 is reduced by both a decrease in available O2 and by an increase in mitochondrial ROS generation that can oxidize Fe(II) (Guzy et al., 2005), resulting in rapid HIF-1a protein instability and binding to HIF-1b. Many HIF-1 target genes have been implicated in pulmonary hypertension. One of the most noteworthy is endothelin1 (ET-1) which is a 21-amino acid vasoconstricting peptide produced primarily in the endothelium and having a key role in vascular homeostasis. In normal conditions, ET-1 is produced in very low levels but has a marked upregulation in hypoxic conditions (Abraham et al., 2004). Other HIF-1 target genes involved in pulmonary hypertension include angiotensin converting enzyme (ACE), vascular endothelial growth factor (VEGF), inducible, endothelial and neuronal forms of nitric oxide synthase (NOS), and erythropoietin (EPO) (Melillo et al., 1997, Lam et al., 2008). HIF-1 involvement has been demonstrated in all three vascular cell types of the pulmonary artery. In the fibroblast, it is directly linked with p38 MAP kinase phosphorylation, which is essential for the hypoxic proliferation of these cells (Welsh et al., 2001). In smooth muscle cells, the gene expression of the previously discussed Kv channels responsible for vasoconstriction are HIF-1 dependent (Whitman et al., 2008), and in endothelial cells, HIF-1 has been implicated in the generation of plexiform lesions in association with VEGF overproduction (Tuder, 2001). The importance of HIF-1 in the hypoxic response has not just been confined to in vitro cells studies. Work using HIF-1 + /- mice have demonstrated protection from hypoxia-induced pulmonary hypertension that was linked with decreased ET-1 levels in the KO mice compared to wild-type litter mates (Brusselmans et al., 2003). HIF-1 mRNA levels show a dramatic increase in response to hypoxia. One possible explanation for this is that HIF-1 mRNA production is controlled by microRNAs (miRs) (Rane et al., 2009). miRNA Control of Gene Regulation to Hypoxia: Hypoxamirs MicroRNAs are small RNAs consisting of about 22 nucleotides. Unlike protein-coding genes, miRs exhibit extraordinary gene regulatory functions, silencing gene expression via interaction with the 3¢ UTR of the transcript (Loshikhes et al., 2007). Recent studies looking at gene regulation in hypoxic conditions showed these genes to be under the control of hypoxic-inducible miRs, which have been termed hypoxamirs (Huang et al., 2010, Chan et al., 2011, Qin et al., 2010). One particular hypoxamir of interest is miR-210 that is regulated by HIF-1 and binds directly to a HRE on the proximal miR-210 promoter (Camps et al., 2008). Mir-210 has been shown to be produced in many cells exposed to hypoxic conditions (Devlin et al., 2011) and can inhibit proteins that are crucial for cell cycle progression, such as members of the MAP kinase family of proteins (Mohhankumer et al., 2007). In addition to this, MIR-210 has been shown, in hypoxic conditions, to suppress mitochondrial metabolism (Chen et al., 2010), induce angiogenisis via VEGF signaling (Hu et al., 2010), and stall DNA repair (Crosby et al., 2009), all pathological processes involved in the development of pulmonary
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WELSH AND PEACOCK Address correspondence to: Prof. Andrew Peacock Scottish Pulmonary Vascular Unit Regional Heart and Lung Centre Glasgow, G81 4HX United Kingdom E-mail:
[email protected] Received February 1, 2013; accepted in final form February 6, 2013