1. Introduction
Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to hypoxia is thought to be a key component of the vascular remodeling that occurs in chronic hypoxic pulmonary hypertension. Many pulmonary disorders, including chronic obstructive pulmonary disease, are associated with chronic hypoxia, and when pulmonary hypertension and right heart failure develop due to pulmonary vascular remodeling, patient survival is impaired. Hypoxia is one of the factors known to cause secondary pulmonary hypertension and pulmonary vascular remodeling [1]. According to a WHO statement in 1996, there were approximately 140 million people living at altitudes above 2500m and there are several areas of permanent habitation at altitudes in excess of 4000 m. After several weeks of exposure to high altitude, lowlanders develop pulmonary hypertension, which is not completely reversed by supplemental oxygen [2], suggesting development of vascular remodeling of the lung [3]. Secondary pulmonary hypertension is characterized by proliferation of vascular smooth muscle cells and pulmonary arterial fibroblasts in small pulmonary vessels [4-6]. These results suggest that hypoxic enhancement of PASMC proliferation contributes to the progression of hypoxia-associated small pulmonary arterial remodeling and secondary pulmonary hypertension. In animals, hypoxia has been shown to cause pulmonary vascular wall thickening by inducing PASMC proliferation [7-9]. Most commonly, pulmonary vascular remodeling is studied in rats or mice exposed to 10% oxygen hypoxia for 2 to 8 weeks [9,10]. In the animals exposed to chronic hypoxia, muscular arteries increase the thickness and distal extension and migration of PASMC into normally non-muscular arteries can be observed (Figure 1).
In addition, many
Although HIF-1α regulates various transcriptional genes for angiogenic factors, severe hypoxia and iron depletion induce cell growth arrest. In contrast to severe hypoxia, moderate hypoxia can also enhances the proliferation of airway-smooth muscle cells, lung fibroblasts and mesangial cells [17,18]. Proliferation of PASMCs, which causes pulmonary vascular remodeling, requires re-entry of the cells into the cell cycle. We confirmed that the cultured PASMC cell cycle progresses more quickly in hypoxia and that severe hypoxia or iron depletion using an iron chelator, which mimics anoxia, caused inhibition of cell cycle progress compared to the normoxic conditions (Figure 2).
2. Cell cycle regulation of hypoxic PASMC proliferation
Under normal physiological conditions, the majority of the pulmonary vascular cells are in a quiescent state. The most important molecular event necessary for progression of the cell cycle is phosphorylation of the retinoblastoma protein by cyclin-dependent kinase (CDK)-cyclin complexes. Cell cycle progression requires the coordinated interaction of CDK and its regulatory subunits, the cyclins, to drive cells through G1 into S phase to ultimately result in cell division. Cyclin–CDK complexes activate transcription factors important in cell cycle progression. The cyclin–CDK complexes include cyclin D–CDK4/ CDK6 and cyclin E–CDK2 which inactivate retinoblastoma, an antitumor and antiproliferative protein which limits E2F-mediated gene transcription [19]. CDK inhibitors are proteins that bind cyclin–CDK complexes, inhibit hyperphosphorylation of retinoblastoma, cause G1 arrest, and suppress cell proliferation [20]. CDK activity can be inhibited by CDK inhibitors, which arrest the cell cycle at each corresponding phase and inhibit cell proliferation. Two families of CDK inhibitors have been shown to regulate vascular smooth muscle cell proliferation, p21 and p27. The loss of CDK inhibitors has been implicated in tumor development [21,22], and is closely related with the state of the tumor suppressor p53 [23,24].
2.1. Role of tumor suppressor p53 and CDK inhibitor p21
The endogenous CDK inhibitor p21 plays an important role in PASMC proliferation via induction of the tumor suppressor p53 [23,25], and has been identified as a key regulator of the cell cycle in cells exposed to hypoxia and oxidative stress [26-28]. In tumors expressing wild-type p53, apoptosis occurs in hypoxic regions, whereas tumors expressing mutant p53 exhibit lower levels of apoptosis in hypoxic regions [29]. p53-/- mouse embryo fibroblasts are more resistant to hypoxia-induced apoptosis, and have selective growth advantages compared to wild-type p53 cells [30]. In addition, hypoxic p53 accumulation has been linked to the hypoxia-inducible factor-1α (HIF-1α), which is known as a central transcriptional factor operating during hypoxia toward angiogenesis [31,32]. We recently reported that hypoxic p53 accumulation has been linked to the hypoxia inducible factor-1α (HIF-1α) [9]. These results support the view that the p53 protein opposes cell proliferation under hypoxia, and p53 plays a critical role as a modulator of hypoxia-induced small pulmonary arterial remodeling. Decreased expression of p21 and increased expression of HIF-1α via suppression of p53 protein may mitigate hypoxic pulmonary arterial remodeling and PASMC proliferation. Recently, several groups identified microRNAs (miRNAs) regulated by p53 [33,34]. miRNAs are non-coding RNA molecules which modulate gene expression by binding to complementary sequences in the coding - or the 3`-untranslated region of target mRNAs. miRNAs can regulate cell proliferation, differentiation and apoptosis [35,36]. The miR34a has been shown to be the most significant miRNA induced by p53, which is closely related to induction of apoptosis and cell cycle arrest in cancer cells [37]. We have shown that this miRNA is also associated with HIF-1α expression both in animal and human lung tissues [9,38,39].
p21 has been shown to regulate cell cycle progression through both p53-dependent and -independent pathways [24,25,40]. However, it is known that nitric oxide (NO) donors suppress proliferation of cultured PASMC via the expression of p53 and p21 [23]. NO is synthesized from L-arginine via nitric oxide synthase (NOS), and endothelial NOS plays an important regulatory role in hypertrophic and hyperplastic growth of PASMC
In our recent data from p53 knockout mice, chronic hypoxia increased p21 expression and induced medial wall thickening of small pulmonary arteries in wild type mice, and the deletion of the p53 gene prevented the hypoxic induction of p21 expression [9]. These results indicate that under hypoxic conditions, induction of the p53-p21 signaling pathway serves as a negative feed-back to prevent excessive vascular cell proliferation and vascular remodeling. Using cultured PASMCs, we confirmed that the anti-proliferative NO pathway was intact in the hypoxic condition and the protein expression of p21 was associated with HPASMC proliferation (Figure 3).
2.2. CDK inhibitor p27
The suppressive effect of hypoxia on p27 expression has been demonstrated in mice with pulmonary hypertension induced by hypoxia [8]. However, the expression of p27, which blocks the cell cycle at the G0/1 phase, is regulated by several mechanisms including transcription, protein degradation and translation [43-45]. We reported that hypoxia-induced down-regulation of p27 was not mediated by hypoxia
Previous findings have suggested that p27 mRNA stability is controlled by interactions between MAPK [46] and Rho-dependent translation[47]. Further, cAMP induces cell relaxation through Rho GTPase activation [48,49], which might be an important target of hypoxic pulmonary vascular remodeling [50,51]. These reports imply that the Rho and MAPK interaction contributes to p27 mRNA stability during exposure to agents that elevate cAMP and hypoxia. These interactions may be also possible in smooth muscle cells. The Rho inhibitor Y-27632 inhibited human aortic smooth muscle proliferation in response to platelet derived growth factor and markedly suppressed neointima formation associated with decreased expression of p27 in rat carotid artery [52]. Regarding the effect of Rho on the expression of p27 in PASMC, we simply measure the p27 protein expression using Rho inhibitor in cultured PASMC and found that the p27 protein expression was increased by Y-27632 (Figure 5).
3. Conclusion
It is well accepted that hypoxia is a cause of pulmonary vascular remodeling and PASMC proliferation. In the aggregate, research conducted by us and others suggests that decreased oxygen levels affect PASMC proliferation. Decreased expression of p27 and signal transduction via p53 and p21 play critical roles in the fine-tuning of hypoxic PASMC proliferation.
However, the hypoxia induced remodeling of the pulmonary circulation including PASMC proliferation is a highly complex process, which may have numerous interactions between the vascular cells, especially between endothelial cells and lung fibroblasts. Because of that, it is difficult to explain the
Acknowledgement
We wish to thank Prof. Norbert F Voelkel, Virginia Commonwealth University, Richmond, VA, USA and Dr. Herman J. Bogaard, VU University Medical Center, Amsterdam, the Netherlands, for their critical reading of this manuscript.
References
- 1.
Cogo A. Napolitano G. Michoud M. C. Barbon D. R. Ward M. Martin J. G. 2003 Effects of hypoxia on rat airway smooth muscle cell proliferation. J. Appl. Physiol.94 1403 1409 - 2.
Groves B. M. Reeves J. T. Sutton J. R. Wagner P. D. Cymerman A. Malconian M. K. Rock P. B. Young P. M. Houston C. S. 1987 Operation Everest II: elevated high-altitude pulmonary resistance unresponsive to oxygen. J. Appl. Physiol.63 521 530 - 3.
Hainsworth R. Drinkhill M. J. 2007 Cardiovascular adjustments for life at high altitude. Respir Physiol Neurobiol.158 204 211 - 4.
Biernacki W. Flenley D. C. Muir A. L. Mac W. Nee 1988 Pulmonary hypertension and right ventricular function in patients with COPD. Chest.94 1169 1175 - 5.
Mac W. Nee 1994 Pathophysiology of cor pulmonale in chronic obstructive pulmonary disease Part One. Am. J. Respir. Crit. Care Med.150 833 852 - 6.
Semmens M. Reid L. 1974 Pulmonary arterial muscularity and right ventricular hypertrophy in chronic bronchitis and emphysema. Br J Dis Chest.68 253 263 - 7.
Yu A. Y. Shimoda L. A. Iyer N. V. Huso D. L. Sun X. Mc Williams R. Beaty T. Sham J. S. Wiener C. M. Sylvester J. T. Semenza G. L. 1999 Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha. J. Clin. Invest.103 691 696 - 8.
Yu L. Quinn D. A. Garg H. G. Hales C. A. 2005 Cyclin-dependent kinase inhibitor27Kip1 but not p21WAF1/Cip1, is required for inhibition of hypoxia-induced pulmonary hypertension and remodeling by heparin in mice. Circ. Res. 97: 937-945. - 9.
Mizuno S. Bogaard H. J. Kraskauskas D. Alhussaini A. Gomez-Arroyo J. Voelkel N. F. Ishizaki T. 2011 53 Gene deficiency promotes hypoxia-induced pulmonary hypertension and vascular remodeling in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 300: L753-61. - 10.
Bogaard H. J. Natarajan R. Mizuno S. Abbate A. Chang P. J. Chau V. Q. Hoke N. N. Kraskauskas D. Kasper M. Salloum F. N. Voelkel N. F. 2010 Adrenergic receptor blockade reverses right heart remodeling and dysfunction in pulmonary hypertensive rats. Am. J. Respir. Crit. Care Med.182 652 660 - 11.
Cooper A. L. Beasley D. 1999 Hypoxia stimulates proliferation and interleukin-1alpha production in human vascular smooth muscle cells. Am. J. Physiol. 277: H1326 37 - 12.
Kadowaki M. Mizuno S. Demura Y. Ameshima S. Miyamori I. Ishizaki T. 2007 Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of27kip1 Respir. Res. 8: 77. - 13.
Eddahibi S. Fabre V. Boni C. Martres M. P. Raffestin B. Hamon M. Adnot S. 1999 Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells Relationship with the mitogenic action of serotonin. Circ. Res.84 329 336 - 14.
Stiebellehner L. Frid M. G. Reeves J. T. Low R. B. Gnanasekharan M. Stenmark K. R. 2003 Bovine distal pulmonary arterial media is composed of a uniform population of well-differentiated smooth muscle cells with low proliferative capabilities. Am. J. Physiol. Lung Cell Mol. Physiol. 285: L819 28 - 15.
Tamm M. Bihl M. Eickelberg O. Stulz P. Perruchoud A. P. Roth M. 1998 Hypoxia-induced interleukin-6 and interleukin-8 production is mediated by platelet-activating factor and platelet-derived growth factor in primary human lung cells. Am. J. Respir. Cell Mol. Biol.19 653 661 - 16.
Frank D. B. Abtahi A. Yamaguchi D. J. Manning S. Shyr Y. Pozzi A. Baldwin H. S. Johnson J. E. de Caestecker M. P. 2005 Bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension. Circ. Res.97 496 504 - 17.
Krick S. Hänze J. Eul B. Savai R. Seay U. Grimminger F. Lohmeyer J. Klepetko W. Seeger W. Rose F. 2005 Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1alpha and an autocrine angiotensin system. FASEB J.19 857 859 - 18.
Sahai A. Mei C. Pattison T. A. Tannen R. L. 1997 Chronic hypoxia induces proliferation of cultured mesangial cells: role of calcium and protein kinase C. Am. J. Physiol. 273: F954 60 - 19.
Shackney S. E. Shankey T. V. 1999 Cell cycle models for molecular biology and molecular oncology: exploring new dimensions. Cytometry.35 97 116 - 20.
Sherr C. J. Roberts J. M. 2004 Living with or without cyclins and cyclin-dependent kinases. Genes Dev.18 2699 2711 - 21.
Lloyd R. V. Erickson L. A. Jin L. Kulig E. Qian X. Cheville J. C. Scheithauer B. W. 1999 27kip1 a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am. J. Pathol. 154: 313-323. - 22.
Gartel A. L. Tyner A. L. 1998 The growth-regulatory role of21 WAF1/CIP1). Prog. Mol. Subcell. Biol. 20: 43-71. - 23.
Mizuno S. Kadowaki M. Demura Y. Ameshima S. Miyamori I. Ishizaki T. 2004 42 Mitogen-activated protein kinase regulated by p53 and nitric oxide in human pulmonary arterial smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 31: 184-192. - 24.
Mizuno S. Bogaard H. J. Voelkel N. F. Umeda Y. Kadowaki M. Ameshima S. Miyamori I. Ishizaki T. 2009 Hypoxia regulates human lung fibroblast proliferation via53 and-independent pathways. Respir. Res. 10: 17. - 25.
O’Reilly M. A. Staversky R. J. Watkins R. H. Reed C. K. de Mesy K. L. Jensen J. N. Finkelstein P. C. Keng 2001 The cyclin-dependent kinase inhibitor21 protects the lung from oxidative stress. Am. J. Respir. Cell Mol. Biol. 24: 703-710. - 26.
Adachi S. Ito H. Tamamori-Adachi M. Ono Y. Nozato T. Abe S. Ma Ikeda F. Marumo M. Hiroe 2001 Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis in cardiomyocytes. Circ. Res.88 408 414 - 27.
Mc Grath-Morrow S. A. Stahl J. 2001 Growth arrest in A549 cells during hyperoxic stress is associated with decreased cyclin B1 and increased21 Waf1/Cip1/Sdi1) levels. Biochim. Biophys. Acta. 1538: 90-97. - 28.
Roy S. Khanna S. Bickerstaff A. A. Subramanian S. V. Atalay M. Bierl M. Pendyala S. Levy D. Sharma N. Venojarvi M. Strauch A. Orosz C. G. Sen C. K. 2003 Oxygen sensing by primary cardiac fibroblasts: a key role of21 Waf1/Cip1/Sdi1). Circ. Res. 92: 264-271. - 29.
Graeber T. G. Osmanian C. Jacks T. Housman D. E. Koch C. J. Lowe S. W. Giaccia A. J. 1996 Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature.379 88 91 - 30.
Yu J. Wang Z. Kinzler K. W. Vogelstein B. Zhang L. 2003 PUMA mediates the apoptotic response to53 in colorectal cancer cells. Proc. Natl. Acad. Sci. U.S.A. 100: 1931-1936. - 31.
An W. G. Kanekal M. Simon M. C. Maltepe E. Blagosklonny M. V. Neckers L. M. 1998 Stabilization of wild-type53 by hypoxia-inducible factor 1alpha. Nature. 392: 405-408. - 32.
Sano M. Minamino T. Toko H. Miyauchi H. Orimo M. Qin Y. Akazawa H. Tateno K. Kayama Y. Harada M. Shimizu I. Asahara T. Hamada H. Tomita S. Molkentin J. D. Zou Y. Komuro I. 2007 53 inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature. 446: 444-448. - 33.
Brosh R. Shalgi R. Liran A. Landan G. Korotayev K. Nguyen G. H. Enerly E. Johnsen H. Buganim Y. Solomon H. Goldstein I. Madar S. Goldfinger N. A. Børresen-Dale L. Ginsberg D. Harris C. C. Pilpel Y. Oren M. Rotter V. 2008 53 miRNAs are involved with E2F in a feed-forward loop promoting proliferation. Mol. Syst. Biol. 4: 229. - 34.
Hermeking H. 2007 53 enters the microRNA world. Cancer Cell. 12: 414-418. - 35.
Kloosterman W. P. Plasterk R. H. A. 2006 The diverse functions of microRNAs in animal development and disease. Dev. Cell.11 441 450 - 36.
Bushati N. Cohen S. M. 2007 microRNA functions. Annu. Rev. Cell Dev. Biol.23 175 205 - 37.
Tarasov V. Jung P. Verdoodt B. Lodygin D. Epanchintsev A. Menssen A. Meister G. Hermeking H. 2007 Differential regulation of microRNAs by53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle. 6: 1586-1593. - 38.
Mizuno S. Yasuo M. Bogaard H. J. Kraskauskas D. Natarajan R. Voelkel N. F. 2011 Inhibition of histone deacetylase causes emphysema. Am. J. Physiol. Lung Cell Mol. Physiol. 300: L402 13 - 39.
Mizuno S. Bogaard H. J. Gomez-Arroyo J. Alhussaini A. Kraskauskas D. Cool C. D. Voelkel N. F. 2012 MicroRNA-199a-5p is associated with hypoxia inducible factor-1α expression in the lung from COPD patients. Chest. in press. - 40.
Datto M. B. Li Y. Panus J. F. Howe D. J. Xiong Y. Wang X. F. 1995 Transforming growth factor beta induces the cyclin-dependent kinase inhibitor21 through a p53-independent mechanism. Proc. Natl. Acad. Sci. U.S.A. 92: 5545-5549. - 41.
Mitani Y. Maruyama K. Sakurai M. 1997 Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation.96 689 697 - 42.
Ananthakrishnan M. Barr F. E. Summar M. L. Smith H. A. Kaplowitz M. Cunningham G. Magarik J. Zhang Y. Fike C. D. 2009 L-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets. Am. J. Physiol. Lung Cell Mol. Physiol. 297: L506 11 - 43.
Eto I. 2006 Nutritional and chemopreventive anti-cancer agents up-regulate expression of27Kip1 a cyclin-dependent kinase inhibitor, in mouse JB6 epidermal and human MCF7, MDA-MB-321 and AU565 breast cancer cells. Cancer Cell Int. 6: 20. - 44.
Loda M. Cukor B. Tam S. W. Lavin P. Fiorentino M. Draetta G. F. Jessup J. M. Pagano M. 1997 Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor27 in aggressive colorectal carcinomas. Nat. Med. 3: 231-234. - 45.
Philipp-Staheli J. K. Kim H. Liggitt D. Gurley K. E. Longton G. Kemp C. J. 2004 Distinct roles for53 p27Kip1, and p21Cip1 during tumor development. Oncogene. 23: 905-913. - 46.
Sakakibara K. Kubota K. Worku B. Ryer E. J. Miller J. P. Koff A. Kent K. C. Liu B. 2005 PDGF-BB regulates27 expression through ERK-dependent RNA turn-over in vascular smooth muscle cells. J. Biol. Chem. 280: 25470-25477. - 47.
Vidal A. Millard S. S. Miller J. P. Koff A. 2002 Rho activity can alter the translation of27 mRNA and is important for RasV12-induced transformation in a manner dependent on p27 status. J. Biol. Chem. 277: 16433-16440. - 48.
Dong J. M. Leung T. Manser E. Lim L. 1998 cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha. J. Biol. Chem.273 22554 22562 - 49.
Laufs U. Marra D. Node K. Liao J. K. 1999 Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of27 Kip1). J. Biol. Chem. 274: 21926-21931. - 50.
J. Hyvelin M. Howell K. Nichol A. Costello C. M. Preston R. J. Mc Loughlin P. 2005 Inhibition of Rho-kinase attenuates hypoxia-induced angiogenesis in the pulmonary circulation. Circ. Res.97 185 191 - 51.
Connolly M. J. Aaronson P. I. 2011 Key role of the RhoA/Rho kinase system in pulmonary hypertension. Pulmonary Pharmacology & Therapeutics.24 1 14 - 52.
Sawada N. Itoh H. Ueyama K. Yamashita J. Doi K. Chun T. H. Inoue M. Masatsugu K. Saito T. Fukunaga Y. Sakaguchi S. Arai H. Ohno N. Komeda M. Nakao K. 2000 Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries. Circulation.101 2030 2033 - 53.
Stenmark K. R. Mecham R. P. 1997 Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu. Rev. Physiol.59 89 144