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• W2098840758 abstract "Midkine (MK) is expressed in a precise temporal-spatial pattern during lung morphogenesis; however, its role in pulmonary homeostasis is unknown. Increased MK staining and mRNA expression were observed in the lungs of hypoxia-susceptible CAST/eiJ mice during hypoxia. MK expression was induced by hypoxia in cell lines in vitro. Because the transcription factor hypoxiainducible factor-1α (HIF-1α) modulates cellular responses to hypoxia, we tested whether increased expression of MK in the lung was mediated by HIF-1α. HIF-1α enhanced the transcription of MK, acting on HIF-1α regulatory elements located in the MK gene promoter. Site-directed mutagenesis of the 3′ HIF response element in the MK promoter blocked the stimulatory effects of HIF-1α. To directly assess the role of MK on lung morphogenesis, transgenic mice were generated in which MK was expressed in the respiratory epithelial cells of the developing lung. MK increased muscularization of small pulmonary arteries, increasing α-smooth muscle actin and caldesmon staining and the expression of myocardin. MK directly enhanced the expression of myocardin and the smooth muscle-specific genes α-smooth muscle actin, calponin, and SM-22 in vascular smooth muscle precursor cells. Expression of MK in the respiratory epithelium is regulated by hypoxia and HIF-1α. These data provide a model wherein the respiratory epithelium responds to hypoxia via HIF-1α-dependent regulation of MK, enhancing myocardin expression to influence pulmonary vascular gene expression. Midkine (MK) is expressed in a precise temporal-spatial pattern during lung morphogenesis; however, its role in pulmonary homeostasis is unknown. Increased MK staining and mRNA expression were observed in the lungs of hypoxia-susceptible CAST/eiJ mice during hypoxia. MK expression was induced by hypoxia in cell lines in vitro. Because the transcription factor hypoxiainducible factor-1α (HIF-1α) modulates cellular responses to hypoxia, we tested whether increased expression of MK in the lung was mediated by HIF-1α. HIF-1α enhanced the transcription of MK, acting on HIF-1α regulatory elements located in the MK gene promoter. Site-directed mutagenesis of the 3′ HIF response element in the MK promoter blocked the stimulatory effects of HIF-1α. To directly assess the role of MK on lung morphogenesis, transgenic mice were generated in which MK was expressed in the respiratory epithelial cells of the developing lung. MK increased muscularization of small pulmonary arteries, increasing α-smooth muscle actin and caldesmon staining and the expression of myocardin. MK directly enhanced the expression of myocardin and the smooth muscle-specific genes α-smooth muscle actin, calponin, and SM-22 in vascular smooth muscle precursor cells. Expression of MK in the respiratory epithelium is regulated by hypoxia and HIF-1α. These data provide a model wherein the respiratory epithelium responds to hypoxia via HIF-1α-dependent regulation of MK, enhancing myocardin expression to influence pulmonary vascular gene expression. Midkine (MK) 1The abbreviations used are: MK midkine; HRE, HIF-1 response element; α-SMA, α-smooth muscle actin; E, embryonic day; PN, postnatal day; HIF-1α/β, hypoxia-inducible factor α or β; TTF-1, thyroid transcription factor-1; SRF, serum response factor; CArG, cis-regulatory DNA element (CC(A/T)6GG); FiO2, fraction of inspired oxygen; JEG-3, human placental adenocarcinoma; MFLM-4, mouse fetal lung mesenchyme cell line; H-441, human pulmonary adenocarcinoma cell line; rtTA, reverse tetracycline transactivator; RVH, right ventricular hypertrophy; LRP, low density lipoprotein receptor-related protein; RV, right ventricle; RT, reverse transcription; CMV, cytomegalovirus. is a retinoic acid-responsive, heparin binding growth factor expressed in various cell types during embryo-genesis (1Kadomatsu K. Tomomura M. Muramatsu T. Biochem. Biophys. Res. Commun. 1988; 151: 1312-1318Crossref PubMed Scopus (473) Google Scholar, 2Tomomura M. Muramatsu K. Matsubara S. Muramatsu T. J. Biol. Chem. 1990; 295: 10765-10770Abstract Full Text PDF Google Scholar). In vitro studies demonstrated that MK promotes angiogenesis (3Choundhuri R.I. Zhang H.T. Domini S. Ziche M. Bicknell B. Cancer Res. 1997; 57: 1814-1819PubMed Google Scholar), cell growth (4Muramatsu H. Muramatsu T. Biochem. Biophys. Res. Commun. 1991; 177: 652-658Crossref PubMed Scopus (213) Google Scholar), and cell migration (5Takada T. Toriyama K. Muramatsu H. Song X.-J. Torii S. Muramatsu T. J. Biochem. (Tokyo). 1997; 122: 453-458Crossref PubMed Scopus (143) Google Scholar). MK mRNA was detected in the developing lung (6Mitsiadis T.A. Salmivirta M. Muramatsu T. Muramatsu H. Rauvala H. Lehtonen E. Jalkanen M. Thesleff I. Development. 1995; 121: 37-51Crossref PubMed Google Scholar, 7Kaplan F. Comber J. Sladek R. Hudson T.J. Muglia L.J. Macrae T. Gagnon S. Asada M. Brewer J.A. Sweezy N.B. Am. J. Respir. Cell Mol. Biol. 2003; 28: 33-41Crossref PubMed Scopus (48) Google Scholar, 8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). In the mouse, MK was detected as early as embryonic day (E) 10 in the primordial lung buds (9Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncog. 1995; 6: 151-177PubMed Google Scholar). Exogenous MK enhanced growth of the pulmonary mesenchyme during branching morphogenesis of embryonic lung in vitro (10Toriyama K. Muramatsu H. Hoshino T. Torii S. Muramatsu T. Differentiation. 1996; 61: 161-167Crossref Scopus (28) Google Scholar). The temporal-spatial expression of MK in the developing lung and its regulation by a lung-specific homeobox containing transcription factor, thyroid transcription factor-1 (TTF-1), were recently demonstrated (8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). Although the biological functions of MK are not fully understood, MK protein and splice variants of MK mRNA have been detected in several carcinomas (9Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncog. 1995; 6: 151-177PubMed Google Scholar, 11Garver R.I. Chan C.S. Milner P.G. Am. J. Respir. Cell Mol. Biol. 1993; 9: 463-466Crossref PubMed Scopus (117) Google Scholar). These data suggest that MK may play a role in tumorigenesis, perhaps mediated by its effects on angiogenesis. In the mouse lung MK is expressed in a bimodal manner, with high levels of expression observed at E13-16.5 and postnatal days (PN) 5-12 (8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). Elevated MK expression in the postnatal period was coincident with alveolarization and pulmonary vascular development and remodeling. MK mRNA and protein expression are absent in the mouse lung after the alveolar stage of lung development (PN15). Because of its precise pattern of expression and its potential role in vasculogenesis and angiogenesis, we hypothesized that MK may play a role in the formation and differentiation of the pulmonary vascular bed. The pulmonary vasculature is highly responsive to changes in ambient oxygen. Chronic hypoxia initially induces vasoconstriction followed by increased muscularization of the vasculature associated with pulmonary hypertension (12Thomas B.J. Wanstall J.C. Eur. J. Pharmacol. 2003; 477: 153-161Crossref PubMed Scopus (34) Google Scholar). The effects of hypoxia have been recently surveyed in several strains of mice. CAST/eiJ is an inbred strain of mice in which chronic hypoxia caused increased muscularization of small pulmonary arteries. CAST/eiJ mice develop right ventricular hypertrophy and pulmonary hypertension, whereas FVB/N mice were unresponsive to chronic hypoxia. 2W. Nichols, unpublished observations. Cellular responses to hypoxia are mediated in part by the regulator of cellular and systemic oxygen homeostasis, hypoxia-inducible factor-1 (HIF-1) (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2209) Google Scholar,14Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1205) Google Scholar). HIF-1 is an αβ-heterodimeric protein that was initially identified as a DNA binding factor that mediates hypoxia-inducible activity of the erythropoietin 3′ enhancer (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2209) Google Scholar). Although HIF-1β is constitutively expressed, HIF-1α is rapidly induced by hypoxia. Under normoxic conditions, HIF-1α is regulated by hydroxylation, subsequent ubiquitination, and proteasomal degradation (15Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1849) Google Scholar). During hypoxia HIF-1β binds HIF-1α, prohibiting proteasomal degradation, and the complex is transported to the nucleus, where it binds HIF response elements (HREs, 5′-RCGTG-3′). The binding of HIF-1α/HIF-1β to HREs assists in the recruitment of coactivator molecules that form transcription initiation complexes to enhance the expression of genes that mediate cellular and physiologic responses to hypoxia (16Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). Epithelial-mesenchymal interactions, mediated by autocrine-paracrine signals, play a critical role in directing morphogenesis by providing signals that induce genetic programs in pulmonary cells (17Costa R.H. Kalinichenko V.V. Lim L. Am. J. Physiol. 2001; 280: L823-L838Crossref PubMed Google Scholar). The process of alveolarization occurs primarily during the postnatal period and is completed by 3-4 weeks of age in the mouse. During this period alveoli are increasingly septated, and alveolar walls thin to generate an efficient gas exchange compartment. Also required in this process is the formation of the pulmonary vasculature. Muscularization of pulmonary arterioles develops late in gestation and is mediated by autocrine-paracrine signaling, which directs smooth muscle cell-specific gene expression (18Veyssier-Belot C. Cacoub P. Cardiovasc. Res. 1999; 44: 274-282Crossref PubMed Scopus (75) Google Scholar). Smooth muscle differentiation is directed by a serum response factor (SRF) coactivator, myocardin (19Wang Z. Wang D.-Z. Pipes G.C.T. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7129-7134Crossref PubMed Scopus (432) Google Scholar), which forms protein-DNA complexes consisting of dimerized SRF bound to CArG box sequences (20Kumar M.S. Owens G.K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 737-747Crossref PubMed Scopus (158) Google Scholar). Although epithelial-mesenchymal interactions are known to be critical to lung morphogenesis, the molecular mechanisms by which epithelial and mesenchymal cells communicate during lung formation or a response to hypoxia remain poorly understood. In the present study we demonstrate that MK expression was induced by HIF-1α during hypoxia. Furthermore, increased expression of MK in the respiratory epithelium increased the expression of myocardin, smooth muscle cell markers and increased the muscularization of small pulmonary arteries. These data provide a mechanism by which hypoxia influences gene expression in the respiratory epithelium to generate midkine, a paracrine signal, that influences the formation of the pulmonary vasculature. Mouse Models and Animal Husbandry—CAST/eiJ mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and housed in a temperature-controlled room (22-25 °C) under a 12-h light/12-h dark cycle. Animal husbandry followed protocols approved by the Institutional Animal Care and Use Committee at Cincinnati Children's Hospital Research Foundation. Human surfactant protein C (SP-C) is selectively expressed in endodermally derived epithelial cells in the embryonic lung and is expressed in subsets of alveolar and bronchiolar epithelial cells after birth (21Wert S.E. Glasser S.W. Korfhagen T.R. Whitsett J.A. Dev. Biol. 1993; 156: 426-443Crossref PubMed Scopus (273) Google Scholar). The SP-C-rtTA+/tg transgene was generated by inserting 3.7 kilobases of the human SP-C promoter upstream of the reverse tetracycline transactivator (rtTA) gene (22Tichelaar J.W. Lu W. Whitsett J.A. J. Biol. Chem. 2000; 275: 11858-11864Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The (tetO)7-CMV-MK+/tg or -tg/tg transgene was generated by inserting a 696-bp mouse MK cDNA downstream of a minimal CMV promoter containing seven concatamerized tetracycline receptor binding sites. Transgenic mice were generated by pronuclear injection using standard techniques, and genotypes were determined by PCR. (tetO)7-CMV-MK+/tg or -tg/tg mice were identified by amplification of a 559-bp sequence of the transgene by using the following primers: forward (5′-GCC ATC CAC GCT GTT TTG AC-3′) and reverse (5′-GGT CTT TGA CTT GGT CTT GGA GG-3′). PCR parameters included heating at 95 °C for 5 min followed by 30 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s followed by a 7-min extension at 72 °C. SP-C-rtTA+/tg mice were identified by PCR amplification as previously described (23Mucenski M.L. Wert S.E. Nation J.M. Loudy D.E. Huelsken J. Birchmeier W. Morrisey E.E. Whitsett J.A. J. Biol. Chem. 2003; 278: 40231-40238Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Double transgenic mice (SP-C-rtTA+/tg, (tetO)7-CMV-MK+/tg or -tg/tg) were created by crossing the SP-C-rtTA+/tg transgenic mice with (tetO)7-CMV-MK+/tg or -tg/tg transgenic mice. MK expression was induced by feeding animals doxycycline-containing food (625 mg/kg, Harlan Teklad, Madison, WI). Transgenic and control mice were housed as described above. MK transgene expression was verified by reverse transcription (RT)-PCR in three separate transgenic lines. The experiments described here utilize mice from a single founder line that expressed MK under control of doxycycline. Immunohistochemistry—Immunohistochemical staining for MK was performed as previously described (8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). Immunohistochemical staining for α-smooth muscle actin (α-SMA) (1:20,000, Sigma) and caldesmon (1:500, Sigma) were performed using 5-μm paraffin sections. Sections were treated with 3% hydrogen peroxide in methanol for 15 min. Staining was performed following the manufacturer's instructions using a “Mouse on Mouse” (M.O.M.) monoclonal antibody kit (Vector Laboratories, Burlingame, CA), and sections were development in 3,3-diamino-benzidine reagent (Sigma). Bromodeoxyuridine immunostaining was performed as previously described (23Mucenski M.L. Wert S.E. Nation J.M. Loudy D.E. Huelsken J. Birchmeier W. Morrisey E.E. Whitsett J.A. J. Biol. Chem. 2003; 278: 40231-40238Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In Vitro Exposure to Hypoxia—Human placental adenocarcinoma (JEG-3), mouse fetal lung mesenchyme (MFLM-4) (24Akeson A.L. Wetzel B. Thompson F.Y. Brooks S.K. Paradis H. Gendron R.L. Greenberg J.M. Dev. Dyn. 2000; 217: 11-23Crossref PubMed Scopus (63) Google Scholar), and human pulmonary adenocarcinoma (H-441) cell lines were maintained in minimal essential medium, Dulbecco's modified Eagle's medium, and RPMI, respectively, containing 10% fetal calf serum, 2 mm glutamine, and antibiotics. Cells were routinely cultured in 5% CO2, 95% air (normoxic conditions) at 37 °C. At 80-90% confluence, cells were split and plated in 35-mm dishes and allowed to grow in normoxia for 18 h. Cells were placed into an incubator in which FiO2 was regulated (Thermo Forma Series II Water Jacketed CO2 Incubator, model 3100, Marietta, OH). Cells were exposed to a mixture of 5% O2, 90% N2, and 5% CO2 for 4 h. Following the manufacturer's instructions, cells were rapidly lysed, and total RNA was isolated using the Absolutely RNA® RT-PCR Miniprep kit (Stratagene, La Jolla, CA). In Vitro Addition of MK Protein—MFLM-4 cells were maintained as outlined above. At 80-90% confluence, cells were split and plated in 35-mm dishes. After 6 h, 10 μg/ml recombinant human MK (rhMK 258-MD, R&D Systems, Minneapolis, MN) in Dulbecco's modified Eagle's medium containing 3% fetal calf serum was added. Cells were lysed, and RNA was isolated as outlined previously after 24 h in culture. RT-PCR Analysis—Total RNA was isolated from wild type and double transgenic lung using Trizol® reagent (Invitrogen) and from cells grown in culture by using the Absolutely RNA® RT-PCR Miniprep Kit (Stratagene) following the manufacturers' instructions. 2-μg aliquots of total RNA were treated with 1 μl each of RNasin (Promega, Madison, WI) and DNase I (Invitrogen) at room temperature for 15 m. The DNase I was heat-denatured at 90 °C for 10 min, and reverse transcription was performed with SuperScript™II-RT (Invitrogen) according to the manufacturer's protocol. PCR was performed using 2-μl aliquots of the generated cDNA using Taq polymerase (Roche Applied Science). Products were electrophoresed on a 1.5% agarose gel with appropriate molecular weight standards. Primers used for the PCR reactions include MK (forward (5′-GCC TGG AAA GTG GGA CAA GAT G-3′) and reverse (5′-GGA AGA CAA AAG GCA CTG GTG G-3′)), HIF-1α (forward (5′-GTT TCT GCT GCC TTG TAT AGG AGC AAT TA-3′) and reverse (5′-AAA GTT CAC CTG AGC CTA ATA GTC CCA G-3′)), calponin (25Miano J.M. Olson E.N. J. Biol. Chem. 1996; 271: 7095-7103Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), SM-22 (26Li L. Miano J.M. Cserjesi P. Olson E.N. Circ. Res. 1996; 78: 188-195Crossref PubMed Scopus (340) Google Scholar), α-SMA (27Arakawa E. Hosegawa K. Yanai N. Obinata M. Matsuda Y. FEBS Lett. 2000; 481: 193-196Crossref PubMed Scopus (43) Google Scholar), myocardin (28Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (310) Google Scholar), and glyceraldehyde-3-phosphate dehydrogenase (28Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (310) Google Scholar). PCR parameters included an initial heating at 95 °C for 5 min. MK was amplified by 35 cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s. HIF-1α was amplified by 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 m. Both were followed by a 7-min extension at 72 °C. All other primer pairs were utilized as outlined (25Miano J.M. Olson E.N. J. Biol. Chem. 1996; 271: 7095-7103Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 26Li L. Miano J.M. Cserjesi P. Olson E.N. Circ. Res. 1996; 78: 188-195Crossref PubMed Scopus (340) Google Scholar, 27Arakawa E. Hosegawa K. Yanai N. Obinata M. Matsuda Y. FEBS Lett. 2000; 481: 193-196Crossref PubMed Scopus (43) Google Scholar, 28Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (310) Google Scholar). Real-time RT-PCR—Total RNA was isolated from mouse lungs and reverse-transcribed to cDNA. Oligonucleotide SYBR green primer pairs for myocardin (28Yoshida T. Sinha S. Dandre F. Wamhoff B.R. Hoofnagle M.H. Kremer B.E. Wang D.Z. Olson E.N. Owens G.K. Circ. Res. 2003; 92: 856-864Crossref PubMed Scopus (310) Google Scholar) and β-actin (5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′) were generated. Quantitative fluorogenic amplification of cDNA was performed in the Smart Cycler® processing block, model SC1000-1 (Cepheid, Sunnyvale, CA) and by using the LightCycler-DNA Master SYBR Green I kit (Roche Applied Science). The relative abundance of mRNA was determined from standard curves generated from the amplification from serially diluted standard pools of cDNA and normalized to β-actin mRNA. Plasmid Construction and Mutagenesis—The entire 2.5-kilobase (p2.5MK-luc) and a 5′-truncated 1.7-kilobase (p1.7MK-luc) mouse MK promoter was directionally cloned into the pGL3-basic reporter plasmid (Promega) and verified by sequencing as previously described (8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). Site-directed mutagenesis of the two putative HREs in the p2.5MK-luc reporter construct was performed by using the QuikChange™ site-directed mutagenesis kit (Stratagene). Synthetic oligonucleotides containing the desired mutation (RCGTG → CTTGC) were extended during a PCR reaction to generate promoters that contained a mutant 5′ HRE, p2.5MK(Δ5′HRE)-luc, and a mutant 3′ HRE, p2.5MK(Δ3′HRE)-luc. All constructs were verified by sequencing. Transfection and Reporter Gene Assays—Functional assays of reporter gene constructs were performed in triplicate using transient transfection of JEG-3 as previously outlined (8Reynolds P.R. Mucenski M.L. Whitsett J.A. Dev. Dyn. 2003; 227: 227-237Crossref PubMed Scopus (39) Google Scholar). Plasmid concentrations included 500 ng/μl pRSV-βGAL, 100 ng/μl p2.5MK-luc or p1.7MK-luc, 80-480 ng/μl pCMV-HIF-1α, and pCDNA control vector to bring the total amount of DNA in each transfection to 1.08 μg. β-Galactosidase assays were performed as previously described (29Bohinski R.J. DiLauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar). Reporter assays were normalized for transfection efficiency based on the β-galactosidase activity. Morphometric Analysis—Mice were killed as previously described (23Mucenski M.L. Wert S.E. Nation J.M. Loudy D.E. Huelsken J. Birchmeier W. Morrisey E.E. Whitsett J.A. J. Biol. Chem. 2003; 278: 40231-40238Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). At the time of necropsy, hearts were removed and dissected. The right ventricle (RV) was separated from the left ventricle and septum (LV+S) and weighed on an analytical balance. Ratios of RV weight/LV+S weight and RV weight/body weight are represented. Lungs were inflation fixed, washed in phosphate-buffered saline, dehydrated in a series of alcohols, and embedded in paraffin (30Wert S.E. Yoshida M. LeVine A.M. Ikegami M. Jones T. Ross G.F. Fisher J.H. Korfhagen T.R. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5972-5977Crossref PubMed Scopus (362) Google Scholar). To assess muscularization of the peripheral pulmonary vessels, lung sections were immunohistochemically stained with an α-SMA monoclonal antibody as described above. In each randomized 60× field, vessels (15-50-μm external diameter) were classified as fully muscularized (actin staining >75% of the circumference), partially muscularized (actin staining 25-50% of the circumference), or non-muscularized (actin staining <25% of the circumference) as previously described (31Zaidi S.H. You X.M. Ciura S. Husain M. Rabinovitch M. Circulation. 2002; 105: 516-521Crossref PubMed Scopus (139) Google Scholar). The percentage of peripheral pulmonary blood vessels that were fully or partially muscularized was calculated, and t tests were performed to demonstrate significant differences between double transgenic and non-transgenic littermates at the p ≤ 0.05 level. In Vivo Exposure to Hypoxia—Double transgenic and non-transgenic mice were placed in a normobaric chamber and exposed to FiO2 of 0.10 for 5 weeks. Normobaric hypoxia was achieved by displacement with N2 regulated by a Pro-Ox Model 110 unit (Biospherix, Ltd., Redfield, NY). The chamber was changed regularly with activated carbon (Fisher), Drierite (Hammond Drierite Co, Ltd., Xenia, OH), and Baralyme® (Allied Healthcare Products, Inc., St. Louis, MO) to prevent the toxic accumulation of ammonia, excess humidity, and carbon dioxide. Once a week, mice were exposed to room air for less than 20 min to obtain body weights, clean cages, and/or replenish food and water. Pulmonary Arteriograms, Histology, and Arterial Density Counts—Adult mice were sacrificed with a sodium pentobarbital (26%) euthanasia solution (Fort Dodge Animal Health, Fort Dodge, IA), and lungs were infused with a heated solution of gelatin and barium through the pulmonary artery as previously described (32Le Cras T.D. Hardie W.D. Fagan K. Whitsett J.A. Korfhagen T.R. Am. J. Physiol. 2003; 285: L1046-L1054PubMed Google Scholar). Pulmonary arterial architecture was imaged by x-ray radiography. The left lungs were subsequently embedded in paraffin and sectioned as previously described. To determine arterial density, barium-filled pulmonary arteries were counted by a blinded observer in randomly selected high-powered (60×) fields of distal lung. Fields containing large airways and/or large vessels were excluded. All vessels were counted, and five high-powered fields were counted per animal. Western Blot Analysis—Western blot analysis was performed with the following primary antibodies: a rabbit polyclonal antibody to Tie-2 diluted 1:2000 and a goat polyclonal antibody to platelet endothelial cellular adhesion molecule diluted 1:2000 (Santa Cruz Biotechnology, Santa Cruz, CA). Lung homogenates (25 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked overnight at 4 °C in 5% nonfat dried milk. Immunodetection was performed by incubating the membranes with the primary antibody diluted in blocking buffer for 1 h at room temperature. After washing, a secondary horseradish peroxidase-conjugated antibody (Santa Cruz) was diluted in blocking buffer (1:10,000) and applied for 60 min. After three washes, ECL Plus detection (Amersham Biosciences) was performed, and auto-radiographs were taken. Increased MK Expression during Hypoxia in Vivo and in Vitro—CAST/eiJ mice, an inbred strain, develop vascular remodeling and cor pulmonale during hypoxia (FiO2 of 0.10), whereas FVB/N mice are resistant to hypoxia. MK was not induced in the lungs of adult FVB/N mice during hypoxia (Fig. 1, A and B). Although MK was barely detectable in the lungs of adult CAST/eiJ mice housed in normoxic conditions, intense staining of MK was observed in alveolar epithelial cells and pulmonary vasculature of adult CAST/eiJ mice exposed to hypoxia (FiO2 of 0.10) for 4 weeks (Fig. 1, C and D). MK staining was observed in the peripheral pulmonary epithelium and smooth muscle of the pulmonary vasculature (Fig. 1, E and F). Intensity of staining with MK increased during exposure to hypoxia (Fig. 1, G and L). MK staining was induced in the epithelial cells lining proximal bronchioles after 1 week in hypoxia (Fig. 1, G and I). When hypoxic exposure was lengthened to 2 weeks MK staining was observed in the alveolar regions of the lung parenchyma (Fig. 1J). After exposure to hypoxia for 3 and 4 weeks MK staining was observed throughout the lung and was detected in epithelial cells of the conducting and peripheral airways and in the smooth muscle cells of the peripheral pulmonary blood vessels (Fig. 1, K and L). MK mRNA was detected in the lungs of CAST/eiJ mice after hypoxic exposure for 1 week, and expression steadily increased as the hypoxic exposure lengthened to 4 weeks (Fig. 2). Increasing MK expression was associated with increased immunostaining for α-SMA (Fig. 1F) and increased α-SMA mRNA (not shown). Thus, hypoxia induced MK expression in the lungs of hypoxemia-susceptible CAST/eiJ mice.Fig. 2MK mRNA expression in CAST/eiJ mice during hypoxia. CAST/eiJ mice were exposed to hypoxia (FiO2 of 0.10) for 0, 1, 2, 3, or 4 weeks, and whole lung RNA was isolated and reverse-transcribed to cDNA. MK mRNA was detected by RT-PCR, and relative expression levels were compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.View Large Image Figure ViewerDownload (PPT) The effect of hypoxia on MK expression was assessed in various cell lines including human placental adenocarcinoma (JEG-3), mouse fetal lung mesenchyme (MFLM-4), and human pulmonary adenocarcinoma (H-441). HIF-1α and MK mRNA expression were increased by hypoxia in all of the cell lines tested (Fig. 3). HIF-1α Regulates MK Transcription in Vitro—Because HIF-1α is a known transcriptional mediator of response to hypoxia, effects of HIF-1α on MK transcription were assessed. JEG-3 cells were co-transfected with a luciferase-reporter plasmid containing 2.5 kilobases of the 5′-region of the mouse MK gene (p2.5MK-luc) and an expression vector containing HIF-1α cDNA. HIF-1α activated the MK promoter (Fig. 4) and acted in an additive manner when co-transfected with TTF-1 (Fig. 4B). There are two potential HREs in the 2.5MK-luc plasmid, located at -1701 to -1697 (5′-HRE) and +48 to +52 (3′-HRE). Deletion of the 5′-HRE in the promoter (p1.7MK-luc) did not inhibit HIF-1α responses (Fig. 4C). To identify HREs that mediate HIF-1α-induced MK transcription, site-directed mutagenesis was performed on the HREs located at -1701 to -1697 and +48 to +52, generating p2.5MK(Δ5′HRE)-luc and p2.5MK(Δ3′HRE)-luc, respectively (Fig. 4D). Mutation of the 5′-HRE did not significantly inhibit transactivation by HIF-1α. In contrast, HIF-1α did not induce MK transcription when the 3′-HRE was mutated (Fig. 4D), demonstrating that the proximal HRE regulates HIF-1α-induced expression of MK. Inducible Expression of MK in the Murine Lung—To assess the effects of chronic MK expression during lung morphogenesis, mouse MK was expressed in the respiratory epithelium of transgenic mice using the human surfactant protein C promoter (21Wert S.E. Glasser S.W. Korfhagen T.R. Whitsett J.A. Dev. Biol. 1993; 156: 426-443Crossref PubMed Scopus (273) Google Scholar). The human surfactant protein C promoter mediates respiratory epithelial cell-specific expression of the rtTA, which binds doxycycline and induces MK expression from tet-O binding sites that activate a minimal CMV promoter (Fig. 5A). Expression and regulation of the trans" @default.
• W2098840758 created "2016-06-24" @default.
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• W2098840758 creator A5009991971 @default.
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• W2098840758 date "2004-08-01" @default.
• W2098840758 modified "2023-10-17" @default.
• W2098840758 title "Midkine Is Regulated by Hypoxia and Causes Pulmonary Vascular Remodeling" @default.
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