Inhibitor of Differentiation 1 Promotes Endothelial Survival in a Bleomycin Model of Lung Injury in Mice
2007; Elsevier BV; Volume: 171; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.070226
ISSN1525-2191
AutoresHuimin Zhang, William E. Lawson, Vasiliy V. Polosukhin, Ambra Pozzi, Timothy S. Blackwell, Ying Litingtung, Chin Chiang,
Tópico(s)Cancer-related molecular mechanisms research
ResumoThe Id family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in diverse cellular processes such as proliferation, apoptosis, differentiation, and migration. However, the specific role of Id1 in lung injury has not been investigated. Bleomycin has been widely used to generate animal models of acute lung injury and fibrogenesis. In this study we found that, on bleomycin challenge, Id1 expression was significantly up-regulated in the lungs, predominantly in endothelial cells, as revealed by double immunolabeling and quantitative flow cytometric analysis. Mice with Id1 loss-of-function (Id1−/−) displayed increased vascular permeability and endothelial apoptosis in the lungs after bleomycin-induced injury. Cultured Id1−/− lung microvascular endothelial cells also showed decreased survival when exposed to bleomycin. We detected a decrease in the level of Bcl-2, a primary anti-apoptotic protein, in Id1−/− endothelial cells, suggesting that down-regulated Bcl-2 may promote endothelial apoptosis in the lung. Therefore, we propose that Id1 plays a crucial role in promoting endothelial survival in the adult lung on injury. In addition, bleomycin-exposed Id1−/− mice showed increased lung collagen accumulation and fibrogenesis, suggesting that Id1 up-regulation in the lung may play a critical role in lung homeostasis. The Id family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in diverse cellular processes such as proliferation, apoptosis, differentiation, and migration. However, the specific role of Id1 in lung injury has not been investigated. Bleomycin has been widely used to generate animal models of acute lung injury and fibrogenesis. In this study we found that, on bleomycin challenge, Id1 expression was significantly up-regulated in the lungs, predominantly in endothelial cells, as revealed by double immunolabeling and quantitative flow cytometric analysis. Mice with Id1 loss-of-function (Id1−/−) displayed increased vascular permeability and endothelial apoptosis in the lungs after bleomycin-induced injury. Cultured Id1−/− lung microvascular endothelial cells also showed decreased survival when exposed to bleomycin. We detected a decrease in the level of Bcl-2, a primary anti-apoptotic protein, in Id1−/− endothelial cells, suggesting that down-regulated Bcl-2 may promote endothelial apoptosis in the lung. Therefore, we propose that Id1 plays a crucial role in promoting endothelial survival in the adult lung on injury. In addition, bleomycin-exposed Id1−/− mice showed increased lung collagen accumulation and fibrogenesis, suggesting that Id1 up-regulation in the lung may play a critical role in lung homeostasis. The Id family of genes consists of four members (Id1 to Id4) encoding helix-loop-helix transcriptional regulators that lack the basic domain. They function primarily by binding to and inhibiting the transcriptional activities of basic helix-loop-helix or Ets transcription factors.1Ruzinova MB Benezra R Id proteins in development, cell cycle and cancer.Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar One function of Id proteins is to inhibit myofibroblast differentiation by disrupting transcription complex formation involving basic helix-loop-helix proteins.2Sun XH Copeland NG Jenkins NA Baltimore D Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins.Mol Cell Biol. 1991; 11: 5603-5611Crossref PubMed Scopus (532) Google Scholar Another important role of Id proteins is their active engagement in cell-cycle regulation as well as promoting cell survival and delaying onset of cellular senescence.3Zebedee Z Hara E Id proteins in cell cycle control and cellular senescence.Oncogene. 2001; 20: 8317-8325Crossref PubMed Google Scholar Id proteins have been implicated in embryonic development and cancer by playing key roles in cellular processes such as proliferation, apoptosis, differentiation, or migration of various cell types, notably endothelial and neural precursor cells.1Ruzinova MB Benezra R Id proteins in development, cell cycle and cancer.Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 4Benezra R Rafii S Lyden D The Id proteins and angiogenesis.Oncogene. 2001; 20: 8334-8341Crossref PubMed Google Scholar, 5Wong YC Wang X Ling MT Id-1 expression and cell survival.Apoptosis. 2004; 9: 279-289Crossref PubMed Scopus (100) Google Scholar, 6Lyden D Young AZ Zagzag D Yan W Gerald W O'Reilly R Bader BL Hynes RO Zhuang Y Manova K Benezra R Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts.Nature. 1999; 401: 670-677Crossref PubMed Scopus (781) Google Scholar, 7Engel I Murre C The function of E- and Id proteins in lymphocyte development.Nat Rev Immunol. 2001; 1: 193-199Crossref PubMed Scopus (165) Google Scholar Several lines of evidence have suggested that Id proteins play a key role in endothelial function and homeostasis. For example, Id proteins can regulate angiogenesis and promote endothelial survival during embryonic development and in tumor progression.4Benezra R Rafii S Lyden D The Id proteins and angiogenesis.Oncogene. 2001; 20: 8334-8341Crossref PubMed Google Scholar Mice lacking Id1 and Id3 functions displayed brain hemorrhage during embryonic development and defects in tumor-promoted angiogenesis.6Lyden D Young AZ Zagzag D Yan W Gerald W O'Reilly R Bader BL Hynes RO Zhuang Y Manova K Benezra R Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts.Nature. 1999; 401: 670-677Crossref PubMed Scopus (781) Google Scholar Deregulated Id1 expression in endothelial cells substantially affected angiogenesis and tumor growth in various tumor models.8Iavarone A Lasorella A Id proteins in neural cancer.Cancer Lett. 2004; 204: 189-196Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 9Li H Gerald WL Benezra R Utilization of bone marrow-derived endothelial cell precursors in spontaneous prostate tumors varies with tumor grade.Cancer Res. 2004; 64: 6137-6143Crossref PubMed Scopus (70) Google Scholar, 10de Candia P Benera R Solit DB A role for Id proteins in mammary gland physiology and tumorigenesis.Adv Cancer Res. 2004; 92: 81-94Crossref PubMed Scopus (49) Google Scholar, 11Ling MT Lau TC Zhou C Chua CW Kwok WK Wang Q Wang X Wong YC Overexpression of Id-1 in prostate cancer cells promotes angiogenesis through the activation of vascular endothelial growth factor (VEGF).Carcinogenesis. 2005; 26: 1668-1676Crossref PubMed Scopus (107) Google ScholarIn vitro studies also demonstrated that overexpression of Id1 reduced human endothelial cell apoptosis rate.12Nishiyama K Takaji K Kataoka K Kurihara Y Yoshimura M Kato A Ogawa H Kurihara H Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis.Circulation. 2005; 112: 2840-2850Crossref PubMed Scopus (54) Google Scholar Id1 has also been shown to delay endothelial senescence by suppressing CDK inhibitor expressions and may be an important component of the cellular stress response pathway.13Alani RM Young AZ Shifflett CB Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a.Proc Natl Acad Sci USA. 2001; 98: 7812-7816Crossref PubMed Scopus (185) Google Scholar, 14Sharpless NE Bardeesy N Lee KH Carrasco D Castrillon DH Aguirre AJ Wu EA Horner JW DePinho RA Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.Nature. 2001; 413: 86-91Crossref PubMed Scopus (670) Google Scholar, 15Tang J Gordon GM Nickoloff BJ Foreman KE The helix-loop-helix protein id-1 delays onset of replicative senescence in human endothelial cells.Lab Invest. 2002; 82: 1073-1079Crossref PubMed Scopus (62) Google Scholar Transplantation of Id1-overexpressing human umbilical vein endothelial cells into mice increased capillary density and limb salvage rate, indicating involvement of Id1 in endothelial repair.12Nishiyama K Takaji K Kataoka K Kurihara Y Yoshimura M Kato A Ogawa H Kurihara H Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis.Circulation. 2005; 112: 2840-2850Crossref PubMed Scopus (54) Google Scholar Up-regulation of Id1 expression in endothelial cells was also detected during hypoxic vascular remodeling in pulmonary hypertension, suggesting a contribution of Id1 in maintaining endothelial homeostasis.16Frank DB Abtahi A Yamaguchi DJ Manning S Shyr Y Pozzi A Baldwin HS Johnson JE de Caestecker MP Bone morphogenetic protein 4 promotes pulmonary vascular remodeling in hypoxic pulmonary hypertension.Circ Res. 2005; 97: 496-504Crossref PubMed Scopus (120) Google Scholar Id1 is highly expressed in the lung mesenchyme during embryogenesis,17Jen Y Manova K Benezra R Expression patterns of Id1, Id2, and Id3 are highly related but distinct from that of Id4 during mouse embryogenesis.Dev Dyn. 1996; 207: 235-252Crossref PubMed Scopus (167) Google Scholar but its expression was detected at basal level in the adult murine lung (this work),18Chambers RC Leoni P Kaminski N Laurent GJ Heller RA Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching.Am J Pathol. 2003; 162: 533-546Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar suggesting that Id1 might play a role in regulating adult lung homeostasis. Bleomycin has been widely used in animal models of acute lung injury and fibrosis, and mice treated with a single dose of bleomycin intratracheally displayed massive epithelial and endothelial cell damage followed by fibrogenesis.19Chua F Gauldie J Laurent GJ Pulmonary fibrosis: searching for model answers.Am J Respir Cell Mol Biol. 2005; 33: 9-13Crossref PubMed Scopus (225) Google Scholar, 20Zhang HY Gharaee-Kermani M Zhang K Karmiol S Phan SH Lung fibroblast alpha-smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis.Am J Pathol. 1996; 148: 527-537PubMed Google Scholar, 21Cutroneo KR Phan SH TGF-beta1-induced Smad 3 binding to the Smad 7 gene: knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF-β1 secretion: bleomycin acts through TGF-β1.J Cell Biochem. 2003; 89: 474-483Crossref PubMed Scopus (18) Google Scholar, 22Desmoulière A Geinoz A Gabbiani F Gabbiani G Transforming growth factor-β1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.J Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1880) Google Scholar, 23Nakao A Fujii M Matsumura R Kumano K Saito Y Miyazono K Iwamoto I Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice.J Clin Invest. 1999; 104: 5-11Crossref PubMed Scopus (387) Google Scholar, 24Bonniaud P Margetts PJ Kolb M Schroeder JA Kapoun AM Damm D Murphy A Chakravarty S Dugar S Higgins L Protter AA Gauldie J Progressive transforming growth factor β1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor.Am J Respir Crit Care Med. 2005; 171: 889-898Crossref PubMed Scopus (223) Google Scholar, 25Chen J Stubbe J Bleomycins: towards better therapeutics.Nat Rev Cancer. 2005; 5: 102-112Crossref PubMed Scopus (502) Google Scholar Up-regulation of Id1 in bleomycin-treated rat lungs has been reported although its detailed tissue distribution and specific functions were not investigated.18Chambers RC Leoni P Kaminski N Laurent GJ Heller RA Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching.Am J Pathol. 2003; 162: 533-546Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar In this study, we found that on bleomycin-induced injury, Id1 is up-regulated predominantly in endothelial cells, suggesting a potential role of Id1 in these cell types. Loss of Id1 function in the lung endothelium resulted in increased vascular permeability and endothelial cell death after bleomycin instillation. In agreement, we found that bleomycin-treated Id1−/− lung microvascular endothelial cells showed decreased survival in culture with significant reduction in the level of anti-apoptotic protein Bcl-2. Lung fibrosis is characterized by an initial accumulation of inflammatory cells, epithelial and endothelial injury and apoptosis, fibroblast proliferation, myofibroblast accumulation, and increased deposition of extracellular matrix proteins resulting in irreversible distortion of lung architecture.26Phan SH Fibroblast phenotypes in pulmonary fibrosis.Am J Respir Cell Mol Biol. 2003; 29: S87-S92PubMed Google Scholar, 27Phan SH The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (410) Google Scholar, 28Kuhn III, C Boldt J King Jr, TE Crouch E Vartio T McDonald JA An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.Am Rev Respir Dis. 1989; 140: 1693-1703Crossref PubMed Scopus (377) Google Scholar, 29Zhang K Rekhter MD Gordon D Phan SH Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study.Am J Pathol. 1994; 145: 114-125PubMed Google Scholar, 30Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3159) Google Scholar, 31Selman M King TE Pardo A Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med. 2001; 134: 136-151Crossref PubMed Scopus (1546) Google Scholar Loss of Id1 function also resulted in increased susceptibility to fibrogenesis possibly because of increased endothelial damage. Collectively, our studies reveal a new function of Id1 in the lung endothelium in promoting the survival of pulmonary endothelial cells on bleomycin-induced lung injury. Id1-null mice (Id1−/−) (gift of Dr. Robert Benezra, Memorial Sloan-Kettering Cancer Center, New York, NY) and Tie1-Cre mice32Gustafsson E Brakebusch C Hietanen K Fassler R Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice.J Cell Sci. 2001; 114: 671-676PubMed Google Scholar were bred in the C57BL/6J background, and ShhCre-ZEG mice33Li Y Zhang H Litingtung Y Chiang C Cholesterol modification restricts the spread of Shh gradient in the limb bud.Proc Natl Acad Sci USA. 2006; 103: 6548-6553Crossref PubMed Scopus (129) Google Scholar were bred in the C57BL/6J;129 background. For the Id1 time-course study, C57BL/6J mice (8 to 10 weeks of age) were purchased from the Jackson Laboratory (Bar Harbor, ME). Id1−/− mice were backcrossed for five generations to C57BL/6 background, and pairs of wild-type and Id1−/− littermates from Id1+/− matings were used for studies. Mice were treated with either saline or bleomycin (0.08 U) by intratracheal injection in a total volume of 50 μl of saline.34Lawson WE Polosukhin VV Zoia O Stathopoulos GT Han W Plieth D Loyd JE Neilson EG Blackwell TS Characterization of fibroblast-specific protein 1 in pulmonary fibrosis.Am J Respir Crit Care Med. 2005; 171: 899-907Crossref PubMed Scopus (153) Google Scholar The experimental protocol was reviewed and approved by the Institutional Animal Care and Utilization Committee at Vanderbilt University. ShhCre-GFP embryonic lungs were fixed in 4% paraformaldehyde for 5 hours at 4°C and embedded in OCT cryoprotectant embedding medium. Cryosections at 15 μm were collected and immunostained with PECAM-1 antibody (BD Pharmingen, San Diego, CA) followed by Alexa 568-conjugated secondary antibody (Molecular Probes, Eugene, OR) for signal visualization. ShhCre-GFP adult lungs were perfused using phosphate-buffered saline (PBS), then inflated and fixed in 4% paraformaldehyde for 5 hours at 4°C. OCT was subsequently injected intratracheally into fixed lung to preserve the lung architecture. Lungs were embedded in OCT and 15-μm sections were collected and green fluorescent protein (GFP) fluorescence visualized using a BX60F5 microscope (Olympus, Center Valley, PA). Adult lungs were perfused, inflated, excised, and fixed in 4% paraformaldehyde at 4°C overnight. Subsequently, lungs were embedded in paraffin blocks and 5-μm sections were collected and processed for immunolabeling. Antibodies against Id1 (clone C-20; Santa Cruz Biotechnology, Santa Cruz, CA), smooth muscle α-actin (α-SMA; Sigma Chemical, St. Louis, MO), CD34 (Labvision, Fremont, CA), and β-galactosidase (LacZ) (Sigma) were used for immunostaining. For general immunolabelings, slides were antigen-retrieved using citrate buffer (pH 6.0) and incubated at 4°C overnight with primary antibody. Alexa-conjugated secondary antibodies or horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and horseradish peroxidase detection kit (Labvision) were used for signal visualization. For Id1 immunolabeling, lungs were perfused with PBS and fixed in EFA solution (100% ethanol, 37% formaldehyde, and 100% acetic acid at v/v/v ratio of 6:3:1) at 4°C for 5 hours. Lungs were subsequently dehydrated and embedded in paraffin blocks and 5-μm sections were collected and processed for immunolabeling. Slides were incubated at 4°C overnight with primary antibody, Id1, at 1:6000. Detection was performed using polymer-horseradish peroxidase secondary antibodies (Zymed, South San Francisco, CA) diluted at 1:4 and visualized using the TSA Plus Fluorescence System (Perkin-Elmer, Emeryville, CA) diluted at 1:200. Slides were counterstained with TO-PRO-3 (Invitrogen, Carlsbad, CA) to highlight nuclei. For double labelings involving Id1, sequential immunostainings were performed instead of a one-step double labeling. Confocal images were taken using the Zeiss Upright LSM510 confocal microscope (Carl Zeiss, Thornwood, NY) at the Vanderbilt Cell Imaging Core. Regular images were taken using the Olympus BX60F5 microscope. Left lungs of bleomycin-treated wild-type and Id1−/− mice were harvested and homogenized in radioimmunoprecipitation assay lysis buffer at pH 7.4. Protein lysates of 100 μg each were resolved on sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA). Primary antibodies against Id1 or α-SMA were used for detection. Equal loading of protein samples was monitored and normalized to the level of α-tubulin (Calbiochem-EMD Biosciences, La Jolla, CA). Blots were analyzed using QuantityOne software (Bio-Rad). For Western blotting using fluorescence-activated cell sorting (FACS)-sorted cells, cells were immediately frozen in liquid nitrogen after collection. Sorted cells were lysed in radioimmunoprecipitation assay buffer and loaded at 50 μg/lane on sodium dodecyl sulfate-polyacrylamide gels. Additional antibodies used for blotting were PECAM-1 (BD Pharmingen), TTF-1 (Labvision), Bcl-2, Bcl-xL (Santa Cruz Biotechnology), p-MEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 (Cell Signaling Technology, Beverly, MA). Blots were scanned and quantified as described above. Vascular permeability was examined using the Evans blue extravasation method as previously described.35Londhe VA Belperio JA Keane MP Burdick MD Xue YY Strieter RM CXCR2 is critical for dsRNA-induced lung injury: relevance to viral lung infection.J Inflamm (Lond). 2005; 2: 4Crossref PubMed Scopus (28) Google Scholar In brief, Evans blue dye at 20 mg/kg body weight was injected into each animal via the retro-orbital sinus. Three hours after injection, lungs were perfused and homogenized in saline. Evans blue was extracted and quantified by dual wavelength at 620 and 740 nm using Bio-Rad Smartspec3000. Corrected pulmonary Evans blue absorbance at 620 nm was calculated as A620 nm (1.426 × A740 nm + 0.03). Permeability index was generated by dividing the corrected pulmonary Evans blue absorbance by the plasma Evans blue absorbance at 620 nm. Adult lung microvascular endothelial cells were isolated and cultured as previously described.36Pozzi A Moberg PE Miles LA Wagner S Soloway P Gardner HA Elevated matrix metalloprotease and angiostatin levels in integrin α1 knockout mice cause reduced tumor vascularization.Proc Natl Acad Sci USA. 2000; 97: 2202-2207Crossref PubMed Scopus (349) Google Scholar In brief, lungs were perfused with 0.25% trypsin (Mediatech, Herndon, VA) and 2 μg/ml collagenase (Roche Applied Science. Indianapolis, IN). Perfused lungs were incubated at 37°C for 20 minutes. Then lung lobes were trimmed with a sterile scalpel and washed 10 to 20 times with 1 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Detached cells were collected and centrifuged at 2000 rpm. The cell pellet was washed once and resuspended in EGM2-MV medium supplemented with vascular endothelial growth factor (Cambrex, Walkersville, MD) and 2% fetal bovine serum. Cells were grown in six-well dishes (BD Falcon, Franklin Lakes, NJ) or coverslips (Fisher, Pittsburgh, PA) for 3 days before bleomycin treatment. Vascular endothelial growth factor and fetal bovine serum were removed during bleomycin incubation. All cells were maintained at 37°C and 5% CO2 in a Hera cell incubator unit (Kendro Laboratories, Waltham, MA). Cells from triplicate wells were harvested for immunodetection by Western blotting as above. For cell death detection in lung tissue sections, paraformaldehyde-fixed paraffin sections were boiled in citrate buffer (pH 6.0) for 20 minutes, and apoptotic cells were detected by TUNEL using the In Situ cell death detection kit (Chemicon, Temecula, CA) according to the manufacturer's protocol. Detection was performed using a horseradish peroxidase-conjugated secondary antibody and visualized using the TSA Plus fluorescence system (Perkin-Elmer). Slides were subsequently double-stained with CD34 to mark endothelial cells. For TUNEL staining of cultured endothelial cells, freshly isolated lung microvascular endothelial cells were grown in culture dishes for 3 days before treatment with 250 ng/ml bleomycin for 6 hours. The bleomycin-containing media were then removed and replaced with fresh EGM2-MV media for 3 hours. The cells were subsequently stained with TUNEL according to the manufacturer's protocol. For FACS analysis on freshly isolated murine lung microvascular endothelial cells, saline- or bleomycin-treated lungs were perfused with 25 U/ml dispase (BD Biosciences) plus 2 μg/ml collagenase (Roche Applied Science) then incubated in digestive solution at 37°C for 20 minutes. The lungs were then minced, and a single-cell suspension was obtained by passing cells through a 40-μm cell strainer (BD Falcon). After centrifugation at 1000 rpm for 5 minutes, collected cells were resuspended in red cell lysis buffer37Lorimore SA Coates PJ Scobie GE Milne G Wright EG Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?.Oncogene. 2001; 20: 7085-7095Crossref PubMed Scopus (319) Google Scholar and incubated at room temperature for 10 minutes. The cells were then washed twice with PBS and stained with apoptotic marker using the annexin-5 apoptosis detection kit (Biovision, Mountain View, CA) according to the manufacturer's protocol. For FACS analysis on cultured lung microvascular endothelial cells, freshly isolated lung microvascular endothelial cells were grown in a culture dish for 3 days before treatment with 250 ng/ml bleomycin for 6 hours. Cells were collected by dispase/collagenase digestion and labeled with annexin-5 to mark apoptotic cells. FACS sorting was performed at the Vanderbilt Flow Cytometry Facility. Lungs were perfused, inflated (20 cmH2O), excised, and fixed in 4% paraformaldehyde at 4°C overnight. Subsequently, lungs were embedded in paraffin blocks, and 5-μm sections were collected and processed for hematoxylin and eosin (H&E). Slides of lung tissue were randomized and evaluated on 10 sequential, nonoverlapping fields (magnification, ×30) of lung parenchyma for each specimen. Evaluation of parenchymal distortion on H&E-stained lung sections was done by a pathologist blinded to the genotype and treatment group, using a 0 to 4 point scale, with a score of 0, normal architecture; 1, increased thickness of up to 50% of interalveolar septa; 2, thickening of >50% of interalveolar septa without formation of fibrotic foci; 3, thickening of the interalveolar septa with formation of isolated fibrotic foci; and 4, formation of multiple fibrotic foci with total or subtotal distortion of parenchymal architecture.38Lawson WE Polosukhin VV Stathopoulos GT Zoia O Han W Lane KB Li B Donnelly EF Holburn GE Lewis KG Collins RD Hull WM Glasser SW Whitsett JA Blackwell TS Increased and prolonged pulmonary fibrosis in surfactant protein C-deficient mice following intratracheal bleomycin.Am J Pathol. 2005; 167: 1267-1277Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar The left lungs of wild-type and Id1−/− mice, harvested at 2 weeks after bleomycin, were analyzed for hydroxyproline content as previously described.39Reddy GK Enwemeka CS A simplified method for the analysis of hydroxyproline in biological tissues.Clin Biochem. 1996; 29: 225-229Crossref PubMed Scopus (1065) Google Scholar In brief, the left lung lobes were weighed and homogenized in distilled water. The samples were mixed well and digested with 2 N sodium hydroxide in a total volume of 100 μl at 120°C for 20 minutes. After digestion, 900 μl of chloramine T (1.27 g of chloramine T, 20 ml of 50% n-propanol, and citrate-acetate buffer in 100 ml) was added to each sample, mixed, and left at room temperature for 25 minutes. Then, 1 ml of Ehrlich's solution (15 g of 4-dimethylaminobenzaldehyde in 100 ml of n-propanol and 70% perchloric acid at a volume ratio of 2:1) was added to each sample, mixed, and incubated for 20 minutes at 65°C. Samples were cooled for 10 minutes and then read at 550 nm on a spectrophotometer. Concentrations were calculated against a hydroxyproline standard curve. Trichrome staining of lung sections for collagen content was performed by the Vanderbilt Immunohistochemistry Core Laboratory. To assess differences among groups, statistical analyses were performed using a one-way analysis of variance with Microsoft Excel (Microsoft Corporation, Redmond, WA) and significance accepted at P < 0.05. Results are presented as mean ± SEM. Although Id1 is highly expressed in the embryonic lung mesenchyme during a period of epithelial-mesenchymal interaction,17Jen Y Manova K Benezra R Expression patterns of Id1, Id2, and Id3 are highly related but distinct from that of Id4 during mouse embryogenesis.Dev Dyn. 1996; 207: 235-252Crossref PubMed Scopus (167) Google Scholar its expression is not detectable in normal adult lung tissue sections by immunohistochemistry (Figure 1A, saline 1 week). By Western blot analysis, which is a more sensitive detection method using whole lung homogenates, we detected weak Id1 expression (Figure 1B, saline). To investigate whether Id1 expression is up-regulated on pulmonary insult, we treated 8-week-old adult wild-type mice with a single 0.08-U dose of bleomycin intratracheally and harvested lungs at 1 week after bleomycin for Id1 immunohistochemistry. Interestingly, we found significant induction of nuclear Id1 expression in the bleomycin-treated wild-type lung compared with saline control (Figure 1A, Bleo 1 week). The specificity of Id1 antibody staining was confirmed using Id1−/− lung as a negative control (Figure 1A; Bleo 1 week, Id1−/−). To evaluate the level and time course of Id1 induction in wild-type mice, we examined Id1 protein levels from lung samples collected at 1, 2, and 3 weeks after bleomycin instillation (n = 3, Figure 1B). As shown by Western blotting, Id1 expression is significantly up-regulated at 1 week after bleomycin compared with saline control, and its up-regulation is maintained for 2 and 3 weeks after bleomycin (Figure 1B, data not shown). We found that a large proportion of Id1 expression localized to lung endothelial cells as revealed by double immunolabeling with Id1 and endothelial marker CD34, which labels the capillary bed.40Balyasnikova IV Visintine DJ Gunnerson HB Paisansathan C Baughman VL Minshall RD Danilov SM Propofol attenuates lung endothelial injury induced by ischemia-reperfusion and oxidative stress.Anesth Analg. 2005; 100: 929-936Crossref PubMed Scopus (60) Google Scholar As shown in Figure 2A, Id1-positive cells displayed nuclear Id1 expression surrounded by membrane and cytoplasmic expression of CD34 (Figure 2A, arrows). There were only a few Id1-expressing cells that appeared to be CD34-negative (Figure 2A, arrowhead). To determine the fraction of Id1 protein expression level derived from endothelial cells, we performed FACS analysis of Tie1Cre-GFP-labeled endothelial cells to quantify the relative level of endothelial-derived Id1 expression by Western blotting. Tie-1 is a receptor tyrosine kinase expressed during early stages of vascular development, and Tie-1 promoter-driven Cre-GFP reporter expression specifically marks endothelial cells in the adult mice.32Gustafsson E Brakebusch C Hietanen K Fassler R Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice.J Cell Sci. 2001; 114: 671-676PubMed Google Scholar By crossing Tie1Cre to ZEG mouse, which contains a transgene harboring a conditional GFP reporter
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