Inhibitor of Differentiation Promotes Endothelial Survival in a Bleomycin Model of Lung Injury in Mice

【摘要】  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 (Id1C/C) displayed increased vascular permeability and endothelial apoptosis in the lungs after bleomycin-induced injury. Cultured Id1C/C 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 Id1C/C 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 Id1C/C mice showed increased lung collagen accumulation and fibrogenesis, suggesting that Id1 up-regulation in the lung may play a critical role in lung homeostasis.
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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.1 One function of Id proteins is to inhibit myofibroblast differentiation by disrupting transcription complex formation involving basic helix-loop-helix proteins.2 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.3 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.1,4-7
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.4 Mice lacking Id1 and Id3 functions displayed brain hemorrhage during embryonic development and defects in tumor-promoted angiogenesis.6 Deregulated Id1 expression in endothelial cells substantially affected angiogenesis and tumor growth in various tumor models.8-11 In vitro studies also demonstrated that overexpression of Id1 reduced human endothelial cell apoptosis rate.12 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.13-15 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.12 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.16
Id1 is highly expressed in the lung mesenchyme during embryogenesis,17 but its expression was detected at basal level in the adult murine lung (this work),18 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.19-25 Up-regulation of Id1 in bleomycin-treated rat lungs has been reported although its detailed tissue distribution and specific functions were not investigated.18 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 Id1C/C 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.26-31 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.

【关键词】  inhibitor differentiation promotes endothelial survival bleomycin

Materials and Methods

Mice and Bleomycin Treatment

Id1-null mice (Id1C/C) (gift of Dr. Robert Benezra, Memorial Sloan-Kettering Cancer Center, New York, NY) and Tie1-Cre mice32 were bred in the C57BL/6J background, and ShhCre-ZEG mice33 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). Id1C/C mice were backcrossed for five generations to C57BL/6 background, and pairs of wild-type and Id1C/C littermates from Id1+/C 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.34 The experimental protocol was reviewed and approved by the Institutional Animal Care and Utilization Committee at Vanderbilt University.

Immunohistochemistry

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 -actin (-SMA; Sigma Chemical, St. Louis, MO), CD34 (Labvision, Fremont, CA), and ß-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.

Western Blotting

Left lungs of bleomycin-treated wild-type and Id1C/C 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 -SMA were used for detection. Equal loading of protein samples was monitored and normalized to the level of -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.

Pulmonary Vascular Permeability Assay

Vascular permeability was examined using the Evans blue extravasation method as previously described.35 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 x 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.

Pulmonary Endothelial Cell Culture

Adult lung microvascular endothelial cells were isolated and cultured as previously described.36 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.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL)

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.

Quantification of Apoptotic Endothelial Cells by FACS Analysis

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 buffer37 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.

Histology and Pathology Scoring

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, x30) 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.38

Hydroxyproline Assay and Trichrome Staining

The left lungs of wild-type and Id1C/C mice, harvested at 2 weeks after bleomycin, were analyzed for hydroxyproline content as previously described.39 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.

Statistics

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.

Results

Although Id1 is highly expressed in the embryonic lung mesenchyme during a period of epithelial-mesenchymal interaction,17 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 Id1C/C lung as a negative control (Figure 1A ; Bleo 1 week, Id1C/C). 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).

Figure 1. Id1 expression is significantly up-regulated in the adult lung on bleomycin injury. A: Representative photographs showing sections from 8-week-old wild-type and Id1C/C lungs treated with saline or 0.08 U of bleomycin and collected after 1 week. Sections were immunostained with Id1 (red), and nuclei were counterstained with TO-PRO3 (green). Note the nuclear Id1 expression in wild-type lungs treated with bleomycin but not in saline controls. The specificity of Id1 staining is confirmed by using bleomycin-treated Id1C/C lungs as negative control. B: a: Protein extracts from 8-week-old C57BL/6 wild-type lungs or Id1C/C lungs treated with saline or 0.08 U bleomycin and collected after 1 and 2 weeks were immunoblotted with Id1 antibody. -Tubulin was used as a loading control. b: Densitometric measurements of Id1 bands were performed and normalized to the density of -tubulin bands. Original magnifications, x400.

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.40 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.32 By crossing Tie1Cre to ZEG mouse, which contains a transgene harboring a conditional GFP reporter that is activated in the presence of Cre recombinase,41 mice were indelibly marked by GFP in all lung endothelial cells. The FACS-sorted endothelial fraction was confirmed by Western blotting with endothelial marker, PECAM-1 (Figure 2B) . By Id1 Western blot analysis, we found that the GFP-positive endothelial cell population expressed a relatively much higher level of Id1 compared with the nonendothelial cell population. At 1 week after bleomycin, we determined that Id1 expression level in the lung endothelial population was up-regulated up to 11-fold compared with saline control based on normalized densitometric measurements (Figure 2B) . We also determined that 86% of total Id1 expression level was derived from the lung endothelial fraction (Figure 2B) . In contrast, bleomycin-treated nonendothelial cells displayed only approximately threefold up-regulation. This significant contribution in Id1 expression level by the endothelial cell population persisted at 2 weeks after bleomycin (Figure 2C) .

Figure 2. Id1 expression is up-regulated prominently in the injured lung endothelium. A: Representative photographs showing sections from 8-week-old wild-type lungs at 1 and 2 weeks after bleomycin. Sections were double-stained with Id1 (red) and endothelial marker CD34 (green) antibodies. Arrows in the figures point to representative Id1-expressing endothelial cells; note the membrane and cytoplasmic CD34 staining that wraps around the nuclear Id1 staining. Arrowheads point to Id1-expressing nonendothelial cells. B: Eight-week-old wild-type Tie1Cre-ZEG lungs were treated with saline or 0.08 U of bleomycin and collected after 1 week; lung cells were sorted into endothelial (GFP+) and nonendothelial (GFPC) group by FACS. Protein extracts were immunoblotted with Id1 and -tubulin as the loading control. The endothelial fraction was confirmed by immunoblotting with endothelial marker, PECAM-1. Densitometric measurements of Id1 bands were performed and normalized to the density of -tubulin bands and numeric values indicating differential levels of expression are shown at the bottom. Note the dramatic up-regulation of Id1 protein level in endothelial cells from bleomycin-treated lungs compared with the nonendothelial fraction and saline controls. C: Eight-week-old wild-type Tie1Cre-ZEG lungs were collected at 2 weeks after bleomycin or saline and then subjected to FACS sorting and Western blotting. Protein extracts were immunoblotted with Id1 and -tubulin. Densitometric measurements of Id1 bands were performed and normalized to the density of -tubulin bands. Original magnifications: x400 (A, low magnification); x1000 (A, high magnification).

To examine Id1 expression in the alveolar epithelium, we took advantage of the Sonic hedgehog (Shh)-Cre mouse line that we had generated, which marks all lung cells of epithelial origin (Figure 3A) .33 Shh is expressed early in the epithelium of the embryonic lung primordium.42-45 Mice generated by crossing Shh-Cre to ROSA26R mouse, which contains a transgene harboring a conditional LacZ reporter that is activated in the presence of Cre recombinase, are indelibly marked in all lung epithelial cells by LacZ reporter expression. Subsequently, all lung epithelial cells are marked by LacZ as shown at embryonic stages E11.5, E15.5, and in the normal adult bronchial and alveolar epithelium (Figure 3A) . By double immunolabeling, we observed that Id1-expressing cells showed very few co-localization with LacZ+ epithelial cells both in the bronchial epithelium and in the alveolar bed (Figure 3B , arrow). Most Id1-expressing cells do not co-localize with epithelial LacZ expression (Figure 3B , arrowheads). By counting 10 nonoverlapping fields of Id1 and LacZ double-stained sections, we determined that only 3% of all Id1-expressing cells were LacZ-positive. Similar to the strategy used in Figure 2 , we used ShhCre-ZEG reporter mice to selectively identify GFP-positive epithelial cells in the lung by FACS analysis; the epithelial fraction was confirmed by Western blotting with epithelial marker TTF-146 (Figure 3C) . As shown in Figure 3B , bleomycin-treated GFP-positive epithelial cells displayed less than twofold up-regulation of Id1, whereas the nonepithelial fraction displayed up to ninefold increase in Id1 expression level. These results indicate that Id1 is not significantly up-regulated in epithelial cells of bleomycin-treated lungs, which is consistent with the immunohistochemical data (Figure 3B) .

Figure 3. Id1 expression is not significantly up-regulated in bleomycin-exposed lung fibroblasts and ShhCre-GFP-marked epithelium. A: Distribution of GFP-labeled epithelial cells (green) in embryonic lungs at E11.5 and E15.5 and in the adult lung at 6 weeks. Embryonic lung sections were double-stained with endothelial marker PECAM-1 (red). Note that GFP expression is strictly confined in epithelial cells throughout development and is excluded from the PECAM-1-positive endothelial domain. GFP expression was detected in the adult bronchial epithelium (Br) and alveolar epithelial cells. Vessel (V) was negative. B: Representative photographs showing sections from 8-week-old wild-type ShhCre-R26R lungs at 1 week after bleomycin. Sections were double-stained with Id1 (red) and epithelial marker Shhcre-LacZ (green). Arrows in the figures point to representative Id1-expressing epithelial cells. Arrowheads point to Id1-expressing nonepithelial cells. C: Eight-week-old wild-type ShhCre-ZEG lungs collected at 1 week after bleomycin or saline were subjected to FACS sorting and Western blotting. Protein extracts were immunoblotted with Id1 and -tubulin. Densitometric measurements of Id1 bands were performed and normalized to the density of -tubulin bands. The epithelial fraction was confirmed by immunoblotting with epithelial marker, TTF-1. D: Representative photographs showing sections from 8-week-old wild-type lungs at 2 weeks after bleomycin. Sections were double-stained with Id1 (red) and myofibroblast marker, smooth muscle -actin (SMA) (green). Original magnifications: x200 (A); x600 (B); x400 (D).

We observed a few fibroblastic-like cells expressing Id1 as has been reported previously in the rat lung.18 However, Id1 expression was not detected in the majority of -SMA-positive cells located in fibrotic foci at 2 weeks after bleomycin (Figure 3D) . Collectively, our data suggest that 80% of total Id1 protein expression level in the lung, at 1 and 2 weeks after bleomycin exposure, is derived from the endothelial cell population, and 20% is derived from other lung cell types, possibly immune cells that are involved in lung injury by bleomycin and have been shown to express Id1.47 Our finding indicates that epithelial cells and -SMA-positive fibroblasts do not contribute to the bulk of Id1 expression in bleomycin-injured lungs. In sum, our finding strongly supports a critical role of Id1 in lung endothelial cells on bleomycin-induced injury; however, this study does address the potential role of Id1 in other cell types.

Because lung endothelial cells comprised a large fraction of Id1-expressing cells after bleomycin exposure, we investigated the functional role of Id1 in endothelial cells. To assess endothelial dysfunction in Id1C/C mutant lungs, we first evaluated change in vascular permeability by measuring the extravasation of Evans blue dye, which, when injected via the retro-orbital sinus, can immediately complex with circulating albumin (see Materials and Methods). Accumulation of albumin-dye complexes within the lung parenchyma was quantified spectrophotometrically and used as an indicator of vascular macromolecular leakage.35 We found that wild-type lungs showed significant leakage of Evans Blue at 1 week after bleomycin when compared with saline-treated lungs (Figure 4A) . However, vascular leakage is more pronounced in Id1C/C mutants, showing a 58% increase in dye content when compared with bleomycin-treated wild-type lungs (n = 6, P < 0.001; Figure 4A ), indicating increased disruption of the endothelial barrier in the absence of Id1 function.

Figure 4. Bleomycin-injured Id1C/C lungs display increased endothelial barrier dysfunction and elevated endothelial apoptosis. A: Increased vascular permeability in bleomycin-challenged Id1C/C lungs. Pulmonary vascular permeability of saline or bleomycin-treated 8-week-old wild-type or Id1C/C lungs was determined by Evans blue extravasation assay (n = 6 for each group). Note 58% increase in Evans Blue dye leakage in injured Id1C/C lungs compared with wild-type lungs. Asterisks denote a significant difference (P < 0.001) between wild-type and Id1C/C lungs at 1 week after bleomycin. B: Endothelial cell death detection in wild-type and Id1C/C lungs by TUNEL (red) and CD34 (green) double-labeling. Representative sections are shown for lungs at 1 week after bleomycin or saline. C: Flow cytometric counting of apoptotic endothelial cells in wild-type or Id1C/C Tie1Cre;ZEG lungs at 1 week after bleomycin or saline. a: Representative dotspot graphs showing cell sorting results of 10,000 lung cells, using annexin-5 as the cell death marker and GFP as the endothelial marker. The top right quadrant represents apoptotic endothelial cells. b: Statistical representation of the sorting results from six independent experiments (n = 6). Note there is an average 37.6% increase in endothelial cell death in injured Id1C/C lungs compared with wild-type lungs. Asterisks denote a significant difference (P < 0.05) between wild-type and Id1C/C lungs. Original magnifications, x400.

Id1 has been shown to attenuate apoptotic cell death in human umbilical vein endothelial cells.12 Therefore, it is conceivable that an increase in endothelial permeability in Id1C/C lungs is, at least in part, contributed by an increase in endothelial cell death. To determine the level of endothelial cell death in Id1C/C and wild-type lungs at 1 week after bleomycin instillation, we first performed TUNEL and CD34 double immunolabeling. We found that there is a significantly higher number of apoptotic endothelial cells in Id1C/C compared with wild-type mice (Figure 4B) . For quantitative analysis, we performed FACS to measure the percent increase in apoptotic endothelial cells that are double-positive for Tie1-GFP (endothelial) and annexin-5 (apoptotic) in Id1C/C lungs compared with wild type. In bleomycin-treated lungs, FACS analysis indicated that wild-type and Id1C/C mice contained, respectively, an average of 11.7% and 16.1% apoptotic endothelial cells that were double-positive for GFP and annexin-5 (Figure 4C) . Therefore, there was an average increase of 37.6% in apoptotic endothelial cells in Id1C/C relative to the wild-type level (n = 6). Saline-treated lungs showed a relatively low level of apoptotic endothelial cells and were not significantly different in both genotypes (Figure 4C) . This result clearly indicates a significant rise in apoptotic endothelial cells in Id1C/C mutant mice after bleomycin treatment, suggesting a key role of Id1 in attenuating endothelial cell death on lung injury.

Because Id1 function has been linked to cell proliferation, we also examined the change in endothelial proliferation in wild-type and Id1C/C lungs after bleomycin treatment. Results from double labeling with proliferating cell nuclear antigen and CD34, to detect proliferating endothelial cells, revealed that the lung endothelium was relatively quiescent and did not appear to become highly proliferative on bleomycin treatment. In addition, we did not find significant differences in endothelial proliferation in Id1C/C compared with wild-type lungs at 1 week after bleomycin, suggesting that Id1 is unlikely to play a major role in endothelial cell proliferation after lung injury (data not shown). Collectively, our findings indicate that Id1 plays a key role in the survival of cells of the lung endothelium after exposure to bleomycin, and loss of Id1 function results in increased endothelial apoptosis and damage to the lung endothelium.

To examine further the role of Id1 in endothelial cell function and cell death induced by bleomycin, we performed cell culture experiments using freshly isolated lung microvascular endothelial cells (LMVECs) from both wild-type and Id1C/C lungs. Cultured wild-type LMVECs expressed a moderate level of Id1, and this expression was further increased after bleomycin challenge (Figure 5A) . We also observed that although freshly dissociated wild-type LMVECs could be passaged three times in culture, it was not possible to passage Id1C/C LMVECs after first plating, which is probably because of their reduced growth potential. This is consistent with a slight increase in apoptotic cells in saline-treated Id1C/C LMVECs compared with Id1 wild type (Figure 5B , 3.11 versus 2.03%). Wild-type and Id1C/C LMVEC cultures grown for 3 days were treated with 250 ng/ml bleomycin for 6 hours and analyzed by TUNEL or double-labeled with Tie1Cre-GFP (endothelial reporter) and annexin-5-Cy3 (apoptotic marker) and sorted by FACS analysis to quantify the level of apoptotic endothelial cells in culture (n = 6). Consistent with TUNEL staining, which showed significantly more apoptotic cells in Id1C/C LMVECs (Figure 5B) , Id1C/C LMVECs displayed an average of 17.3% apoptotic endothelial cells compared with 8.2% in wild type (Figure 5C) . These results indicate that Id1 plays a crucial role in LMVEC survival in culture and are consistent with the functional role of Id1 in bleomycin-injured lungs in vivo.

Figure 5. Id1C/C lung microvascular endothelial cells display reduced survival in culture. A: Id1 is up-regulated in endothelial cells by bleomycin in vitro. Western blotting showing Id1 level from wild-type primary LMVECs treated with saline or 250 ng/ml bleomycin for the indicated time points. Values represent relative fold change of Id1 protein level normalized to the density of -tubulin bands. B: Cell death detection of wild-type and Id1C/C primary LMVECs by TUNEL (red) and Tie1Cre-GFP (green) double labeling. Representative photographs are shown for cell cultures treated with 250 ng/ml bleomycin for 6 hours followed by no bleomycin for 3 hours. C: Flow cytometric counting of apoptotic endothelial cells in wild-type or Id1C/C Tie1Cre-ZEG LMVECs treated with saline or 250 ng/ml bleomycin for 6 hours. a: Representative dotspot graphs showing the results of cell sorting, at 10,000 cells, each using annexin-5 as the cell death marker and GFP as the endothelial marker. The top right quadrant represents apoptotic endothelial cells. b: Statistical representation of the sorting results from six independent experiments (n = 6). Note that there is nearly twofold increase in endothelial cell death in Id1C/C LMVECs compared with wild type. Asterisks denote a significant difference (P < 0.05) between wild-type and Id1C/C cells. Original magnifications, x100.

We reasoned that increased endothelial cell apoptosis may be linked to decreased levels of Bcl-2 family proteins, specifically Bcl-2 and Bcl-xL, which have been shown to be regulated by Id1 in several cancer cell lines.48-50 Because Bcl-2 proteins are well-known anti-apoptotic molecules, they may serve as a potential downstream effector of Id1 in the process of endothelial maintenance. Hence, we examined the expressions of Bcl-2 and Bcl-xL in bleomycin-exposed wild-type and Id1C/C LMVECs by Western blotting. Although we did not detect a significant difference in Bcl-xL levels in Id1C/C endothelial cell population compared with wild type, Bcl-2 expression was significantly higher in wild-type LMVECs as early as 6 hours after bleomycin exposure (data not shown). This difference was even more pronounced after 12 hours of bleomycin treatment (Figure 6) . This finding strengthens the notion that Id1 can inhibit cell apoptosis by modulating Bcl-2 expression. In addition, we examined the MEK/ERK pathway, which is involved in maintaining cell survival by regulating Bcl-2 expression in diverse cell types51 and during lung injury.52 By Western blotting using antibodies against phosphorylated-MEK and phosphorylated-ERK, we found that MEK/ERK pathway activity was also decreased, concomitant with reduced Bcl-2 protein level (Figure 6, A and B) . Collectively, our results indicate that up-regulation of Id1 expression seems to attenuate lung endothelial cell death by inducing Bcl-2 expression, which may be modulated by the MEK/ERK pathway.

Figure 6. Id1C/C lung microvascular endothelial cells show reduced Bcl-2 level and MEK/ERK activity. Western blotting of cell lysates collected from wild-type or Id1C/C primary LMVECs treated with 250 ng/ml bleomycin for or 12 hours. Note that Bcl-2 level is significantly decreased in LMVECs in the absence of Id1 function. The MEK/ERK signaling pathway is also affected in Id1C/C endothelial cells, based on lower levels of phosphorylated MEK/ERK.

Because endothelial injury and the resulting increase in vascular permeability may be involved in the pathogenesis of pulmonary fibrosis,53-57 we examined the susceptibility of Id1C/C lungs to bleomycin-induced fibrogenesis. First, we performed morphological analysis of Id1C/C and wild-type lungs by histological staining with H&E of lung sections harvested at 2 weeks after bleomycin instillation to assess the alveolar architecture. As indicated in Figure 7A , Id1C/C lungs showed more parenchymal distortion and fibrotic foci compared with bleomycin-treated wild-type lungs. For semiquantitative analysis of lung fibrosis, H&E-stained sections were evaluated by a pathologist blinded to the genotypes and treatment groups. As shown in Figure 7B , Id1C/C lungs displayed a higher parenchymal distortion, 35% increase, compared with wild type (n = 9, P < 0.001).

Figure 7. Id1C/C lungs are more susceptible to bleomycin-induced fibrogenesis. A: H&E staining of lung sections to show architectural changes. Representative photographs showing foci formation in wild-type and Id1C/C lungs at 2 weeks after bleomycin. B: Increase in the index of architectural distortion in Id1C/C lungs compared with wild-type lungs after bleomycin challenge. Double asterisks denote a significant difference (P < 0.001) between wild-type and Id1C/C lungs at 2 weeks after bleomycin (n = 9 for each group). C: Trichrome staining showing increased collagen deposition in lungs of Id1C/C mice after bleomycin challenge. Representative photographs reveal increased collagen accumulation (blue) in Id1C/C lungs compared with wild type at 2 weeks after bleomycin. D: Quantification of collagen deposition in lungs of wild-type and Id1C/C mice after bleomycin challenge by hydroxyproline assay. Asterisks denote a significant difference (P < 0.01) between wild-type and Id1C/C lungs at 2 weeks after bleomycin (n = 8 for each group). E: Immunostaining of wild-type and Id1C/C lungs with antibodies specific for -SMA. Representative sections are shown for lungs at 2 weeks after bleomycin or saline. Sections were counterstained with hematoxylin to highlight all nuclei. Note elevated expression of -SMA in fibrotic foci of Id1C/C lungs compared with wild type. F: Western blotting showing expression level of -SMA in wild-type and Id1C/C lungs at 2 weeks after bleomycin or saline. Values represent the relative fold change of -SMA protein level normalized to the density of -tubulin bands. Original magnifications, x400.

One of the hallmarks of lung fibrosis is the accumulation of collagen secreted by the large fibroblast population within foci. Indeed, we observed more collagen deposition in Id1C/C lungs at 2 weeks after bleomycin by Trichrome staining (Figure 7C) . In agreement, we detected 90% increase in collagen deposition in Id1C/C lungs compared with wild type, 2 weeks after bleomycin as measured by hydroxyproline content (n = 8, P < 0.01; Figure 7D ). Consistent with elevated collagen secretion, Id1C/C lungs also showed significant increase in the population of myofibroblasts as revealed by -SMA immunolabeling (Figure 7E) . Western blotting of Id1C/C lung homogenates revealed increased levels of -SMA expression compared with wild type (Figure 7F) . Collectively, we demonstrated that Id1C/C mutant lungs are more susceptible to bleomycin-induced fibrogenesis as evidenced by increased lung parenchymal distortion, collagen and hydroxyproline content, and myofibroblast cell population.

Discussion

In this study, we have shown that, although Id1 was expressed at a basal level in the normal adult lung, its protein expression was significantly up-regulated predominantly in cells of the lung endothelium in response to bleomycin injury. This new finding is consistent with the endothelial-specific induction of Id1 in tumors of a variety of tissue origins58 and prompted us to investigate the contributory role of Id1 in bleomycin-injured lung endothelium.

Bleomycin-induced lung injury and fibrogenesis (Figure 8) , like many other types of lung injury, is almost always accompanied by an increase in vascular permeability, suggesting that vascular endothelial integrity and function are compromised. Our finding that bleomycin exposed Id1C/C lungs showed increased vascular permeability suggests that Id1 may play a critical role in maintaining endothelial integrity and function. Consistent with this notion, we demonstrated decreased endothelial cell survival in Id1C/C lungs injured by bleomycin compared with wild type. In addition, bleomycin-treated lung microvascular endothelial cells from Id1C/C lungs also showed significantly reduced survival potential compared with wild-type lung endothelial cells. Furthermore, our results indicate that up-regulation of Id1 expression seems to attenuate lung endothelial cell death by inducing the expression of the anti-apoptotic molecule Bcl-2, which may be modulated by the MEK/ERK pathway. Activated ERK1/2 has been shown to inhibit apoptosis in injured endothelial cells.59,60 Reduction in Bcl-2 expression and MEK/ERK signaling has been documented in acute and chronic phases of injured lungs, respectively.52,61 In addition, enhanced MEK/ERK signaling reduces ischemia/reperfusion (I/R)-induced lung injury, together with an increase in Bcl-2 expression.52 Therefore, MEK/ERK and Bcl-2 pathway may serve as a promising downstream effector of Id1 function in the lung.

Figure 8. Schematic representation of Id1 function in the lung endothelium in acute lung injury. Injury caused by bleomycin results in extensive endothelial apoptosis. The lung responds to tissue damage by up-regulating Id1 expression in endothelial cells (and other cell types). Up-regulation of Id1 in endothelial cells activates the MEK/ERK pathway and elevates expression of anti-apoptotic protein Bcl-2. Increased Bcl-2 protein level reduces the extent of endothelial cell apoptosis, thus alleviating endothelial damage. Acute lung injury often results in pulmonary fibrosis in the chronic phase, which may be attenuated by Id1 function.

The lung microvasculature is intimately associated with the alveolar epithelium for efficient blood-gas exchange and it has been suggested that functional defects of microvessels may play a role in the pathogenesis of lung fibrosis. Pulmonary microvascular lesions and increased vascular permeability have been linked to the pathogenesis of pulmonary fibrosis therefore limiting endothelial cell injury and cell death may alleviate fibrogenesis.54-57,62-66 Several other studies have also implicated a role of the microvasculature in lung fibrosis.53,67-73 Conceivably, increased microvascular damage in Id1C/C lungs may lead to increased extravasation of plasma that may promote fibroblast proliferation. In addition, stimulated/injured endothelial cells can also secrete fibrogenic molecules that may affect fibroblast migration and proliferation.74 Our findings indicate that Id1 plays a critical role in promoting endothelial survival after bleomycin-induced injury and are consistent with a role of the endothelium in lung fibrogenesis. However, less than 20% of Id1 expression was derived from nonendothelial cells in bleomycin-exposed lungs. Our study, therefore, does not exclude the possibility that loss of Id1 function in other lung cell populations may also contribute to increased lung fibrogenesis. Interestingly, Id1 has been shown to be expressed in a subgroup of immune cells such as the macrophages and granulocytes.47 Therefore, it is possible that Id1 may also be involved in the injured lung inflammatory response that may contribute to fibrogenesis. Increasing evidence suggests that rare lung stem cells reside in the bronchioalveolar junction and might be involved in lung repair.75,76 It remains to be determined whether Id1 is involved in the maintenance of rare lung progenitor cells as has been suggested for hematopoietic stem cells.77

Bmp signaling has been strongly implicated in Id gene induction in a number of cell types including endothelial cells,78-80 and vascular endothelial growth factor has also been shown to induce Id1 and Id3 in bone marrow-derived endothelial precursor and hematopoietic cells.81 Both signaling pathways are promising candidates for activating Id1 expression in the lung endothelium on bleomycin injury, although the actual in vivo signals remain to be identified. Signaling mediated by transforming growth factor-ß1 and tumor necrosis factor- are known to play key roles in many types of lung diseases including bleomycin-induced pneumopathy in animal models.21,23,82-86 Interestingly, transforming growth factor-ß1 can exert long-term repression of Id1 expression in a number of cell types,18,87,88 suggesting that transforming growth factor-ß could potentially antagonize Id1 expression in the course of lung pathogenesis. Tumor necrosis factor- has also been implicated in the regulation of Id expression in astrocytes during inflammatory injury of the central nervous system.88-90 Thus, there may be a balance between Id1-inducing and Id1-repressing mechanisms in the bleomycin-injured lung. Therefore, future identification of signals that modulate Id1 expression in bleomycin-induced lung injury will improve our understanding of the regulatory mechanisms underlying Id1 function in lung injury and fibrosis.

Acknowledgements

We thank Dr. Robert Benezra for providing Id1 mutant mice, Dr. Scott Baldwin for providing the Tie1-Cre mice, Dr. Jin Chen for her advice on retro-orbital injection of Evans blue, and the Vanderbilt University Medical Center Cell Imaging Shared Resource for use of the confocal microscope.

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作者单位：From the Departments of Cell and Developmental Biology* and Medicine, and the Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee