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. 2023 Sep 23;24(1):227.
doi: 10.1186/s12931-023-02512-4.

CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s

Jianhui Sun #  1 , Huacai Zhang #  1 , Di Liu  1 , Wenyi Liu  1 , Juan Du  1 , Dalin Wen  1 , Luoxi Li  1 , Anqiang Zhang  1 , Jianxin Jiang  2 , Ling Zeng  3
Affiliations

CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s

Jianhui Sun et al. Respir Res. .

Abstract

Background: Functional alveolar regeneration is essential for the restoration of normal lung homeostasis after acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Lung is a relatively quiescent organ and a variety of stem cells are recruited to participate in lung repair and regeneration after lung tissue injury. However, there is still no effective method for promoting the proliferation of endogenous lung stem cells to promote repair and regeneration.

Methods: Using protein mass spectrometry analysis, we analyzed the microenvironment after acute lung injury. RNA sequencing and image cytometry were used in the alveolar epithelial type 2 cells (AEC2s) subgroup identification. Then we used Sftpc+AEC2 lineage tracking mice and purified AEC2s to further elucidate the molecular mechanism by which CTGF regulates AEC2s proliferation both in vitro and in vivo. Bronchoalveolar lavage fluid (BALF) from thirty ARDS patients who underwent bronchoalveolar lavage was collected for the analysis of the correlation between the expressing of Krt5 in BALF and patients' prognosis.

Results: Here, we elucidate that AEC2s are the main facultative stem cells of the distal lung after ALI and ARDS. The increase of connective tissue growth factor (CTGF) in the microenvironment after ALI promoted the proliferation of AEC2s subpopulations. Proliferated AEC2s rapidly expanded and differentiated into alveolar epithelial type 1 cells (AEC1s) in the regeneration after ALI. CTGF initiates the phosphorylation of LRP6 by promoting the interaction between Krt5 and LRP6 of AEC2s, thus activating the Wnt signaling pathway, which is the molecular mechanism of CTGF promoting the proliferation of AEC2s subpopulation.

Conclusions: Our study verifies that CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of AEC2s subpopulation.

Keywords: Acute lung injury; Acute respiratory distress syndrome; Alveolar epithelial type 1 cells; Alveolar epithelial type 2 cells; Alveolar regeneration; Connective tissue growth factor.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
CTGF promotes the proliferation of AEC2s post ALI. (A) Heatmap of differentially expressed proteins in mouse lungs from 1 to 7 dpi (D1 to D7). (B) Representative histological sections of sham control and CTGF-treated mouse lungs from 3 to 7 dpi. H&E staining. Scale bar, 200 μm. (C) Immunofluorescence (IF) staining of Sftpc and Ki67 in control (ctrl) and CTGF-treated mouse lungs from 3 to 7 dpi (P = 0.0082; P = 0.039; P = 0.0068; P = 0.023; P = 0.042 by two-tailed t test). All bar graphs show the mean ± SEM of three independent experiments. n / HPF, number / high power field. Scale bar, 200 μm. (D) Representative differential interference contrast images of control and 10 ng/ml CTGF-treated AEC2 organoids from Day 1 to Day 13. Scale bar, 100 μm. (E). Representative fluorescence microscopy images of control and CTGF-treated AEC2 organoids at Day 13. Calcein-AM staining was used to stain AEC2 organoids, and organoid size and colony-forming efficiency at Day 13 were analyzed. All bar graphs show the mean ± SEM of three independent experiments. P = 0.001 and P = 0.0011 by two-tailed t test. Scale bar, 50 μm
Fig. 2
Fig. 2
AEC2s mediate alveolar repair via differentiation to AEC1s. (A) High-magnification images of RNAscope staining of Sftpc and AQP5 in AEC2 organoids (left) and lung tissue sections (right). All bar graphs show the mean ± SEM of three independent experiments. P = 0.043; P = 0.028 by two-tailed t test. Scale bar, 50 μm. (B) High-magnification images of the immunofluorescence staining of Sftpc and AQP5 of the AEC2 organoids (left) and lung tissue sections (right). Scale bar, 50 μm
Fig. 3
Fig. 3
CTGF treatment increased the number of AEC2 subpopulations in the mouse lung. (A). Heatmap of the top 30 upregulated genes in CTGF-treated mouse AEC2s compared with nontreated controls. (B). Enriched Gene Ontology classes of AEC2s isolated from CTGF-treated 3 dpi ALI mouse lung tissue compared with control. Red bar, GO class of upregulated genes in AEC2s isolated from CTGF-treated 3 dpi ALI mouse lungs. Black bar, GO class of upregulated genes in AEC2s isolated from sham control mouse lungs. GO terms were ranked by the enrichment P value. (C) STRING analysis of the protein–protein interaction network of highly expressed genes in AEC2s isolated from CTGF-treated 3 dpi ALI mouse lungs compared with the control. (D) The mRNA and protein expression levels of Sftpc, Krt5 and p63. Scale bar, 50 μm. (E) Krt5 expressing AEC2s subpopulations were detected by image cytometry. LysoTracker-pos EpCAM-pos was used as an AEC2 marker protein, and CD45-negative cells could exclude cells from the blood system. Images are representative of 3 independent experiments. (F) Flow cytometry was used to observe the effect of CTGF treatment on the proportion of Krt5 expressing AEC2s in mouse lungs (n = 5; 8.30% ± 2.47% in the control group vs. 14.98% ± 3.72% in the CTGF-treated group, P = 0.0038 by two-tailed t test)
Fig. 4
Fig. 4
Krt5 expressing AEC2s are an important progenitor subpopulation of lung regeneration after ALI. (A) The proliferative potential of Krt5 expressing AEC2s post ALI observed by RNAscope. Scale bar, 50 μm. (B) The proliferative potential of Krt5 expressing AEC2s post ALI observed by immunofluorescence staining. Scale bar, 50 μm
Fig. 5
Fig. 5
AEC2-specific knockout of Krt5 confirmed the essential role of Krt5 in lung repair and regeneration post ALI. (A) Sftpc-CreERT2 and Rosa26-RFP mice were injected with the Krt5 Cre-on AAV-9 vector two weeks after tamoxifen administration. The transcriptional and translational levels of Krt5 in AEC2s were verified by quantitative Western blot and RNAscope analyses. (B) Representative histological sections of control and Krt5 knockout mouse lungs from 3 to 7 dpi (P = 0.0009 for 3 dpi ; P = 0.0001 for 7 dpi by two-tailed t test). H&E staining. Scale bar, 200 μm. (C) High-magnification images of RNAscope staining of control and Krt5 knockout lung tissue sections at 3 and 7 dpi (left) and immunofluorescence (right). Scale bar, 50 μm. (D) Flow cytometry analysis to detect the proportions of proliferating AEC2s post ALI in the control and Krt5 knockout groups (n = 5; 6.48% ± 1.46% for the control group; 3.27% ± 1.12% for the Krt5 knockout group, P = 0.0096 by two-tailed t test)
Fig. 6
Fig. 6
Krt5 promotes the expansion of Krt5 expressing AEC2s by binding to LRP6 and activating the Wnt signaling pathway. (A) Heatmap of the expression levels of key genes in the Wnt signaling pathway in Krt5 expressing AEC2s and Krt5− AEC2s by PCR panel assay. (B) Purified AEC2s were treated with CTGF (10 ng/mL), and confocal FRET analysis was used to verify the Krt5 and LRP6 interaction. Images of the donor (Krt5) and acceptor (LRP6) before and after acceptor photobleaching show a strong recovery of donor fluorescence in the bleached area (marked by white arrow), indicating an interaction between Krt5 and LRP6 on AEC2s. Scale bar, 10 μm. (C) Western blot analysis of the indicated protein expression in Krt5 expressing AEC2s after CTGF (10 ng/ml) stimulation for 7 to 24 h
Fig. 7
Fig. 7
Association between the number of Krt5 expressing AEC2 subpopulations and the severity of ARDS. (A) Representative flow cytometry analysis of Krt5 expressing AEC2 subpopulation percentages in the BALF of control and ARDS patients. (B) Quantitative analysis of Krt5 mRNA and Krt5 expressing AEC2 subpopulation percentages in the BALF of mild, moderate and severe ARDS patients (P = 0.0072 and P = 0.0034 by one-way ANOVA).
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