Identification of transcription factors that regulate re-programming of normal EC into fibrosis-associated EC in IPF lungs
To identify transcriptional regulators that control re-programming of normal EC into fibrosis-associated EC, we performed single cell RNA-sequencing (10X genomics) to compare the transcriptome of endothelial cells in donor and idiopathic pulmonary fibrosis (IPF) lungs (Cincinnati Children’s Hospital Medical Center (CCHMC) datasets, Supplementary Fig. S1a, b). We also downloaded and analyzed the published scRNA-seq data19 from donor and IPF lungs (Northwestern University (NW) datasets, Supplementary Fig. S1c, d). Endothelial cell in both datasets were identified as cells expressing Pecam1 (CD31), Cdh5 (VE cadherin) and lacking Ptprc (CD45) mRNAs, consistent with published studies20. Cross-comparison of two scRNA-seq datasets identified several top differentially expressed endothelial transcription factors. Among universally downregulated transcription factors in IPF endothelial cells were FOXF1, SMAD6, HIF3A, CREB5, TBX3 and GATA2 (Fig. 1a). Universally upregulated transcription factors included ETV6, LEF1, EGR3, MEOX1 and SMAD1 (Fig. 1a). For the follow-up studies, we decided to focus on FOXF1 transcription factor because it’s expression is highly enriched in lung endothelial cells compared to endothelial cells of other organs18,21 and because the FOXF1 loss-of-function gene mutations are linked to ACDMPV, a fatal congenital lung disease with significant fibrotic remodeling13. To verify that identified decrease in FOXF1 is not limited to two datasets, we have also analyzed the publicly available datasets from Yale University and Vanderbilt University6,7. We have confirmed the decreased levels of FOXF1 mRNA in endothelial cells of patients with IPF compared to donor controls (Supplementary Fig. S2a). We sought to determine whether downregulation of FOXF1 in fibrosis-associated EC is essential for lung fibrogenesis.
Expression of FOXF1 is decreased in endothelial cells within fibrotic lesions of human IPF lungs
Using immunostaining with FOXF1, CD31 and αSMA antibodies, we found that FOXF1 protein was undetectable in EC within fibrotic lesions of IPF lungs identified as αSMA-positive regions (Fig. 1b and Supplementary Fig. S2b). While in human donor lungs, FOXF1 protein was detected in approximately 80% of endothelial cells, only 5-10% of endothelial cells expressed FOXF1 in IPF lungs (Fig. 1c). We further verified by qRT-PCR that FOXF1 mRNA was decreased in FACS-sorted EC from IPF lungs compared to FACS-sorted EC from donor lungs (Fig. 1d). Thus, both mRNA and protein levels of FOXF1 are decreased in endothelial cells of IPF lungs.
To identify FOXF1-expressing lung endothelial cells in scRNA-seq CCHMC dataset, endothelial cells from donor and IPF lungs were visualized using uniform manifold approximation and projection (UMAP) after samples integration with Harmony22 (Fig. 1e–g). Both FOXF1 mRNA levels and the total number of FOXF1-expressing EC were decreased in IPF lungs compared to donor lungs (Fig. 1h). To demonstrate that these results are not limited to CCHMC scRNA-seq dataset, we analyzed the publicly available NW scRNA-seq dataset containing endothelial cells from IPF and donor lungs (GSE 12296019). Consistent with the results from CCHMC dataset, both the percentage of FOXF1-positive endothelial cells and FOXF1 mRNA levels were decreased in IPF lungs from NW dataset (Fig. 1i–l).
Based on gene expression signatures, pulmonary endothelial cells from both CCHMC and NW datasets were subdivided into five sub-clusters: arterial, general capillary (gCAP), alveolar capillary (aCAP), venous and lymphatic ECs (Fig. 1e, i, Supplementary Fig. S1a–d). Genes known to be selectively expressed in different sub-types of endothelial cells were used to annotate these sub-clusters23,24. The arterial EC sub-cluster was identified as cells expressing EFNB2, GJA5, DKK2 and HEY1, whereas the venous EC sub-cluster included cells expressing ACKR1, CLU and VWF (Supplementary Fig. S1a, b). The gCAP sub-cluster was enriched in GPIHBP1 and SLC6A4 transcripts, aCAP was enriched in EDNRB, APLN and HPGD, and lymphatic EC sub-cluster expressed PROX1, CCL21 and PDPN (Supplementary Fig. S1a–d). FOXF1 was detected in arterial, venous, and capillary sub-clusters, with the highest expression of FOXF1 in aCAPs and gCAPs (Fig. 1m, n). FOXF1 was undetectable in lymphatic EC (Fig. 1m, n). Also, FOXF1 was not expressed in the COL15+ endothelial cells that were identified in the IPF lungs, but not in the control donor lungs (Supplementary Fig. S2c), confirming the published data6. In both scRNA-seq datasets, FOXF1 expression was decreased in capillary, arterial and venous sub-clusters of IPF lungs compared to donor lungs (Fig. 1m–o). Consistent with scRNA-seq data, RNA in situ hybridization showed that both the percentage of FOXF1-positive EC and expression levels of FOXF1 transcripts were decreased in capillary, arterial and venous sub-clusters of IPF lungs compared to donor lungs (Supplementary Fig. S3).
FOXF1 is decreased in endothelial cells within fibrotic lesions of mouse bleomycin-injured lungs
We next examined expression of FOXF1 in endothelial cells in a mouse model of pulmonary fibrosis. Three weekly intratracheal (IT) injections of bleomycin were used to induce lung fibrosis in wild type mice (Supplementary Fig. S4a). Bleomycin administration caused a time-dependent increase in collagen depositions as quantified by Sircol assay (Fig. 2a) and confirmed by H&E and Sirius red/Fast green staining (Supplementary Fig. S4b). Increased accumulation of inflammatory cells in bleomycin-treated lungs was shown using flow cytometry analysis for CD45+ cells (Supplementary Fig. S4c). Next, we FACS-sorted CD31+/CD45− lung endothelial cells at different time points after bleomycin treatment and measured endothelial Foxf1 expression by qRT-PCR. A decrease in endothelial Foxf1 mRNA was observed as early as day 3 after the first bleomycin treatment (Fig. 2b). The percentage of FOXF1-positive ECs in lung tissue was also decreased as early as day 3 after the first bleomycin treatment as shown by co-localization of FOXF1 with endothelial-specific ERG transcription factor (Supplementary Fig. S4d). Immunostaining for FOXF1 and CD31 showed decreased FOXF1 protein expression in endothelial cells within fibrotic lesions identified as αSMA-positive areas (Fig. 2d, right panel). Consistent with human IPF lungs (Fig. 1b, c), the number of FOXF1+/CD31+ endothelial cells was decreased in murine fibrotic lungs (Fig. 2c).
Next, we performed a single cell RNA-sequencing of bleomycin-treated and control mouse lungs. Foxf1-expressing endothelial cells were visualized using UMAP (Fig. 2e–g). Consistent with human IPF lungs, bleomycin-treated mouse lungs had reduced numbers of Foxf1-positive endothelial cells and decreased expression of Foxf1 mRNA in these cells (Fig. 2h). Mouse lung endothelial cells were further subdivided into arterial, venous, aCAP, gCAP and lymphatic sub-clusters (Supplementary Fig. S5 and Fig. 2e). Endothelial cells expressing Foxf1 transcript were present in arterial, venous, aCAP and gCAP sub-clusters, but were completely absent in lymphatic cells (Fig. 2i). RNA in situ hybridization showed that both the percent of FOXF1-positive endothelial cells as well as the expression levels of FOXF1 transcripts were decreased in arterial, venous, aCAP and gCAP sub-clusters of bleomycin-treated lungs compared to control lungs (Supplementary Fig. S6). Altogether, FOXF1 expression is decreased in most EC types in human and mouse fibrotic lungs.
Deletion of FOXF1 in endothelial cells accelerates pulmonary fibrosis
To determine the role of FOXF1 in endothelial cells during pulmonary fibrosis, we used mice in which the Foxf1 gene was specifically deleted in endothelial cells using Pdgfb-CreER transgene, which does not target other cell types in the adult lung18,25. Since homozygous (Pdgfb-CreER /Foxf1fl/fl mice or endFoxf1−/−) mice developed lung edema and respiratory insufficiency18, we used heterozygous endFoxf1+/− mice for lung fibrosis studies (Supplementary Fig. S7a). Naïve heterozygous Tam-treated endFoxf1+/− mice were phenotypically normal, had normal lung histology and alveolar capillary density as shown by CD31 staining (Supplementary Fig. S7b and18). Since the Pdgfb-Cre transgene contain GFP18, we used flow cytometry to demonstrate that the transgene targets only CD45−CD31+ (endothelial), but CD45+CD31− (hematopoietic) or CD45−CD31− (non-endothelial, non-hematopoietic) cell types in the lung (Supplementary Fig. S7c, d). The scRNA-seq analysis showed co-expression of endothelial Foxf1 and Pdgfb mRNA transcripts in the lung EC (Supplementary Fig. S7f).
Three weekly IT injections of bleomycin were used to induce lung fibrosis in Tam-treated endFoxf1+/− and control Foxf1fl/fl mice (Fig. 3a). Using FACS-sorted lung endothelial cells, we have shown that bleomycin treatment caused more profound decrease of Foxf1 mRNA in endFoxf1+/− endothelial cells compared to control endothelial cells (Supplementary Fig. S8a). Decreased Foxf1 in endothelial cells was associated with more severe fibrosis in bleomycin-treated endFoxf1+/− mice compared to bleomycin-treated control mice, as shown by quantifying collagen depositions in the lung tissue using Sircol assay (Fig. 3b). Compared to controls, bleomycin-treated endFoxf1+/− mice had increased Ashcroft scores (Supplementary Fig. S8b, c), decreased body weights (Fig. 3c), reduced lung capacity and compliance (Fig. 3d, e), impaired lung mechanics (Supplementary Fig. S8d–j) and arterial oxygenation (Supplementary Fig. S8k). Lung fibrosis in endFoxf1+/− mice developed faster and was more severe compared to control mice, as shown by Sirius red/fast green (Fig. 3f), Trichrome (Fig. 3g), and immunostaining for αSMA (Fig. 3h). Increased fibrotic depositions in bleomycin-treated endFoxf1+/− lungs were also supported by increased Acta2, Col1a1, Col3a1, Ctgf, Cthrc1, Vim and Fn1 mRNAs (Fig. 3i). Thus, FOXF1 deficiency in murine endothelial cells exacerbates bleomycin-induced pulmonary fibrosis.
FOXF1-deficient endothelial cells increase myofibroblast activation
To identify molecular mechanisms by which FOXF1-deficient ECs promote lung fibrogenesis, we performed bulk RNA-seq using FACS-sorted endothelial cells from bleomycin-treated control and endFoxf1+/− lungs (Supplementary Fig. S9). Gene Set Enrichment Analysis (GSEA) of RNA-seq data showed that the most enriched functional categories in endFoxf1+/− endothelial cell were wound healing, extracellular matrix organization, cell migration, blood vessel remodeling, inflammation, myeloid leukocyte migration, and RHO GTPase signaling (Supplementary Fig. S9a–d). Consistent with inactivation of one Foxf1 allele, Foxf1 mRNA was decreased 2.1-fold in ECs of bleomycin-treated endFoxf1+/− lungs compared to control lungs (Supplementary Fig. S9a), a finding confirmed by qRT-PCR of FACS-sorted ECs (Fig. 4a). FOXF1-deficiency in endFoxf1+/− ECs was associated with increased expression of pro-fibrotic and pro-inflammatory genes, including Il6, Tnfα, Ccl2, Cxcl1 and Thbs1 (Supplementary Fig. S9a), findings validated by qRT-PCR (Fig. 4a).
Next, we determined the effect of FOXF1 deficiency on secretion of profibrotic and proinflammatory mediators by endothelial cells in vitro. Human endothelial cells, HUVEC, were infected with lentiviruses carrying either FOXF1 shRNA or control shRNA. The shFOXF1-treated HUVECs exhibited decreased FOXF1 mRNA as shown by qRT-PCR (Fig. 4b), demonstrating approximately similar efficiency of FOXF1 inhibition compared to haploinsufficient endothelial cells from endFoxf1+/− mice (Fig. 4a). Conditioned media (CM) from control and FOXF1-deficient HUVECs were collected. Invasion and proliferation of human CCD-19Lu lung fibroblasts were measured in the presence of CM in vitro. CM from FOXF1-deficient HUVECs increased invasion and proliferation of lung fibroblasts compared to CM from control cells (Fig. 4c, d). CM from FOXF1-deficient HUVECs also increased expression of ACT2, VIM, FN1 and COL3A1 in human CCD-19Lu fibroblasts (Fig. 4e). To verify that the results are not limited to HUVECs, we also used human pulmonary arterial endothelial cells (HPAEC) and human pulmonary microvascular endothelial cells (HPMEC) to confirm that CM from FOXF1-deficient HPAEC and HPMEC cells increased invasion, proliferation as well as expression of pro-fibrotic genes in lung fibroblasts (Supplementary Figs. S10a–d and S11a–d). Thus, FOXF1-deficient endothelial cells induced the pro-fibrotic phenotype in lung fibroblasts, possibly, through secretion of soluble mediators.
To identify the differentially changed soluble mediators in the CM from FOXF1-deficient HUVECs compared to control HUVECs, we used the Proteome Profiler Human Cytokine Array. CCL2, CXCL1, G-CSF, GM-SCF, ICAM-1 and IL-6 proteins were increased in CM from FOXF1-deficient HUVECs, whereas CXCL12, IL-8, IL-13 and IL-16 were unchanged (Fig. 4f). Since TNFα and IL-6 increase activation of fibroblasts during lung fibrosis26, we used blocking antibodies to inhibit increased levels of IL-6 or TNFα in CM from FOXF1-deficient HUVECs (Fig. 4g). Inhibition of either IL-6 or TNFα significantly decreased migration of CCL-19LU fibroblasts into the bottom chamber of Transwells containing CM from FOXF1-deficient HUVECs (Fig. 4g). To verify that the results are not limited to HUVEC cells, we also used HPAEC cells and confirmed that inhibition of either IL-6 or TNFα significantly decreased migration of CCL-19LU fibroblasts into the bottom chamber of Transwells containing CM from FOXF1-deficient HPAEC (Supplementary Fig. S10e, f). Thus, FOXF1-deficient endothelial cells stimulate the migration of lung fibroblasts in vitro by secreting IL-6 and TNFα.
FOXF1 deficiency in endothelial cells increases the number of macrophages in the fibrotic lungs
Since numerous cytokines from the protein array, including IL-6, TNFα, CCL2 and CXCL1, regulate recruitment of macrophages during lung injury26, we examined the number of macrophages in Foxf1-deficient lungs. At day 21 after bleomycin injury, accumulation of macrophages in fibrotic lesions of bleomycin-treated endFoxf1+/− lungs was increased compared to control lungs as shown by immunostaining for F4/80 (Fig. 5a) and Mac3 (Supplementary Fig. S12a). We also used flow cytometry analysis to demonstrate that the number of macrophages (CD45+ CD11clow/+ CD64+) was increased in bleomycin-injured endFoxf1+/− lungs compared to control bleomycin-injured lungs (Fig. 5b and Supplementary Fig. S12b–c). Of note, no changes in the number of inflammatory cells, including macrophages, were reported in uninjured endFoxf1+/− and control lungs18. We next determined whether increased secretion of pro-inflammatory mediators by FOXF1-deficient endothelial cells is important for macrophage migration in vitro. CM from control or FOXF1-deficient HUVECs was added to the bottom chambers of Transwells, and the migration of human macrophages from the upper chambers towards CM was assessed in the presence of blocking antibodies specific to CCL2, CXCL1, IL-6 or TNFα (Fig. 5c). Inhibition of these pro-inflammatory cytokines decreased migration of macrophages towards CM from FOXF1-deficient HUVECs compared to CM from control HUVECs (Fig. 5c and Supplementary Fig. S12d). Thus, FOXF1-deficient endothelial cells stimulate migration of macrophages by secreting multiple proinflammatory mediators.
R-Ras is a direct transcriptional target of FOXF1
FACS-sorted endothelial cells from bleomycin-treated endFoxf1+/− lungs exhibited decreased expression of Rras (Fig. 4a), a critical mediator of endothelial barrier function and vascular repair after injury27,28. Consistent with decreased expression of Rras in endothelial cells, bleomycin-treated endFoxf1+/− lungs displayed increased endothelial permeability as determined by Evans blue dye (Supplementary Fig. S13a). Since increased endothelial permeability contributes to lung fibrosis29 and endFoxf1+/− mice had exacerbated lung fibrosis after bleomycin injury (Fig. 3), we examined whether FOXF1 regulates R-RAS in pulmonary endothelial cells. Based on scRNA-seq data analysis, R-RAS mRNA was decreased in human IPF endothelial cells compared to donor endothelial cells (Fig. 6a). We also FACS-sorted endothelial cells from IPF lungs and used qRT-PCR to demonstrate that R-RAS mRNA was decreased in IPF endothelial cells, coinciding with decreased expression of FOXF1 mRNA (Fig. 6b). Immunostaining for R-RAS, FOXF1 and CD31 showed that FOXF1 co-localized with R-RAS in endothelial cells of donor lungs, indicating that both proteins are co-expressed in normal ECs (Fig. 6c). In contrast, neither R-RAS nor FOXF1 were detected in endothelial cells within fibrotic lesions of IPF lungs (Fig. 6c). In vitro shRNA-mediated knockdown of FOXF1 in HUVECs decreased expression of R-RAS (Fig. 6d). These findings were confirmed in HPAEC and HPMEC cells (Supplementary Fig. S14a, b). In agreement with human data, shRNA-mediated knockdown of Foxf1 in mouse MFLM 91U endothelial cells also decreased expression of Rras (Fig. 6e). Next, we used publicly available ChIP-seq dataset25 to show that FOXF1 protein directly bound to the Rras promoter region in endothelial cells (Fig. 6f). The FOXF1-binding region in Rras promoter had H3K4me3 but not H3K27me3 marks (Fig. 6f), suggesting that FOXF1 transcriptionally activates Rras gene promoter. To test this hypothesis, the −762/+13 bp Rras promoter region, containing the FOXF1-binding site identified by ChIP-seq (Fig. 6f), was cloned into the pGL2 luciferase reporter plasmid (Fig. 6g, upper schematic diagram). In co-transfection experiments, CMV-Foxf1 expression vector increased transcriptional activity of the −762/+13 Rras promoter region compared to CMV-empty vector (Fig. 6g). Thus, Rras is a direct transcriptional target of FOXF1 in endothelial cells.
Next, we examined whether R-RAS is involved in FOXF1-mediated regulation of CCL2, CXCL1, IL-6 and TNFα. We over-expressed R-Ras in FOXF1-deficient MFLM 91U endothelial cells in vitro (Fig. 6h and Supplementary Fig. S14a, b). Overexpression of R-Ras decreased Ccl2 and TNFα mRNAs in mock-transfected cells and prevented upregulation of Ccl2 and TNFα in cells transfected with Foxf1-specific siRNA (Fig. 6h). Overexpression of R-Ras did not prevent upregulation of Cxcl1 and IL-6 in FOXF1-deficient endothelial cells (Supplementary Fig. S14c). Altogether, our data suggest that FOXF1 in lung endothelium stimulates transcription of Rras, which inhibits expression of CCL2 and TNFα. FOXF1 inhibits CXCL1 and IL-6 independently of R-Ras.
Transgenic overexpression of FOXF1 in endothelial cells decreases lung fibrosis after bleomycin-induced injury
Since conditional deletion of FOXF1 in endothelial cells increased pulmonary fibrosis after chronic bleomycin injury (Fig. 3), we examined whether overexpression of FOXF1 in endothelial cells was sufficient to inhibit lung fibrosis. To test this hypothesis, we generated an inducible, EC-specific FOXF1 overexpression mouse model (Pdgfb-CreERtg/+; LSL-rtTAtg/+; TetO-Foxf1tg/+ mice; abbreviated as endFoxf1OE) (Supplementary Fig. S15a). Upon tamoxifen administration, Cre-mediated recombination of LoxP-floxed stop codon (LSL) results in rtTA expression in endothelial cells. In the presence of doxycycline, rtTA binds to and activates the TetO7-CMV promoter driving expression of the mouse Foxf1 transgene (Supplementary Fig. S15a). Thus, combined administration of tamoxifen (Tam) and doxycycline (Dox) causes increased expression of FOXF1 in endothelial cells of endFoxf1OE mice (Fig. 7a). FACS-sorted endothelial cells from Tam- and Dox-treated endFoxf1OE lungs had a 4-fold increase in Foxf1 mRNA compared to controls (Fig. 7b). Without bleomycin injury, the Tam- and Dox-treated endFoxf1OE mice had normal lung architecture and capillary density as shown by H&E staining and immunostaining for CD31 (PECAM-1) (Supplementary Fig. S15b). To induce pulmonary fibrosis, Tam- and Dox-treated endFoxf1OE and single transgenic (control) mice were given three weekly IT injections of bleomycin (Fig. 7a). While bleomycin treatment decreased Foxf1 mRNA in FACS-sorted endothelial cells from both groups of mice, Foxf1 expression was higher in endFoxf1OE ECs compared to control ECs (Fig. 7b). Transgenic overexpression of Foxf1 increased survival of mice after bleomycin injury with 95% of endFoxf1OE mice surviving the injury, but only 20% of control single-transgenic littermates being alive 25 days after the last bleomycin treatment (Fig. 7c). Fibrotic lung remodeling was decreased in bleomycin-treated endFoxf1OE mice compared to controls as demonstrated by Ashcroft score, Trichrome staining, Sirius red/Fast green staining and immunostaining for αSMA (Fig. 7d–h). Biochemical quantification of lung collagen amounts using Sircol assay was consistent with decreased fibrosis in endFoxf1OE lungs (Fig. 7i). Moreover, endothelial over-expression of Foxf1 decreased recruitment of macrophages into bleomycin-treated lungs and decreased expression of pro-inflammatory genes in total lung RNA (Fig. 7j, k). Thus, overexpression of FOXF1 in endothelial cells prior to bleomycin injury attenuates pulmonary fibrosis and improves survival after bleomycin-induced lung injury.
Next, we assessed whether increasing endothelial FOXF1 in already established lung fibrosis will improve the outcomes. Overexpression of FOXF1 in endothelial cells at day 10 after bleomycin injury decreased body weight loss, improved mice survival, and decreased collagen depositions in endFoxf1OE mice compared to bleomycin-injured control mice (Fig. 8a–i). Based on Flexivent measurements of lung mechanics, many functional parameters in endFoxf1OE mice were improved, including increased lung compliance, total lung capacity and decreased tissue resistance (Supplementary Fig. S16a–h). Finally, overexpression of FOXF1 in endothelial cells at day 10 after bleomycin injury, decreased expression of pro-fibrotic genes in total lung RNA and inhibited recruitment of macrophages to the lung tissue (Fig. 8j, k). Altogether, overexpression of FOXF1 in endothelial cells either prior to or after bleomycin injury attenuates pulmonary fibrosis and improves mice survival after the injury.
Nanoparticle delivery of non-integrating Foxf1 expression-vector into the lung endothelium attenuates pulmonary fibrosis
Since genetic overexpression of Foxf1 in endothelial cells attenuated pulmonary fibrosis, we next tested whether nanoparticle delivery of Foxf1 cDNA into endothelial cells can be a therapeutic approach to inhibit lung fibrosis. Mouse Foxf1 cDNA was cloned into non-integrating self-replicating episomal EEV vector to generate EEV-Foxf1 construct (Fig. 9a). Transfection of EEV-Foxf1 plasmid into Foxf1-negative HEK-293T cells increased the FOXF1 protein levels in vitro as shown by Western blot (Fig. 9b). To deliver EEV-Foxf1 plasmid to endothelial cells in vivo, we utilized the poly β-amino esters (PBAE) polymer30,31, which formed stable nanoparticles in complex with plasmid DNA (Fig. 9c and Supplementary Fig. S17a, b). The hydrodynamic average diameter of the PBAE nanoparticles loaded with EEV-Foxf1 plasmid DNA (Nano-Foxf1) was 146.27 ± 9.54 nm (Supplementary Fig. S17c), whereas the average surface charge of the Nano-Foxf1 was 28.3 ± 1.71 mV (Supplementary Fig. S17d). In vitro treatment of HEK-293T cells with nanoparticles containing EEV-Foxf1 plasmid resulted in the efficient expression of red fluorescent protein (RFP) in vast majority of cells (Supplementary Fig. S17e). Next, fluorescently-labeled PBAE nanoparticles with either EEV-Foxf1 or EEV-Empty (control) plasmids were delivered to bleomycin-treated mice (Fig. 9d, e and Supplementary Fig. S17f, g) via tail vein on the same day as bleomycin administration. Using FACS analysis of enzymatically digested lung tissue, nanoparticles were detected in ~92% of lung endothelial cells (CD31+/CD45−) (Fig. 9e). Nanoparticles-mediated targeting of epithelial (CD326+/CD45−/CD31−), immune (CD45+/CD31−) and mesenchymal (CD31−/CD45−/CD326−) cells was ineffective (Fig. 9d, e). Nanoparticles were still detected in ~50–70% of pulmonary endothelial cells at day 28 after single nanoparticle delivery (Fig. 9e and Supplementary Fig. S17g). Administration of EEV-Foxf1-nanoparticles increased Foxf1 mRNA in FACS-sorted lung endothelial cells as demonstrated by qRT-PCR (Fig. 9f). Treatment with nanoparticles containing pEEV-Foxf1 vector on the same day as bleomycin injury significantly increased survival of mice as shown by Kaplan–Meier curve (Supplementary Fig. S18a) and decreased collagen depositions in the lung tissue as shown by H&E and Sirius red staining (Supplementary Fig. S18b).
Next, we assessed the therapeutic efficacy of EEV-Foxf1-nanoparticles after lung injury. Mice were treated with EEV-Foxf1-nanoparticles at day 10 after bleomycin administration (Fig. 9f) when the fibrosis lung remodeling is already present (Fig. 2a). Nanoparticle delivery of Foxf1 into endothelial cells improved mice survival (Fig. 9g), prevented the loss of body weight after bleomycin injury (Supplementary Fig. S19a), and decreased lung collagen depositions quantified by Sircol assay (Fig. 9h). Decreased collagen amounts in EEV-Foxf1-treated lungs coincided with increased Foxf1 mRNA in FACS-sorted lung endothelial cells (Fig. 9i). Decreased fibrotic remodeling in EEV-Foxf1-treated lungs was detected and quantified based on Sirius red/Fast green staining, Trichrome staining, and immunostaining for αSMA (Fig. 9j–m). In addition, nanoparticle delivery of Foxf1 into endothelial cells at day 10 after bleomycin injury, reduced macrophage infiltration into fibrotic regions (Fig. 9j, right panels and Supplementary Fig. S19l), decreased expression of pro-fibrotic genes in total lung RNA (Fig. 9n), decreased Ashcroft score (Fig. 9o), improved lung compliance and total lung capacity in EEV-Foxf1-treated mice (Supplementary Fig. S19b–j) and arterial oxygenation (Supplementary Fig. S19k). Altogether, nanoparticle delivery of Foxf1 cDNA into endothelial cells on the day of bleomycin injury or during fibrotic stage after the injury inhibits pulmonary fibrosis and improves mice survival.