Research ArticlePulmonary fibrosis

Caveolin-1–derived peptide limits development of pulmonary fibrosis

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Science Translational Medicine  11 Dec 2019:
Vol. 11, Issue 522, eaat2848
DOI: 10.1126/scitranslmed.aat2848
  • Fig. 1 Suppression of profibrogenic responses in fLfs by CSP or its deletion peptides.

    (A) Human non-fLfs (hnLfs) and hfLfs (hfLfs) isolated from the lung tissues of healthy donors and patients with IPF or (B) mouse non-fLfs (mnLfs) and mouse fLfs (mfLfs) from uninjured mice and mice with BLM-induced PF, respectively, were treated with PBS, CSP (10 μM), or CP for 48 hours. Conditioned media and cell lysates were immunoblotted. (C) A series of overlapping deletions were made in CSP, and hfLfs were treated with these peptides and assessed for p53 and profibrogenic proteins to identify the minimum amino acids required for the CSP effects. (D) A series of truncations/mutations were made in CSP7 and tested in hfLfs and mfLfs using αSMA expression to assess bioactivity. (E) Total RNA from hfLfs and mfLfs treated with CSP7 or truncated or mutated CSP7 were analyzed for COL1, αSMA, FN, and TN-C mRNA by qRT-PCR. The values are presented as bar graphs after normalization with β-actin transcript. (F) hnLfs and hfLfs treated with PBS, CSP7, or CP for 48 hours were subjected to MTT assay to assess proliferation. The experiments were repeated two (A to D) or three times (E and F). Data in (E) and (F) are represented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained by one-way analysis of variance (ANOVA) with Turkey’s multiple comparison test.

  • Fig. 2 Intraperitoneal injection of CSP7 delivered daily limits 1× BLM-induced PF in mice.

    Mice exposed to saline or BLM by intranasal instillation were euthanized 7 to 21 days later. (A) Total lung hydroxylproline (n = 4 to 5 per group). (B) H&E- and trichrome-stained lung sections. BLM-exposed mice received intraperitoneal injections with or without CSP7 or CP (1.5 mg/kg) daily for 7 days starting d14 after BLM injury. (C) Percent survival during 7-day treatment relative to number of mice (n = 19 per group) on d14 after BLM. (D) Representative micro-CT images on d21 after BLM. Lung volumes (E) were measured by quantitative-CT renditions (n = 4 to 6 per group), and lung compliance (F) and elastance (G) were measured using a flexiVent system (n = 3 per group). (H) Lung weights presented as a bar graph (n = 3 per group). (I) H&E- and trichrome-stained lung sections. Lung homogenates from mice treated with CSP7 from two sources were tested for (J) hydroxyproline (n = 10 per group) and (K) soluble collagen (n = 15 per group), (L) profibrogenic proteins, or (M) mRNA (n = 3 per group). Lung homogenates were tested for TGF-β and CTGF (N) protein, (O and P) mRNA (n = 3 per group), and (Q) MPO (n = 4 to 5 per group). Total (R) and differential (S) counts from BALF were determined (n = 3 per group). The experiments were repeated two to three times. Bars indicate means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained by one-way ANOVA with Turkey’s multiple comparison and log-rank tests, respectively. Scale bars, 100 μm (B and I).

  • Fig. 3 Intraperitoneal injection of CSP7 delivered daily inhibits progression of TGF-β1–induced established PF.

    Mice were intranasally instilled with Ad-TGF-β1 to induce PF. Control mice were exposed to Ad-Ev. After 14 days, Ad-TGF-β1–treated mice received intraperitoneal injections of CSP, CSP7, or CP (1.5 mg/kg). (A) The percent survival 14 to 28 days after-Ad-TGF-β1 presented as a line graph relative to total number mice (n = 13 per group) at the beginning of CSP7 treatment. (B) Representative micro-CT images at 28 days after Ad-TGF-β1. (C) Lung volumes were measured using quantitative-CT renditions (n = 3 to 7 per group). Lung (D) compliance and (E) elastance were obtained by the flexiVent system (n = 3 to 9 per group). (F) Lung weights presented as bar graph (n = 3 per group). (G) Lung sections stained with H&E and trichrome. Scale bars, 100 μm. Whole-lung homogenates analyzed for (H) total hydroxyproline and (I) soluble collagen content (n = 5 to 6 per group). (J) Total protein and (K) RNA (n = 3 per group) from lung homogenates tested for profibrogenic marker protein and mRNA, respectively. (L) Neutrophil accumulation measured by MPO assay in lung homogenates (n = 4 per group). Lung lavage (M) total and (N) differential numbers of leukocytes (n = 3 per group). The experiments were repeated two times. Bars represent means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained by log-rank and one-way ANOVA with Turkey’s multiple comparison tests, respectively.

  • Fig. 4 PF induced by chronic repetitive-dose BLM was attenuated by CSP or CSP7 treatment in mice.

    Mice were exposed to BLM (2 U/kg) once every 2 weeks for a total of 8× in 4 months by intranasal instillation. Two weeks after the final dose of BLM, BLM-treated mice (n = 10 per group) received intraperitoneal injections of CSP, CSP7, and CP (1.5 mg/kg) daily for 2 weeks. (A) The percent survival during 2 weeks of CSP7 treatment presented as a line graph. (B) Representative micro-CT images 2 weeks after peptide treatment. (C) Lung volumes were measured using quantitative-CT renditions (n = 3 to 4 per group). Lung (D) compliance and (E) elastance were obtained by the flexiVent (n = 3 to 5 per group). (F) Lung weights presented as bar graph (n = 3 per group). (G) Lung sections stained with H&E and trichrome to assess PF. Scale bars, 100 μm. The lung homogenates were tested for (H) hydroxyproline and (I) soluble collagen (n = 6 to 10 per group). The lung protein and RNA were analyzed for profibrogenic (J) proteins and (K) mRNAs (n = 3 per group). (L) Neutrophil accumulation was measured by MPO assay in lung homogenates (n = 4 per group). (M) Total and (N) differential counts of lung lavage leukocytes (n = 3 per group). The experiments were repeated twice. Bars depict means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were obtained by one-way ANOVA with Turkey’s multiple comparison test and log-rank tests, respectively.

  • Fig. 5 Effect of CSP7 modification and nebulization on antifibrotic activity and peptide structure.

    WT mice with 1× BLM-induced PF were treated with capped CSP7 (using acetyl group at the N termini) or uncapped CSP7 and CP daily for 7 days, starting d14 after BLM injury. The lung homogenates were immunoblotted for (A) profibrogenic proteins or analyzed for (B) total hydroxyproline content (n = 4 to 5 per group). (C) hfLfs treated with PBS, CSP, or CSP7 with (CSP7-M) or without (CSP7) mannitol for 48 hours were immunoblotted for profibrogenic proteins. (D) The lung homogenates from mice treated with CSP7 or CSP7-M as in (A) were immunoblotted for profibrogenic proteins. (E) Percent recovery of nebulized CSP7. (F) Mass spectroscopy of fluids collected after nebulization. (G) Immunoblots of lysates of hfLfs exposed to DPBS, CSP7, CP, or CSP7 nebulized using Aeroneb Pro (CSP7N) analyzed for protein expression. m/z, mass/charge ratio. (H) Immunoblots of lysates from two hfLf lines (#9 and #17) treated with DPBS containing lactose solution (PBS) or formulated with CSP7 or CP (50 μM) in DPBS containing lactose for 48 hours analyzed for profibrogenic proteins. The experiments were repeated two times. Bars show mean ± SD. ****P < 0.0001 were obtained by one-way ANOVA with Turkey’s multiple comparison test.

  • Fig. 6 Nebulization of CSP7 delivered daily resolved single-dose BLM–induced PF.

    WT mice were exposed to saline or BLM (8 U/kg). After 14 days, mice exposed to BLM were treated with/without placebo, formulated CSP7, or CP containing lactose monohydrate stabilizer for 2 hours daily for 7 days using a nose-only nebulization tower. (A) The percent survival from 14 to 21 days after BLM injury in each group (n = 20 per group) presented as a line graph relative to total number of mice at the beginning (as 100%). (B) The body weights of treated and untreated animals were measured and represented as a bar graph (n = 5 to 9 per group). All mice were euthanized 21 days after BLM exposure. (C) Lung weights presented as bar graph (n = 4 to 9 per group). Whole-lung homogenates were analyzed for (D) total hydroxyproline (n = 5 to 10 per group) and (E) soluble collagen (n = 5 to 7 per group). (F) Total lung proteins were immunoblotted for profibrogenic proteins. (G) Whole-lung RNA was tested for changes in profibrogenic mRNAs (n = 3 per group). Lung homogenates were tested for TGF-β and CTGF (H) protein and (I and J) mRNA (n = 4 per group). (K) H&E and trichrome staining of lung sections. Magnification, ×20. Scale bars, 100 μm. (L) MPO assay of lung homogenates for pulmonary neutrophil accumulation (n = 3 to 4 per group). The experiments were repeated twice. Bars represent mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained by log-rank and one-way ANOVA with Turkey’s multiple comparison tests, respectively.

  • Fig. 7 DPI of air jet–milled CSP7 mitigates single-dose BLM–induced PF in mice.

    (A) The aerosol performance of micronized CSP7 DP was evaluated by NGI, showing the particle deposit in the NGI stages. CSP7 exhibited high fine particle fraction (FPF) with respirable aerodynamic size (repeated three times). MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation; MOC, micro-orifice collector. WT mice (n = 10 per group) were exposed to either saline or BLM (2.8 U/kg) by intratracheal instillation. After 14 days, BLM-treated mice were left untreated (none) or exposed to air jet–milled CSP7 using a rotating-brush generator, with a consistent chamber concentration of 0.1 mg/liter for 15 min daily for 7 days. Mice exposed to unprocessed CSP7 (1.5 mg kg−1 day−1) by intraperitoneal injection for 7 days were used as positive control. All mice were euthanized 21 days after BLM exposure. (B) Lung weights were measured and presented as bar graph (n = 5 per group). Whole-lung homogenates from mice (n = 3 to 10 per group) were analyzed for (C) total hydroxyproline and (D) soluble collagen contents. Total lung homogenates were immunoblotted for profibrogenic marker and cytokine (TGF-β and CTGF) (E) proteins and (F) mRNAs (n = 3 per group). (G) Pulmonary neutrophil accumulation was measured by MPO assay of lung homogenates (n = 3 per group). (H) Lung sections were H&E- and trichrome-stained. Magnification, ×20. Scale bars, 100 μm. The experiments were repeated two times. Bars indicate mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 were obtained by one-way ANOVA with Turkey’s multiple comparison test.

  • Fig. 8 CSP7 inhibits p53 and apoptosis in AECs of fibrotic lungs.

    (A) CSP deletion fragment sequences. Boxed region shows FTTFTVT. Mice received intraperitoneal injections of CSP deletion fragments or CP (1.5 mg/kg) 1 day after 1× BLM. AECs isolated 3 days later were immunoblotted. (B) Lithium carbonate and SP-C–immunofluorescence staining of uninjured AECs. DAPI, 4′,6-diamidino-2-phenylindole. (C) LDH assay of cultured medium from mouse naïve AECs or AECs treated with BLM (40 μg/ml) alone, BLM + CSP7, or BLM + CP in vitro (n = 3 to 6 per group). (D) Pooled AECs from mice (n = 4 per group) receiving intraperitoneal injections of CSP, CSP7, or CP (1.5 mg/kg) daily for 7 days starting d14 after BLM injury were tested for p53/apoptosis. (E) Lung sections were TUNEL-stained and IHC-stained for apoptosis [cleaved caspase-3 (Cl. cas3)]. (F) Pooled AECs from mice (n = 4 to 5 per group) treated intranasally with Ad-TGF-β1 before daily intraperitoneal injections of CSP7 or CP (1.5 mg/kg) for 2 weeks starting 14 days after transduction were tested for p53/apoptosis. (G) TUNEL and IHC of lung sections. (H) Pooled AECs from mice (n = 4 to 5 per group) exposed to BLM (2 U/kg) by intranasal instillation once every 2 weeks for a total of 8× in 4 months, treated with intraperitoneal injections of CSP7 or CP (1.5 mg/kg) daily for 2 weeks, starting 2 weeks after the final dose of BLM, were tested for p53 and apoptosis. (I) TUNEL and IHC of lung sections. Magnification, ×20. Scale bars, 100 μm. (J) Pooled human normal and IPF lung tissues (n = 4) untreated or treated with CSP7 or CP ex vivo were immunoblotted. (K) AECs isolated from tissues treated as in (J) were immunoblotted. (L) Lysates of AECs isolated from control and IPF tissues treated in vitro as in (J) were immunoblotted. The experiments were repeated twice. Bars represent means ± SD. ***P < 0.001 was obtained by one-way ANOVA with Turkey’s multiple comparison test.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/522/eaat2848/DC1

    Materials and Methods

    Fig. S1. Effect of CSP7 on TGF-β signaling and HuR expression in mouse lung with 1× BLM-induced PF.

    Table S1. In vitro Ames test.

    Table S2. In vitro micronucleus assay.

    Table S3. In vivo micronucleus screening in reticulocytes for clastogenic activity.

    Data file S1. Primary data.

    Reference (37)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Effect of CSP7 on TGF-β signaling and HuR expression in mouse lung with 1× BLM-induced PF.
    • Table S1. In vitro Ames test.
    • Table S2. In vitro micronucleus assay.
    • Table S3. In vivo micronucleus screening in reticulocytes for clastogenic activity.
    • Reference (37)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

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