Research ArticleBiliary Atresia

Large-scale proteomics identifies MMP-7 as a sentinel of epithelial injury and of biliary atresia

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Science Translational Medicine  22 Nov 2017:
Vol. 9, Issue 417, eaan8462
DOI: 10.1126/scitranslmed.aan8462
  • Fig. 1. Study design.

    A total of 175 BA subjects and 70 subjects with IHC were randomized into the discovery cohort (BA = 35 and IHC = 35), validation cohort #1 (BA = 35 and IHC = 35), and validation cohort #2 (BA = 105). Serum proteins were measured by high-throughput proteomic assay SOMAscan) separately for individual cohorts. MMP-7, matrix metalloproteinase-7; ENPP7, ectonucleotide pyrophosphatase/phosphodiesterase family member 7.

  • Fig. 2. Top nine proteins differentially expressed between BA and IHC.

    Data from the discovery cohort categorized into BA (n = 35), IHC (n = 35), and age-matched normal controls (NCs; n = 9). Dots represent the log of relative fluorescent unit (RFU) for individual serum proteins. Bars and whiskers represent median and interquartile range, respectively. Q values from analysis of variance (ANOVA) with multiple hypothesis correction (Benjamini-Hochberg procedure). IGFBP-1, insulin-like growth factor-binding protein 1; ADAM9, disintegrin and metalloproteinase domain-containing protein 9; GCP-2, C-X-C motif chemokine 6; PIGR, polymeric immunoglobulin receptor; SARP-2, secreted frizzled-related protein 1.

  • Fig. 3. Predictive features of MMP-7, ENPP7, and GGT for BA.

    (A) ROC curves for serum MMP-7 and/or ENPP7 in distinguishing BA from IHC in multivariable logistic regression analyses and dot plots of the proteins in the discovery cohort. Data are shown as log of RFU; central horizontal lines and whiskers represent median and interquartile range. (B) Prediction models for MMP-7, ENPP7, and GGT. Sensitivity, specificity, and positive and negative predictive values were calculated at selected optimal cutoff. (C) Serum GGT concentrations in subjects with BA and IHC; normal range is 5 to 59 U/liter. The right panel shows ROC curves for MMP-7 and/or GGT. Q values from ANOVA with multiple hypothesis correction (Benjamini-Hochberg procedure) for (A) and P value from Mann-Whitney test for (C). NPV, negative predictive value; PPV, positive predictive value.

  • Fig. 4. MMP-7 biomarker validation, ROC curves according to serum GGT concentrations, and serum MMP-7 concentration.

    (A) ROC curves in discovery and validation cohort #1 for MMP-7, ENPP7, and GGT. (B) ROC curves (95% CI) for subjects with GGT 100 to 300 U/liter (n = 10 for BA and n = 25 for IHC) and >300 U/liter (n = 50 for BA and n = 13 for IHC). Groups were formed with a combination of subjects with BA and IHC in the discovery cohort and validation cohort #1 based on the availability of GGT. (C) Bar graph of serum MMP-7 in randomly selected samples from patients with BA (n = 10), IHC (n = 10), and NCs (n = 4) measured by antibody-based ELISA compared with (D) the values measured by SOMAscan. Data are presented as median and interquartile range.

  • Fig. 5. MMP-7 expression in human liver, EHBD, and gallbladder.

    (A) Immunohistochemical staining of normal human liver shows minimal or no expression of MMP-7 in hepatocytes and IHBDs but intense expression in the biliary epithelium of gallbladder (GB) and EHBD. Scale bars, 50 μm (in liver, GB, and EHBD) and 20 μm (in IHBD). (B) Variable intensity of expression (0 to 3+) of MMP-7 in the cytoplasm of intrahepatic biliary epithelium of BA subjects. (C) Livers of patients with α1-antitrypsin deficiency had lower MMP-7 expression in the IHBDs. Scale bar, 20 μm for both (B) and (C). (D) Percentage of livers showing different intensities of MMP-7 immunostaining in patients with BA and α1-antitrypsin (A1AT) deficiency. (E) Hepatic mRNA expression of MMP-7 for BA (n = 64), IHC (n = 14), and NC (n = 7). Expression is normalized to NCs and presented as fold change. P values are from Kruskal-Wallis test.

  • Fig. 6. MMP-7 expression in experimental BA.

    (A) Serum MMP-7 measured in mice 7 days after RRV or saline injection (n = 3 to 4 per group). Means ± SEM, P values are from unpaired t test. (B and C) Mmp-7 mRNA expression in the livers (B) and EHBDs (C) at 3, 7, and 14 days after RRV or saline injection (n = 3 to 4 per group and per time point). Values are normalized to Gapdh and are expressed as means ± SEM; P values were for the comparison between two groups at each time point (ANOVA). (D) Liver immunohistochemistry detects MMP-7 in extramedullary hematopoietic cells (arrowheads) in saline-injected mice predominantly at day 3. Hepatic hematopoietic cells decrease after RRV, with MMP-7 being detected in inflammatory cells (arrows) infiltrating the portal tracts at days 7 and 14 after RRV injection. There was no staining of IHBD epithelium. (E) MMP-7 is detected in the duct epithelium in RRV and saline injected groups. At days 7 and 14 after RRV injection, MMP-7 is also detected in inflammatory cells and injured epithelium. Asterisks depict the duct lumen (or lack thereof); scale bars, 20 μm [for (D) and (E)].

  • Fig. 7. Suppression of tissue injury, inflammation, and cytokine expression by batimastat and MMP-7 antibody in experimental BA.

    Neonatal BALB/c mice were injected with RRV in the first day of life and then were injected with batimastat (RRV + batimastat; n = 22), GM6001 (non–MMP-7, RRV + GM6001; n = 17), anti–MMP-7 antibody (RRV + MMP-7 Ab; n = 12), or vehicle in the control group (RRV + vehicle; n = 14). (A) mRNA expression of tissue Mmps in the liver and bile duct at 3 and 7 days after viral injection. (B) Representative liver sections showing variable degrees of portal inflammation and hepatic necrosis in different groups. (C) Representative sections of EHBDs in each group. Asterisks depict the duct lumen (or lack thereof). Scale bars, 100 μm for (B) and 20 μm for (C). (D) Graphs depict the frequency of mice with liver injury, according to the degree of inflammation and hepatocellular necrosis, and frequency of mice with obstructed lumen. (E) Hepatic mRNA expression for cytokines/chemokines 12 days after RRV injection and treatment with batimastat or vehicle (as controls). P values are from ANOVA for (A), chi-square test for (D), and unpaired t test for (E).

  • Table 1. Clinical and biochemical characteristics of subjects.

    Data are presented as the means ± SD. Mann-Whitney test was used. F, female; M, male; GGT, γ-glutamyltranspeptidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TB, total bilirubin.

    VariableDiscovery cohortValidation cohort #1Validation cohort #2
    BAIHCP*BAIHCP*BAP
    Number of patients35353534105
    Age (days)62 ± 2857 ± 310.4367 ± 3066 ± 350.2472 ± 280.07
    Gender (F/M)16/196/2917/1812/2253/52
    AST (U/liter)268 ± 381246 ± 2080.61211 ± 135270 ± 2580.59233 ± 1920.65
    ALT (U/liter)176 ± 177187 ± 1650.80135 ± 90207 ± 2210.60167 ± 1250.77
    TB (mg/dl)8.4 ± 3.28.5 ± 4.40.858.8 ± 3.98.9 ± 4.60.978.3 ± 3.00.99
    GGT (U/liter)619 ± 361179 ± 169<0.0001744 ± 519241 ± 305<0.0001842 ± 6290.11

    *Comparison between BA and IHC.

    †Comparison between BA in the discovery cohort and validation cohort #2

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/417/eaan8462/DC1

      Fig. S1. GGT mRNA expression.

      Fig. S2. Relationship between MMP-7 and hepatic fibrosis at the time of diagnosis of BA.

      Fig. S3. Murine Ggt1 mRNA expression.

      Table S1. Protein results from SOMAscan (provided as an Excel file).

      Table S2. List of 76 serum proteins differentially expressed in BA and IHC.

      Table S3. Prediction of BA by MMP-7 in validation cohort #2.

      Table S4. Primer sequences and PCR product sizes for mouse Mmp-7-, Ggt1-, Th1-, Tnfa-, and IL8-related genes.

    • Supplementary Material for:

      Large-scale proteomics identifies MMP-7 as a sentinel of epithelial injury and of biliary atresia

      Chatmanee Lertudomphonwanit, Reena Mourya, Lin Fei, Yue Zhang, Sridevi Gutta, Li Yang, Kevin E. Bove, Pranavkumar Shivakumar, Jorge A. Bezerra*

      *Corresponding author. Email: jorge.bezerra{at}cchmc.org

      Published 22 November 2017, Sci. Transl. Med. 9, eaan8462 (2017)
      DOI: 10.1126/scitranslmed.aan8462

      This PDF file includes:

      • Fig. S1. GGT mRNA expression.
      • Fig. S2. Relationship between MMP-7 and hepatic fibrosis at the time of diagnosis of BA.
      • Fig. S3. Murine Ggt1 mRNA expression.
      • Table S2. List of 76 serum proteins differentially expressed in BA and IHC.
      • Table S3. Prediction of BA by MMP-7 in validation cohort #2.
      • Table S4. Primer sequences and PCR product sizes for mouse Mmp-7-, Ggt1-, Th1-, Tnfa-, and IL8-related genes.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S1. Protein results from SOMAscan (provided as an Excel file).

      [Download Table S1]

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