Serological Assessment of Activated Fibroblasts by alpha-Smooth Muscle Actin (α-SMA): A Noninvasive Biomarker of Activated Fibroblasts in Lung Disorders

Figures

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  Figure 1. Alignment of the targeted α-SMA sequence in human, mouse, rat, chicken, bovine, and rabbit species (red box). The alignment was performed using Uniprot.

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  Figure 2. Standard curve and native reactivity. A typical standard curve and native reactivity against human serum and urine (A) and rat (B) serum and urine diluted 1:8 for the α-SMA assay, as indicated. Signals are shown as optical density (OD) at 450 nm, subtracting the background at 650 nm, as a function of standard peptide concentration.

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  Figure 3. Assay specificity. Reactivity to the standard peptide (Ac-EEEDSTALV), the elongated peptide (CEEEDSTALV), nonsense peptide (PGEILGHVPG), and nonacetylated α-SMA (EEEDSTALV) was tested for the α-SMA assay. The background signal from the system was tested using a nonsense coating peptide (Biotin-PGEILGHVPG). Signals are shown as optical density (OD) at 450, subtracting the background 650 nm, as a function of standard peptide concentration.

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  Figure 4. α-SMA in the SiaJ model. α-SMA was assessed in supernatant of media (background, n = 18), cells w/o addition of TGF-β (n = 18), and cells with 1 ng/ml TGF-β. Data are presented as Tukey boxplots, and lower limit of detection of the assay is marked with a dotted line. Significance levels: ns = no significance.

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  Figure 5. Western blot showing the specific bands of α-SMA in supernatant from the SiaJ model. Lane 1-4: Supernatant from four replicates from the SiaJ model without addition of TGF-β. Lane 5-8: Supernatant from four replicates from the SiaJ model with addition of 1 ng/ml TGF-β. Bands were observed at 42 kDa for the supernatants with addition of 1 ng/ml TGF-β, corresponding to the molecular weight of α-SMA.

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  Figure 6. Results from cohort 1. Serum α-SMA levels were assessed in healthy controls (n = 20) and in patients with IPF (n = 10), COPD (n = 13), and NSCLC (n = 9). Data were analyzed using a Kruskal-Wallis test adjusted for Dunn's multiple-comparisons test. Data are presented as Tukey boxplots. Significance levels: **P < .01, ***P < .001, and ****P < .0001.

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  Figure 7. ROC curves. ROC curve analysis was used to evaluate the ability of α-SMA to discriminate between (A) healthy controls and IPF patients, (B) healthy controls and COPD patients and, (C) healthy controls and NSCLC patients.

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  Figure 8. Results from cohort 2. (A) Serum α-SMA levels were assessed in healthy controls (n = 20) and NSCLC patients (n = 40). Data were analyzed using a Mann Whitney t test. Data are presented as Tukey boxplots. Significance levels: **P < .01. (B) ROC curve analysis was used to evaluate the ability of α-SMA to discriminate between healthy controls and NSCLC patients in cohort 2.