Lumican expression in diaphragm induced by mechanical ventilation

Abstract

Background: Diaphragmatic dysfunction found in the patients with acute lung injury required prolonged mechanical ventilation. Mechanical ventilation can induce production of inflammatory cytokines and excess deposition of extracellular matrix proteins via up-regulation of transforming growth factor (TGF)-β1. Lumican is known to participate in TGF-β1 signaling during wound healing. The mechanisms regulating interactions between mechanical ventilation and diaphragmatic injury are unclear. We hypothesized that diaphragmatic damage by short duration of mechanical stretch caused up-regulation of lumican that modulated TGF-β1 signaling.

Methods: Male C57BL/6 mice, either wild-type or lumican-null, aged 3 months, weighing between 25 and 30 g, were exposed to normal tidal volume (10 ml/kg) or high tidal volume (30 ml/kg) mechanical ventilation with room air for 2 to 8 hours. Nonventilated mice served as control groups.

Results: High tidal volume mechanical ventilation induced interfibrillar disassembly of diaphragmatic collagen fiber, lumican activation, type I and III procollagen, fibronectin, and α-smooth muscle actin (α-SMA) mRNA, production of free radical and TGF-β1 protein, and positive staining of lumican in diaphragmatic fiber. Mechanical ventilation of lumican deficient mice attenuated diaphragmatic injury, type I and III procollagen, fibronectin, and α-SMA mRNA, and production of free radical and TGF-β1 protein. No significant diaphragmatic injury was found in mice subjected to normal tidal volume mechanical ventilation.

Conclusion: Our data showed that high tidal volume mechanical ventilation induced TGF-β1 production, TGF-β1-inducible genes, e.g., collagen, and diaphragmatic dysfunction through activation of the lumican.

Figure 1. Electron microscopy of the diaphragm.

Representative micrographs (x80,000, longitudinal section and transverse section) of the diaphragmatic sections were from control nonventilated mice and mice ventilated at tidal volume 10 ml/kg or 30 ml/kg for 8 hours with room air (n = 2 per group). Increasing of interfibrillar spacings and disrupted diaphragmatic collagen fibers (black arrow) after ventilation injury, and variation of the cross-section were identified (white arrows). The severity were more aggravated in the lumican-null mice. Scale bars represent 115 nm. (+/+, wild type mice; −/−, lumican-null mice; C, control nonventilated; I, 10ml, ventilated at tidal volume 10 ml/kg; I, 30ml, ventilated at tidal volume 30 ml/kg)

Figure 2. Lumican deficient mice reduced high tidal volume ventilation-induced lumican activation in diaphragm.

(A) The mice were ventilated at 30 ml/kg for 2, 4, and 8 hours with room air. Western blot was performed using an antibody, which recognizes the lumican expression (Top Panel) and an antibody that recognizes glyceraldehydes-phosphate dehydrogenase (GAPDH) expression (Middle Panel). Arbitrary units were expressed as the ratio of lumican to GAPDH (Bottom Panel) (n = 3–5 per group). *P<0.05 versus control, nonventilated mice. (B) Representative photomicrographs (x400) with lumican staining of paraffin diaphragm sections with immunohistochemistry were from control, nonventilated mice and mice ventilated at 10 ml/kg or 30 ml/kg for 8 hours with room air. (n = 3–5 per group). An inset panel showed staining using isotype-matched controls. Positive dark brown diaminobenzidine (DAB) staining of membranes of muscle fiber is identified by arrows. (+/+, wild type; −/−, lum^−/−;C, control nonventilated; I, ventilated injury)

Figure 3. Lumican deficient mice reduced high tidal volume ventilation-induced transforming growth factor-β1 (TGF-β1) production.

TGF-β1 production in bronchoalveolar lavage (BAL) fluid was from control, nonventilated mice and mice ventilated at V_T 30 ml/kg for 2, 4, and 8 hours (A, n = 5 per group) or V_T 30 ml/kg for 8 hours (B, n = 5 per group). * P<0.05 versus control, nonventilated mice; †P<0.05 versus lum^−/− mice. WT: wild type C57BL/6 mice. (+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)

Figure 4. Lumican deficient mice reduced high tidal volume ventilation-induced type I procollagen mRNA expression in diaphragm.

The mice were ventilated at V_T 30 ml/kg at indicated time periods (A, n = 5 per group) or V_T 30 ml/kg for 2 hours (B, n = 5 per group). Reverse transcription-polymerase chain reaction (RT-PCR) was performed for type I procollagen mRNA (Top Panel), GAPDH mRNA (Middle Panel), and arbitrary units (Bottom Panel). Arbitrary units were expressed as the ratios of type I procollagen mRNA to GAPDH. *P<0.05 versus control, nonventilated mice; †P<0.05 versus lum^−/− mice. WT: wild type C57BL/6 mice. (+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)

Figure 5. Lumican deficient mice reduced high tidal volume ventilation-induced type III procollagen mRNA expression in diaphragm.

The mice were ventilated at V_T 30 ml/kg at indicated time periods (A, n = 5 per group) or V_T 30 ml/kg for 2 hours (B, n = 5 per group). RT-PCR was performed for type III procollagen mRNA (Top Panel), GAPDH mRNA (Middle Panel), and arbitrary units (Bottom Panel). Arbitrary units were expressed as the ratios of type III procollagen mRNA to GAPDH. *P<0.05 versus control, nonventilated mice; †P<0.05 versus lum^−/− mice. WT: wild type C57BL/6 mice. (+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)

Figure 6. Lumican deficient mice reduced high tidal volume ventilation-induced fibronectin mRNA expression in diaphragm.

The mice were ventilated at V_T 30 ml/kg at indicated time periods (A, n = 5 per group) or V_T 30 ml/kg for 2 hours (B, n = 5 per group). RT-PCR was performed for fibronectin mRNA (Top Panel), GAPDH mRNA (Middle Panel), and arbitrary units (Bottom Panel). Arbitrary units were expressed as the ratios of fibronectin mRNA to GAPDH. *P<0.05 versus control, nonventilated mice; † P<0.05 versus lum^−/− mice. WT: wild type C57BL/6 mice.(+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)

Figure 7. Lumican deficient mice reduced high tidal volume ventilation-induced α-SMA mRNA expression in diaphragm.

The mice were ventilated at V_T 30 ml/kg at indicated time periods (A, n = 5 per group) or V_T 30 ml/kg for 2 hours (B, n = 5 per group). RT-PCR was performed for α-SMA mRNA (Top Panel), GAPDH mRNA (Middle Panel), and arbitrary units (Bottom Panel). Arbitrary units were expressed as the ratios of α-SMA mRNA to GAPDH. *P<0.05 versus control, nonventilated mice; † P<0.05 versus lum^−/− mice. α-SMA, α-smooth muscle actin; WT: wild type C57BL/6 mice. (+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)

Figure 8. Lumican deficient mice reduced high tidal volume ventilation-induced malondialdehyde (MDA) production in diaphragm.

The mice were ventilated at V_T 30 ml/kg for 8 hours (n = 5 per group). * P <0.05 versus control, nonventilated mice; † P<0.05 versus lum^−/− mice. WT: wild type C57BL/6 mice. (+/+, wild type; −/−, lum^−/−; C, control nonventilated; I, ventilated injury)