Active transforming growth factor-beta1 activates the procollagen I promoter in patients with acute lung injury

Abstract

Objective: Fibroproliferation markers like procollagen I predict mortality in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We sought to determine whether bronchoalveolar lavage fluid (BALF) from patients with lung injury contained mediators that would activate procollagen I promoter and if this activation predicted important clinical outcomes.

Design: Prospective controlled study of ALI/ARDS.

Setting: Intensive care units and laboratory of a university hospital.

Patients and participants: Acute lung injury/ARDS, cardiogenic edema (negative controls) and pulmonary fibrosis (positive controls) patients.

Interventions: Bronchoalveolar lavage fluid was collected within 48 h of intubation from ALI/ARDS patients. BALF was also collected from patients with pulmonary fibrosis and cardiogenic pulmonary edema. Human lung fibroblasts were transfected with a procollagen I promoter-luciferase construct and incubated with BALF; procollagen I promoter activity was then measured. BALF active TGF-beta1 levels were measured by ELISA.

Results: Twenty-nine ARDS patients, nine negative and six positive controls were enrolled. BALF from ARDS patients induced 41% greater procollagen I promoter activation than that from negative controls (p<0.05) and a TGF-beta1 blocking antibody significantly reduced this activation in ARDS patients. There was a trend toward higher TGF-beta1 levels in the ARDS group compared to negative controls (-1.056 log(10)+/-0.1415 vs -1.505 log(10)+/-0.1425) (p<0.09). Procollagen I promoter activation was not associated with mortality; however, lower TGF-beta1 levels were associated with more ventilator-free and ICU-free days.

Conclusions: Bronchoalveolar lavage fluid from ALI/ARDS patients activates procollagen I promoter, which is due partly to TGF-beta1. Activated TGF-beta1 may impact ARDS outcome independent of its effect on procollagen I activation.

Fig. 1

Procollagen I promoter activation. Primary cultures of normal human lung fibroblasts were transiently transfected with plasmids encoding the human procollagen I promoter driving luciferase, which were then incubated with BALF from patients with ARDS, (-) controls, or (+) controls. Luciferase activity was measured in cell lysates 24 h later and is expressed as the percentage of the maximal (0.5 ng/ml TGF-β1) stimulated value. The horizontal bars represents sample means. p<0.001 for comparison between positive and negative controls and p<0.05 for comparison between ARDS and negative controls

Fig. 2

Procollagen I peptide levels in ARDS patients (n=11) compared to negative controls. An ELISA assay was used to measure procollagen I peptide in the BALF from patients with ARDS and negative controls. The horizontal bars represents sample means. p<0.03 for comparison between ARDS patients and controls

Fig. 3

There is a trend toward higher active TGF-β1 levels in patients with ARDS than in controls. An ELISA assay was used to measure active TGF-β1 in the BALF from patients with ARDS or negative controls. All experiments were repeated in duplicate. The horizontal bars represents sample means. p<0.09 for comparison between ARDS patients and controls

Fig. 4

Active TGF-β1 levels correlated with procollagen I peptide levels. The correlation coefficient between active TGF-β1 and procollagen I peptide levels in BALF from ARDS patients (n=11) was 0.73 (p<0.02)

Fig. 5

TGF-β1 in the BALF is largely responsible for procollagen I promoter activation. Primary cultures of normal human lung fibroblasts were transiently transfected with the human procollagen I promoter- luciferase plasmid construct. They were then exposed to BALF from ARDS patients with the highest PIP activity (n=5) in the presence or absence of an antibody to TGF-β1 and luciferase activity was assessed 24 h later. There was a significant fall in procollagen I promoter activation in the presence of TGF-β1 antibody, p=0.008

Fig. 6

Procollagen I promoter activation and active TGF-β1 levels and mortality. BALF procollagen I promoter activation was not different in patients who died from ARDS (28-day all cause mortality) and those who survived, p=0.56 (a). A similar analysis was conducted comparing Log active TGF-β1 levels in patients with ARDS (b). Non-survivors had higher mean levels than survivors, though the difference was not statistically significant, p=0.14

Fig. 7

Survival curves in ARDS patients with high and low TGF-β1 levels. The highest third (High TGF-β1) of TGF-β1 values were compared to the lowest two thirds (Low TGF-β1) to determine the relationship of high TGF-β1 levels and survival (p<0.51)