Mechanisms of Bone Marrow-Derived Fibrocytes Contributing to COPD Patients with Chronic Hypoxemia Induced Pulmonary Hypertension

Pulmonary hypertension is a common complication of chronic obstructive pulmonary disease (COPD). 5–10% of patients with advanced COPD may suffer from severe pulmonary hypertension and present with a progressively downhill clinical course because of right heart failure added to ventilatory handicap. Pulmonary hypertension in COPD progresses over time and its severity correlates with the degree of airflow obstruction and the impairment of pulmonary gas exchange. Animal studies have been shown that a significant accumulation of fibrocytes in the adventitia and media of the remodeled pulmonary arteries. Reduction of fibrocytes in the circulation of chronically hypoxic rats led to a marked attenuation of adventitial thickening and decreased fibrosis. Our recent published paper is one of the first to show directly the relationship between fibrocytes, and chronic airflow obstruction with rapid decline in FEV1 in asthma. There may be a link between fibrocytes and the remodeling process. Less is known at present that fibrocytes may contribute to the vascular remodeling of pulmonary arterioles in COPD with chronic hypoxemia. Our previous studies have shown that there is an increased expression of EGFR in circulating fibrocytes of chronic obstructive asthmatics. TGF-1 may play an important role implicated in the EGFR transactivation contributing to cell proliferation. Oxidant stress may have a synergic effect of TGF-1 inducing EGFR transactivation and the effect can be abolished by an inhibition of ERK and NF-B activity. Therefore, We hope to clarify the higher expression of CCR7, CXCR4, EGFR, ADAM-17, VEGFR and ET-1R on circulating fibrocytes in COPD which are related to chronic hypoxemia induced pulmonary hypertension. In this three year study, non-adherent non-T (NANT) cells will be separated from 80 ml peripheral blood of COPD with (n=30) or without (n=30) chronic hypoxemia and pulmonary hypertension (PH), and healthy subjects with smoker (n=20) and non-smoker (n=20). The proportion of fibrocytes will be indentified by triple staining of ant-CD34, anti-CD45 and anti-collagen I antibodies and determined by flowcytometry. The myofiroblasts are determined by staining of anti--SMA antibody conjugated PE. We will demonstrate the expression of EGFR, ADAM-17, CCR7 and CXCR4 on fibrocytes by triple staining and analyzed by flowcytometry. We will culture the 5105 NANT cells for 14 days to determine whether fibrocytes from COPD with chronic hypoxemia and PH have higher proliferation and transformation into myofibroblasts, contributing to vascular remodeling of PH. To investigate whether the TGF-1 induced EGFR transactivation through the activation of ADAM-17, overexpression of ADAM-17 by transfected with ADAM-17 plasmid and knockout of ADAM-17 by transfected with siRNAs targeting ADAM 17 will evaluate the co-expression of ADAM-17 and EGFR on fibrocytes and determine the proliferation capacity and transformation into myofibroblasts of fibrocytes after 14-day culture. To investigate the molecular mechanisms of TGF-1 or oxidative stress involved in the EGFR transactivation, fibroblasts will be incubated with TGF-1 (10 ng/ml) or/and H2O2 at various concentration or plus inhibitors of ERK, PI-3K, NF-B and Src or inhibitors alone. The expression of Akt, ERK, PI-3K, and NF-kB is measured by Western blot. In some permissive condition, fibrocytes may migrate in into inflammatory tissue and lead to remodeling. Using migration assay, we will determine which chemtaxic factors, such as CCL19, CXCL12, platelet-derived growth factor, may contribute to the homing effect of fibrocytes. We will also use the following blocking or neutralizing antibodies of CCR7, CXCR4, TGF-β, and PDGF to assess the migratory assay of fibrocytes from different COPD patients and normal subjects. In the third year, we will use microarray to study the expression of proliferative or angiogentic genes in the fibrocytes to see any difference between COPD associated with or without chronic hypoxemia and pulmonary hypertension. We will use microarray to study the gene expression from fibrocytes of overexpression of ADAM-17 and CCR7. We will also perform Proteomics analysis to study the different protein or gene expression of fibrocytes stimulated by TGF-1 and oxidative stress, and if so, we will study the mechanisms by which this effect may occur. This research may lead to greater focus on the treatment of airways disease, and to new ways of treating these diseases that is currently unsatisfactory. The results may shed light on the new area of pathogenesis of COPD with chronic hypoxemia and PH, and may advance the scope of therapy for COPD.

Project ID:PC10001-1131
External Project ID:NSC99-2314-B182A-102-MY3