Extracellular matrix composition is modified by ß2-agonists through cAMP in COPD.

Long acting ß2-agonists (LABA) have been reported to modify the extracellular matrix (ECM) composition in the airway wall. Based on our earlier studies we here investigated the mechanism underlying the control of ECM modification by LABA in primary human airway smooth muscle cells. Cells were treated with formoterol or salmeterol (30 min) before TGF-ß1 stimulation (2-3 days) Using RT-PCT, immuno-blotting and ELISA the de novo synthesis and deposition of collagen type-I, -III, -IV and fibronectin were determined. Matrix metalloproteinases (MMP)-2 and -9 were analyzed by zymography. Both LABA activated cAMP and its corresponding transcription factor CREB within 60 min and thus partly reduced TGF-ß1-induced gene transcription of collagen type-I, -III, fibronectin and connective tissue growth factor (CTGF). The inhibitory effect of both LABA on collagen type-I and -III deposition involved a cAMP dependent mechanism, while the inhibitory effect of the two drugs on TGF-ß1-induced fibronectin deposition and on CTGF secretion was independent of cAMP. Interestingly, none of the two LABA reduced CTGF-induced synthesis of collagen type-I or type-III deposition. In addition, none of the two LABA modified collagen type-IV deposition or the expression and activity of MMP-2 or MMP-9. Our results show that LABA can prevent de novo deposition of specific ECM components through cAMP dependent and independent signaling. However, they do not reduce all ECM components by the same mechanism and they do not reduce existing collagen deposits. This might explain some of the controversial reports on the anti-remodeling effect of LABA in chronic inflammatory lung diseases.

The interaction of endothelin-1 and TGF-ß1 mediates vascular cell remodeling.

BACKGROUND: Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-ß1) and connective tissue growth factor. OBJECTIVE: To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC. METHODS: PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-ß1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580. RESULTS: ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-ß1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-ß1. CONCLUSION AND CLINICAL RELEVANCE: ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-ß1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-ß1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

The calcium channel blocker amlodipine exerts its anti-proliferative action via p21(Waf1/Cip1) gene activation.

Proliferation of vascular smooth muscle cells (VSMC) contributes to the progression of atherosclerotic plaques. Calcium channel blockers have been shown to reduce VSMC proliferation, but the underlying molecular mechanism remains unclear. p21(Waf1/Cip1) is a potent inhibitor of cell cycle progression. Here, we demonstrate that amlodipine (10(-6) to 10(-8) M) activates de novo synthesis of p21(Waf1/Cip1) in vitro. We show that amlodipine-dependent activation of p21(Waf1/Cip1) involves the action of the glucocorticoid receptor (GR) and C/EBP-alpha. The underlying pathway apparently involves the action of mitogen-activated protein kinase or protein kinase C, but not of extracellular signal-related kinase or changes of intracellular calcium. Amlodipine-induced p21(Waf1/Cip1) promoter activity and expression were abrogated by C/EBP-alpha antisense oligonucleotide or by the GR antagonist RU486. Amlodipine-dependent inhibition of cell proliferation was partially reversed by RU486 at 10(-8) M (58%+/-29%), antisense oligonucleotides targeting C/EBP-alpha (91%+/-26%), or antisense mRNAs targeting p21(Waf1/Cip1) (96%+/-32%, n=6); scrambled antisense oligonucleotides or those directed against C/EBP-beta were ineffective. The data suggest that the anti-proliferative action of amlodipine is achieved by induction of the p21 (Waf1/Cip1) gene, which may explain beneficial covert effects of this widely used cardiovascular therapeutic drug beyond a more limited role as a vascular relaxant.

Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension.

Primary pulmonary hypertension is a fatal disease causing progressive right heart failure within 3 years after diagnosis. We describe a new concept for treatment of the disease using vasoactive intestinal peptide, a neuropeptide primarily functioning as a neurotransmitter that acts as a potent systemic and pulmonary vasodilator. Our rationale is based on the finding of a deficiency of the peptide in serum and lung tissue of patients with primary pulmonary hypertension, as evidenced by radioimmunoassay and immunohistochemistry. The relevance of this finding is underlined by an upregulation of corresponding receptor sites as shown by Northern blot analysis, Western blot analysis, and immunological techniques. Consequently, the substitution with the hormone results in substantial improvement of hemodynamic and prognostic parameters of the disease without side effects. It decreased the mean pulmonary artery pressure in our eight study patients, increased cardiac output, and mixed venous oxygen saturation. Our data provide enough proof for further investigation of vasoactive intestinal peptide and its role in primary pulmonary hypertension.

Long-term treatment with oral sildenafil in addition to continuous IV epoprostenol in patients with pulmonary arterial hypertension.

OBJECTIVES: To evaluate the effect of long-term oral therapy with sildenafil in patients with pulmonary arterial hypertension receiving long-term IV epoprostenol. DESIGN: Open, uncontrolled trial. SETTING: University hospital. PATIENTS: Two patients with primary pulmonary hypertension and one patient with pulmonary arterial hypertension after surgical closure of an atrial septal defect. All patients were receiving continuous epoprostenol for 1.7 to 7.1 years; two patients also received inhaled iloprost for 1.8 years and 3.8 years, respectively. INTERVENTIONS: Addition of oral sildenafil, up to 200 mg/d, divided in four to six single doses, and hemodynamic measurements and the 6-min walking distance (6MWD) before and after 5 months of treatment with sildenafil. RESULTS: One patient was treated with sildenafil, 200 mg/d; two patients received 75 mg/d due to nausea and headache. Long-term treatment with sildenafil in the three patients reduced mean pulmonary artery pressure by 14%, 41%, and 22%, respectively; in two patients, pulmonary vascular resistance was decreased by 52% and 55%. The 6MWD increased by 34%, 6%, and 29%, respectively. No significant systemic hypotension or decrease of arterial oxygen saturation was seen. CONCLUSION: Sildenafil therapy may be of benefit in patients with pulmonary arterial hypertension receiving long-term infusion of epoprostenol.

Interaction of C/EBPalpha and the glucocorticoid receptor in vivo and in nontransformed human cells.

Belonging to the family of steroid hormones, glucocorticoids are essential for development and survival of vertebrates. The cellular response to glucocorticoids is attributed to the glucocorticoid receptor, which functions as a transcription factor. However, the majority of glucocorticoid-modulated genes lack a DNA binding site for the glucocorticoid receptor, raising the question of which mechanism mediates the responses to glucocorticoids. It has been suggested that besides direct DNA binding of the glucocorticoid receptor, interaction with members of other transcription factor families modulates the effect of the glucocorticoid receptor. However, the significance of such transcription factor interaction is not clear. In cultured human mesenchymal cells and peripheral blood leukocytes of human volunteers treated with glucocorticoids, we detected the formation of a complex between the GR and the CCAAT/enhancer binding protein alpha. In in vitro experiments, this interaction turned out to be responsible for the inhibitory action of glucocorticoids on lymphocytic and mesenchymal cell proliferation. Our results suggest that complex formation of the GR with C/EBPalpha accounts for a novel pathway of glucocorticoid action.