Inflammatory cytokine IL-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated IL-6’s role in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung specific IL-6 over expression transgenic mice (Tg(+)) and compared to wild type (Tg(-)) controls in both normoxic and chronic hypoxic conditions. The Tg(+) mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6-induced arteriopathic changes were accompanied by activation of pro-angiogenic factor, vascular endothelial growth factor, the pro-proliferative kinase, ERK, pro-proliferative transcription factors c-MYC and MAX, the anti-apoptotic proteins survivin and Bcl-2, and down-regulation of the growth inhibitor transforming growth factor-β, and pro-apoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through pro-proliferative anti-apoptotic mechanisms.

MATERIALS AND METHODS HUVEC. HUVECs were obtained from umbilical cords from cesarean sections. The cells were cultured by standard methods in the presence of heparin and endothelial-cell growth supplement (7). They were characterized by their homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. HUVECs were used for these studies after three to five passages.For clonal culture to exclude contamination with hematopoietic stem cells, HUVECs (1-5 x 105 cells per ml) were plated in methylcellulose cultures as described (8), with the modification that half of the fetal bovine serum was replaced with human umbilical cord blood serum. Hematopoietic growth factors were added as follows: rhEpo, 2 units/ml; stem-cell factor, 10 pg/ml; granulocyte/macrophage-colonystimulating factor, 200 units/ml; interleukin 3, 200 units/ml; endothelial-cell growth factor, 20 ktg/ml.Preparation of mRNA. After 1-5 x 107 cells were harvested and washed twice with phosphate-buffered saline, RNA was extracted with guanidinium thiocyanate and lauryl sarcosinate (9). mRNA was adsorbed onto oligo(dT)-cellulose columns (Pharmacia) and, after the columns were washed with high-and low-salt solutions, was eluted with 10 mM Tris'HCl buffer containing 1 mM EDTA (pH 7.4) at 650C. Total amount and concentration of mRNA were determined spectrophotometrically and confirmed by agarose gel electrophoresis.

Erythropoietin is known to be a hematopoietic growth factor with a sinulrly specific action on the proliferation and differentiation of erythroid progenitor cells. We have observed a dose-dependent proliferative action of human recombinant erythropoletin on human umbilical vein endothelial cells and bovine adrenal capillary endothelial cells. Binding studies with radioiodinated recombinant human erythropoietin revealed a large number (s27,000) of an apparent single class of receptors with an affinity in the 10-9 M range.Linkage of the radiolabeled ligand to its receptor via a bifunctional crosslinking agent allowed us to identify an endothelial cell protein of 45 kDa as the principal receptor associated with this mitogenic effect of erythropoietin. Recombinant human erythropoietin also enhanced the migration ofendothelial cells.Human umbilical vein endothelial cells (HUVECs) have been reported to produce a variety of colony-stimulating factors and lymphokines, although not erythropoietin (Epo) (1-5). Epo has been considered unique among the other hematopoietic stimulators because its only generally accepted action has been the proliferation and differentiation of cells of erythroid lineage (6). Colony-stimulating factors, on the other hand, are known to affect a variety of hematopoietic cells, and even cells of nonhematopoietic origin (6)(7)(8). Recombinant human Epo (rHuEpo) has been used therapeutically in thousands of uremic, anemic patients, with the only side effects arising from the vascular system (hypertension and thrombosis) (9).In experiments designed originally to study additional mechanisms of the vascular manifestations (thrombosis and hypertension) of Epo treatment (aside from the ones ascribed to increased blood viscosity), we serendipitously noticed significant cell proliferation when rHuEpo from Integrated Genetics (Framingham, MA) was added to HUVEC cultures. We pursued this observation with a series of new experiments, verifying that rHuEpo from various manufacturers has indeed a mitogenic effect on both HUVECs and bovine adrenal capillary endothelial cells (BACECs). We further analyzed the number and molecular weight of the putative receptor through which this action may be exerted. Since many agents with a mitogenic effect on endothelial cells have also an effect on endothelial cell migration, we investigated this aspect, finding again that the rHuEpo preparations enhanced the chemotaxis of both HUVECs and BACECs. MATERIALS AND METHODSCell Culture. Cesarian section-derived HUVECs were cultured at 37°C and in 5% C02/95% air by standard methodologies (10) in T 25-cm2, 50 ml capacity, tissue culture flasks (Falcon, Becton Dickinson Labware) in the presence of heparin and endothelial cell growth supplement (Sigma). They were characterized by the homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. In general, HUVECs were used for our experiments after two to four passages. rHuEpo, initially from I...

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