Pulmonary microvascular endothelial cells from bleomycin‐induced rats promote the transformation and collagen synthesis of fibroblasts

Abstract

Accumulation and activation of myofibroblasts are the hallmark of progressive pulmonary fibrosis, and the resident fibroblasts are the major source of myofibroblasts. However, the key factors involved in the transformation of fibroblasts are unknown. Pulmonary microvascular endothelial cells (PMVECs), major effector cells against pathogenesis in early stages of the disease, can secrete cytokines to induce the differentiation of mesenchymal cells. We speculated that PMVECs could secrete pro-fibrotic cytokines and promote the transformation of fibroblasts into myofibroblasts. Accordingly, we established a co-culture system with PMVECs and fibroblasts to examine the specific transformation and collagen synthesis of the co-cultured fibroblasts by FACS and Western blot, prior to and after treatment with neutralizing antibodies against transforming growth factor-beta1 (TGF-β1) and connective tissue growth factor (CTGF). We also analyzed expression of TGF-β1 and CTGF in PMVECs. The synthesis and secretion of TGF-β1 and CTGF protein were up-regulated in PMVECs isolated from bleomycin (BLM)-treated rats, most prominently at 7 days post-instillation. We showed that the PMVECs isolated from BLM-induced rats could induce the transformation of normal fibroblasts and their secretion of collagen I, which was inhibited by both neutralizing anti-TGF-β1 and anti-CTGF antibodies. Therefore, up-regulation of TGF-β1 and CTGF in PMVECs plays an important role in activation, transformation, and collagen synthesis of fibroblasts; in particular, these effects in PMVECs are likely to be the key factors for activation and stimulation of static fibroblasts in lung interstitium in early stages of pulmonary fibrosis disease.

Endothelial Cells Recruit Macrophages and Contribute to a Fibrotic Milieu in Bleomycin Lung Injury

“…Reports have cited changes in adhesion molecules (11,13,14), cytokines (10,11,30), vascular mediators (31), and profibrotic molecules (7,8) after the in vitro treatment of endothelial cells with BLM, but many of these mediators were not verified in vivo. Furthermore, the presence of cytokines and chemokines in bronchiolar lavage fluid and serum has been reported in patients and in animals treated with BLM, but the cellular origin of these mediators is either unknown or assumed.…”

“…In vitro, endothelial cells have been shown to secrete profibrotic mediators such as TGF-b, PAI-1, and CTGF, which induce fibroblast growth, differentiation, and the synthesis of collagen (7,8). Recent evidence has also shown that endothelial cells may become fibroblast-like and secrete collagen through the process of EndMT.…”

“…In vitro studies have shown that endothelial cells secrete many profibrotic mediators, and among these, TGF-b (7,8), CTGF (8), and PAI-1 (44) are up-regulated after the stimulation of endothelial cells with BLM in vitro. According to our experiments, CTGF was elevated in endothelial cells between the first and fourth weeks after BLM stimulation, and was significantly elevated during the fourth week.…”

“…Activated endothelial cells are known to secrete cytokines and profibrotic mediators such as transforming growth factor-b (TGF-b), connective tissue growth factor/ CCN family member 2 (CTGF/CCN2), and plasminogen activator inhibitor-1 (PAI-1), which directly recruit and activate fibroblasts to produce collagen. The direct treatment of endothelial cells with BLM in vitro has been shown to induce the secretion of certain profibrotic mediators (7,8), but little is known about the effects of BLM on endothelial cells in vivo. Moreover, recent studies have suggested that a process known as the endothelial-to-mesenchymal transition (EndMT) may be activated during fibrotic processes, creating fibroblast-like cells that contribute to excess collagen production (8,9).…”

“…The direct treatment of endothelial cells with BLM in vitro has been shown to induce the secretion of certain profibrotic mediators (7,8), but little is known about the effects of BLM on endothelial cells in vivo. Moreover, recent studies have suggested that a process known as the endothelial-to-mesenchymal transition (EndMT) may be activated during fibrotic processes, creating fibroblast-like cells that contribute to excess collagen production (8,9). Finally, the activation of endothelial cells may contribute to prolonged tissue injury by promoting a proinflammatory environment.…”