The Research On The Relationship Between The The Phenotype Of Pulmonary Microvascular Endothelial Cells And Pulmonary Fibrosis Induced By Bleomycin In Rats

Purpose:Pulmonary fibrosis is results from abnormal repair and reconstruction of lung tissue after injury. Myofibroblasts (MFbs) marked by increased expression of smooth muscle alpha-actin (alpha-SMA) stress fibers (a contractile phenotype) is one of the primary effective cells involved in the repair and reconstruction of lung tissue. Therefore, accumulation and activation of MFbs are regarded as the hallmark of progressive pulmonary fibrosis. In general, MFbs are mainly derived from transformation of resident lung fibroblasts (Fbs) which will transform to MFbs to play a stronger role in fibrosis by the induction of hypoxia, profibrotic growth factor and so on. Pulmonary microvascular endothelial cells (PMVECs), one of pivotal effective cells against pathopoiesis factor in early stage of disease, are proved to play an important role in mediating inflammatory response, aggravating tissue hypoxia, and secreting cytokines and matrix to regulate the migration, proliferation and differentiation of mesenchymal cells. Many experiments indicated the injury of PMVECs facilitated the progress of pulmonary fibrosis and the accumulation and transformation of fibroblasts was closed correlated with the biological activity of PMVECs in the process of pulmonary fibrosis, so we established a coculture system of PMVECs and Fbs, which can be useful to engineer a more in vivo-like microenvironment for both cells sharing the same culture medium, applying in order to study the phenotype and function of pulmonary microvascular endothelial cell in early stage of progressive pulmonary fibrosis.Methods:SD rats, male, weighing 100±20g (provided by the experimental animal center of the Fourth Military Medical University). The BLM rats were injected intrathecally with 0.125 ml BLM-A5 solution (4 g/L in sterile water, dosage 5mg/kg weight) to induce pulmonary fibrosis, and the control animals were injected intrathecally with 0.125 ml sterile saline. After rats were euthanized at 3, 7, 14, 21and 28 days post-instillation, the peripheral lung tissue was cut for primary cell culture of PMVECs and Fbs, and the remanent lung tissue was used for histological analysis by VG stain.PMVECs and Fbs were isolated from lung tissue of each group rats, and were cultured. PMVECs from each group and Fbs from control group were co-cultured respectively. Immunofluorescence, ELISA, Western Blot(WB), reverse transcription-PCR (RT-PCR), FCM and neutralization test of cytokines were performed to detect the synthesis and secretion of connective tissue growth factor(CTGF) and transformation growth factor-beta1 (TGF-β1) in PMVECs, and to analyze the role of PMVECs in process of pulmonary fibrosis.Results:1,The logarithmic growth phase of PMVECs isolated from BLM-induced rats were ahead of control group.2,Immunofluorescence, Western blot and ELISA analysis revealed a peak of synthesis and secretion of CTGF and TGF-β1 protein in PMVECs at 7-day post-instillation group. RT-PCR analysis revealed that the transcription of TGF-β1 and CTGF were prominently up-regulated in PMVECs of 7-day post-instillation group.3,α-SMA protein was detected in PMVECs of BLM-induced rats by immunofluorescence and western blot analysis.4,MTT, PCNA immunohistologic stain, FCM and western blot analysis revealed the proliferation, the transformation and the function of synthesizing and secreting collagenⅠwere prominently up-regulated in Fbs cocultured with PMVECs of 7, 14, 21, 28-day post-instillation groups, especially in Fbs cocultured with PMVECs of 7-day post-instillation groups.5,Neutralization test of cytokines displayed, about 23.39±2.12% drop in quantity of cocultured Fbs and 4.68±0.86% drop in percent of MFbs in cocultured Fbs were caused by neutralizing of 53.15±8.91% TGF-β1; about 45.16±5.79% drop in quantity of cocultured Fbs and 8.06±1.31% drop in percent of MFbs in cocultured Fbs were caused by neutralizing of 54.65±10.24% CTGF; 73.39±11.89% drop in quantity of cocultured Fbs and 10.15±1.37% drop in percent of MFbs in cocultured Fbs were caused by neutralizing of both TGF-β1 and CTGF.Conclusion:1,The synthesis and secretion of TGF-β1 and CTGF protein were prominently up-regulated in PMVECs in process of BLM-induced rats pulmonary fibrosis, and PMVEC showed secretion phenotype.2,PMVECs isolated from BLM-induced rats can improve the proliferation, the transformation and the function in synthesizing and secreting collagenⅠprotein of control Fbs in vitro, especially in PMVECs of 7-day post-instillation groups.3,Abnormal phenotype in PMVECs, displaying up-regulation of TGF-β1 and CTGF protein expression is the important reason for activation, proliferation and transformation of Fbs, even for excess secretion of extracellular matrix,which is probably the key factor to activate the static Fbs in lung interstitium and stimulate Fb to play its profibrotic role in early stage of disease in vivo.