Study On The Role Of Hypoxia-inducible Factor-1 Alpha In Scar Formation Of Patients With Benign Tracheal Stenosis

Benign airway stenosis is airway occupying or scar contracture stenosis caused by tracheal tuberculosis, trauma, operation, tracheotomy, endotracheal intubation, foreign matter, benign tumor, amyloid, and airway collapsing stenosis caused by thyroid cyst and relapsing polychondriti.With the development of technology of emergency in the intensive care unit and the population of lung transplantation and airway operation, the morbidity rate of airway stenosis gradually increased. The common causes of scar stenosis of airway is trauma. So the tracheal stenosis has become a kind of common disease of respiratory system and benign airway tracheal stenosis has become a common disease in respiratory intervention area. At present, the exact mechanism of tracheal stenosis is still not clear, bronchoscopic interventional therapy and surgical resection are the main methods for the treatment of scar tissue in benign tracheal stenosis. But after the surgical excision of scar tissue, patients had higher incidence of restenosis.Recent studies have found that the damage of tracheal mucosa broke the local mucosal blood circulation, which leads to mucosal ischemia and hypoxia and the subsequent formation of mucosal erosion, ulcer and tracheal cartilage. Karine Deschene’s research showed that the status of the low perfusion caused by mucosal injury induced the changes of temperature, p H and gas exchange that resulted in the formation of hypoxic environment in airway trauma. The change of microenvironment could be conductive to the fibroblasts activation. Studies of animal experiments and clinical cell culture in vitro showed that hypoxia was an important factor leading to the scar formation, fibrosis and inflammation.Hypoxia-inducible factor-1(HIF-1) is a key nuclear transcriptional factor when tissue responds to hypoxia and a kind of oxygen-dependent nuclear transcription factor existed in a mammal and human cells. Under hypoxic conditions, HIF-1 could combine with hypoxia responsing elements of target genes and then activates the transcription of target genes. HIF-1 is a heterodimer comprising an active α-subunit(HIF-1α) and a constitutive β-subunit, and the biological activities are determined by the expression of HIF-1α. Protein stability and transcriptional activity of HIF-1 is regulated by the concentration of oxygen in cells. HIF-1 could regulate some genes including vascular endothelial growth factor( VEGF), transforming growth factor beta(TGF-β), tissue inhibitor of matrix metalloproteinase 1(TIMP-1), matrix metalloproteinase(matrix, metalloproteinases, MMP) and basic fibroblast growth factor( b FGF). These target genes have a relationship with angiogenesis, glucose metabolism, cell survival and apoptosis.Numerous studies of HIF-1α initially focus on the role of growth and metastasis of solid tumors, they thought HIF-1α induced signaling pathway played an important role in the maintenance of characteristics and metastasis of cancer stem cell and may promote tumor development. Studies also show that HIF-1α inhibitor may become promising targeted therapies of solid tumor.In recent years, keloid hypoxic environment and the role of HIF-1α in scar formation and contraction process have received more and more attention. Tracheal stenosis scar is essentially a fibrous proliferative disease, which is characterized by over expression of collagen based extracellular matrix and its composition changes. Xudong Zheng reported that HIF-1αexpression upregulated in keloid fibroblast under hypoxia condition. HIF-1 mediated hypoxia pathway could increase the activity of fibroblasts and participated in the scar formation. In vitro study, Daniele M found that HIF-1α could promote fibroblasts secreting extracellular matrix, and collagen synthesis increase under hypoxic conditions. Zhang et al. confirmed these findings and proposed the increased expression of HIF-1α in keloid was involved in activation of ERK1 / 2 and Akt pathway. Vincent et al found that the biological characteristics of human skin keloid fibroblasts was similar to that of tumor cells, and thought that the unsensitivity of keloid to radiotherapy was closely related to the high expression of HIF-1α in the tissue. Robert M et al. have shown that cardiac fibroblasts could be transformed into myofibroblast in hypoxia environment. In addition, HIF-lα was also involved in renal interstitial fibrosis through increasing the synthesis of extracellular matrix and reduction of its degradation. However, whether HIF-lα is involved in the benign tracheal stenosis scar tissue formation and its related signal transduction pathway has not been reported.The purpose of this study was to investigate the role of HIF-lα in the formation of benign tracheal stenosis scar and the mechanism. The former two parts focused on the expression of HIF-1 and its downstream target genes in benign tracheal stenosis scar, and also the activation of fibroblasts in scar formation process at the histological level. The later two parts further verified the the role of HIF-lα in fibroblast activation and its downstream signaling molecules involved in the scar formation through the identification and cultivation of fibroblasts in vitrohypoxia hypoxia mode. The main contents of this paper are as follows: Part 1 Expression of hypoxia inducible factor-1α(HIF-1α) in thescar tissue of patients with benign tracheal stenosisObjective: We aim to investigate expression of hypoxia inducible factor-1α in the scar tissue of patients with benign tracheal stenosis.Methods: Patients were divided into three groups according to the results of electronic bronchoscope examination(8/group), granulation phase group, proliferative phase group and mature phase group. Real-time PCR and western blot were performed to detect the m RNA and protein expression of HIF-1α in the granulation tissue of the patients with benign tracheal stenosis.Results: HIF-1α m RNA level was the highest in the granulation stage, and there were significant differences(p<0.01) compared with proliferative and mature stages. HIF-1α m RNA level in proliferative stage was significantly higher than that in mature stage(p<0.01).HIF-1α protein expression was also different in three groups, which was the highest in the granulation stage and then reduced gradually with the least level in the mature stage. There was also significant difference(p<0.01) compared with the other two groups.Conclusion: We observed that the protein expression of HIF-1α was the highest in the granulation stage of scar tissue formation,and then gradually reduced to the least level in the mature stage. It suggested that HIF-1α was involved in the development of tracheal stenosis. Part 2 Study on the mechanism of HIF-1α mediated the formation ofscar tissue in patients with benign tracheal stenosisObjective: We aim to investigate the mechanism of HIF-1α mediated the formation of scar tissue in patients with benign tracheal stenosis.Methods: Patients were divided into three groups according to the results of electronic bronchoscope examination(8/group), granulation phase group, proliferative phase group and mature phase group. Western blot were performed to detect the protein expression of α-SMA, Caspase 3, Ⅰ / Ⅲ type collagen, VEGF, b FGF and TGF-β in the airway pathological tissue in patients with benign tracheal stenosis.Results: 1 Compared with granulation and mature stage, the expression of αxpre in proliferative stage is the highest(p<0.05), which is the least in mature stage. 2 Type I/ Ⅲ collagen and TGF-β protein expression showed a similar change tendency withα-SMA in different stages, and the ratio of type I and III collagen showed the same trend. 3 The expression of VEGF decreased gradually with the highest level in granulation stage and least level in mature stage(p<0.05). 4 The expression of b FGF and Caspase-3 in different phases showed the same change trend with VEGF.Conclusion: 1 The expression of type I and Ⅲ collagen and their ratio showed significantly increase in granulation and proliferative phase, and declined in mature phase, which indicated that the proliferation and active secretion of myofibroblast played an important role in the formation of tracheal stenosis. 2 The expression of VEGF decreased gradually with the highest level in granulation stage and least level in mature stage, which suggesting that VEGF played a critical role in the formation of scar. 3 The expression of b FGF showed the highest level in granulation stage and significantly declined in mature stage, which indicated that b FGF participates in the formation of benign tracheal stenosis and this was closely related with angiogenesis stimulating. 4 The expression of Caspase-3 in mature stage was significantly lower than that in granulation and proliferative stage, which indicated that fibroblast apoptosis was inhibited in the development of scar formation in tracheal stenosis and proliferation was increased. Therefore, it showed invasive growth and recurrence in clinic. Part 3 Isolation and identification of fibroblasts from the scar tissueof patients with benign tracheal stenosisObjective: We aim to investigate the isolation and identification of fibroblasts of scar tissue in the patients with benign tracheal stenosis in vitro for research at the cell level.Methods: Scar tissue of the patients with benign tracheal stenosis was cultured with tissue block culture method and fibroblasts was isolated. Then the fibroblast was observed under inverted microscope and identified through HE staining and immunohistochemistry.Results: 1 The adherent cells were typically fusiform with larger cell body, clear boundaries, strong three-dimension and high refraction under the inverted microscope. 2 Immunohistochemistry results showed that vimentin staining was positive and a lot of brown yellow granules in the cytoplasm. 3 HE staining showed that the cells were long spindle shape with clear contour and big nucleolus.Conclusion: Cells cultivated with the modified tissue block culture method were identified as fibroblasts by morphological observation, immunohistochemistry and HE staining. Part 4 Study on the regulation of HIF-1α on fibroblast andactivation and apoptosis and the mechanism under hypoxia.Objective: We aim to investigate the regulation of HIF-1α on fibroblast and activation and apoptosis and the mechanism under hypoxia.Methods: 1 We isolated and cultured fibroblasts from local tracheal stenosis in vitro and conducted the hypoxia-fibroblasts model with three-gas cultural system. The 3th or 4th generation fibroblasts from airway granulation tissue was selected, randomly divided into four groups and cultured for 12 h, 24 h, 48 h and 72 h in the incubator with 4% oxygen concentration. The expression of m RNA and protein of HIF-1α, α-SMA, b FGF, VEGF and TGF- β1 were detected by Real-time PCR and Western blot. 2 The 3th or 4th generation fibroblasts from airway granulation tissue was selected, randomly divided into four groups and cultured in the incubator with 4% 、 8% 、 16% and normal oxygen concentration for 12 h. The expression of m RNA and protein of HIF-1α, α-SMA, b FGF, VEGF and TGF- β were detected by Real-time PCR and Western blot.Results: 1 m RNA and protein expression level of HIF-1α gradually increased with the time extension under 4% oxygen concentration(p<0.05). The expression of m RNA and protein of the target genes including α-SMA, VEGF, b FGF and TGF- β1 increased in varying degrees under 4% oxygen concentration for different time,compared with the control group(p<0.05). 2 HIF-1α m RNA and protein expression gradually increased with the decrease of oxygen concentration that was 16%, 8% and 4%. Each was cultivated for 12h(p<0.05). The expression of m RNA and protein of the target genes includingα-SMA, VEGF, b FGF and TGF- β1 increased in varying degrees under different hypoxia concentration compares with the normal group.Conclusion: Fibroblasts under hypoxia for different time or under reduced oxygen concentration for the same time can promote m RNA and protein expression of HIF-1α and its target genes including α-SMA, b FGF, VEGF, and TGF-β1. Our study shows that HIF-1α can promote the expression of genes associated with angiogenesis and fibroblast proliferation by activation of fibroblast and then participate in the formation of airway scar of the patients with benign tracheal stenosis.