Transforming Growth Factor-β1Induces Matrix Metalloproteinase-9Expression In Rat Vascular Smooth Muscle Cells Via ROS-dependent ERK-NF-κB Pathways

Part ⅠBackground:Expansion of thoracic aortic disease, including thoracic aortic dissection (TAD) and thoracic aortic aneurysm (TAA) are seriously harmful to human health. Matrix metalloproteinases(MMP) decompose extracellular matrix proteins, including elastin and collagen fibers. MMP induce the aortic wall degradation, expansion and rupture. The family of transforming growth factor-β is a multifunctional cytokine family, which play a regulatory role on cell by intracellular Smad transcription factors. Many articles have shown that abnormal TGF-β signaling transduction is related to aortic dissection pathogenesis.Aims:To investigate the molecular mechanisms underlying TGF-β1-induced MMP-9expression in vascular smooth muscle cells(VSMCs).Methods:we primary culture vascular smooth muscle cells from the thoracic aorta of SD rats, and identify its purity by the immunocytochemistry. The different concentrations (0,1,5,10ng/ml) of TGF-β1were used to interfere VSMCs. Medium supernatant, total protein of cells and RNA were extracted at the required time (0、2、4、6、16、24hours). RNA concentration and protein concentration were measured. MMP-9expression was analyzed by gelatin zymography, Western Blot and RT-PCR.Results:The purity of primary cultured VSMC is90%or more after the passage of4th generation. TGF-β1induced MMP-9secretion/expression in a time-and concentration-dependent manner. There was an apparent up-regulation with lOng/ml stimulation after24h, while MMP2expression shows little changes. MMP-9mRNA expression increased time-dependently. There was a significant increase in MMP-9mRNA within6h and sustained over24h during the period of observation.Conclusions:10ng/ml TGF-β1induce VSMCs to produce and secrete large amounts of MMP9. This change occurred at the transcriptional level.Part IIBackground:The reactive oxygen species (ROS) is a general term for several active substances which is formed by oxygen. The oxidative stress is the process of oxidative damage caused by oxygen free radical increasing or clearance reducing in vivo or intracellular. It is well known that oxidative stress closely relate with cardiovascular disease, including aortic aneurysm, hyperlipidemia, hypertension, and diabetes. Studies have shown that oxidative stress plays an important role in the occurrence of aortic dissection. The aortic aneurysm formation can be suppressed through inhibiting the generation of ROS in the animal model of the aortic aneurysm.Aims:To verify whether ROS was involved in TGF-β1induced MMP9expression.Methods:VSMCs were pretreated with NAC for1hour, then incubated with TGF-β1for24h. Medium supernatant, total protein of cells were extracted. MMP-9expression was analyzed by gelatin zymography and Western Blot. VSMCs were replated into the six-hole plate. Cells were incubated DCFH-DA and stimulated with TGF-β1or to combination of NAC. Relative fluorescence intensity was observed and taken photographs with a fluorescence inverted phase contrast microscope. VSMCs were directly exposed to H2O2, NAC and TGF-β1of different concentrations respectively for24h. Medium supernatant, total protein of cells were extracted. MMP-9expression was analyzed by gelatin zymography and Western Blot.Results:5mM NAC significantly suppresses the increase in the MMP9secretion/expression. TGF-β1stimulation increases the intracellular ROS significantly, which reaches its peak value in30min. After allowing NAC to pretreat, the TGF-β1stimulation can be suppressed significantly. H2O2stimulate VSMC expression/secretion of MMP9significantly higher, which can be s suppressed by NAC.Conclusions:TGF-β1induces MMP9expression/secretion depends on the participation of the ROS.Part ⅢBackground:Mitogen-activated protein kinase (MAPK) signal pathway is one of many signaling proteins, which regulates of a variety of cellular activities. MAPK plays a very important role in the process of information from the cellular to the nuclear. Extracellular regulated protein kinase1/2(ERK1/2) transduction pathway is one of the earliest, the most thorough study of four MAPK signal transduction pathway. The ERK signaling pathway is widely involved in the proliferation, differentiation, migration of cardiovascular system cell. Human AAA tissue had significantly elevated levels of pMEKl/2and pERKl/2compared with control tissues.Aims:To verify whether ERK1/2were involved in TGF-β1induced MMP9expression.Methods:VSMCs were pretreated with U0126for1hour, and then incubated with TGF-β1for24h. Medium supernatant, total protein of cells were extracted. MMP-9expression was analyzed by gelatin zymography and Western Blot. TGF-β1was used to interfere VSMCs. Total protein of cells was extracted at the required time (0、3、5、10、15、30min). The phosphorylation of C-Raf, MEK1/2, ERK1/2, P90RSK were analyzed by Western Blot using the corresponding antibody. After pretreatment with NAC and U0126, VSMCs were stimulated with TGF-β1. Total protein of cells was extracted at10min. The phosphorylation of C-Raf, ERK1/2, were analyzed by Western Blot.Results:lOuM U0126significantly suppresses the increase in the MMP9secretion/expression. TGF-β1stimulated the phosphorylation of C-Raf, MEK1/2, ERK1/2and P90-RSK in very short time. They reached their peak values in about10mins and declined afterwards. The phosphorylation of C-Raf (located upstream to MEK1/2) can only be suppressed by NAC, while the phosphorylation of ERK1/2(located downstream to MEK1/2) can be suppressed by both NAC and U0126.Conclusions:TGF-β1induces MMP9expression/secretion involves ROS-dependent MAPK/ERK signal pathways.Part ⅣBackground:NF-κB is the transcription factor as dimer form in the intracellular. NF-κB participates in the regulation of expression of many genes, which exists in almost all eukaryotic cells. Many articles suggest that NF-κB activation, resulting in the expression of MMP-9played an important role in the pathogenesis of aortic aneurysm and aortic dissection.Aims:To verify whether NF-κB was involved in TGF-β1induced MMP9expression.Methods:VSMCs were pretreated with Bay11-7082for1hour, and then incubated with TGF-β1for24h. Medium supernatant, total protein of cells were extracted. MMP-9expression was analyzed by gelatin zymography and Western Blot. TGF-β1was used to interfere VSMCs. Total protein of cells were extracted at the required time(0、1/4、1/2、1、2、4hour) The phosphorylation of NF-κB was analyzed by Western Blot using the corresponding antibody. After pretreatment with NAC、 U0126or Bay11-7082, VSMCs were stimulated with TGF-β1. Total protein of cells was extracted at1hour. The phosphorylation of NF-κB was analyzed by Western Blot. After pretreatment with Bay11-7082, VSMCs were stimulated with TGF-β1. Total protein of cells was extracted at10min. The phosphorylation of ERK1/2was analyzed by Western Blot.Results:luM Bay11-7082significantly suppresses the increase in the MMP9secretion/expression. TGF-β1stimulated the phosphorylation of NF-κB in very short time. They reached their peak values in about1hour. The phosphorylation of P65can be suppressed completely by NAC、U0126and Bay11-7082. The phosphorylation of ERK1/2cannot be suppressed by Bay11-7082.Conclusions:TGF-β1induces MMP9expression in VSMCs involves ROS-dependent MAPK/ERK-NF-κB signal pathways. This study found a new regulatory pathway in VSMC, and there is likely to be found the new treatment of TAA and TAD in the future.