| Research background:The Pulmonary arterial hypertension(PAH) is a common syndrome caused by various diseases, which is a pathophysiological process involved in a kind of multiple factors. PAH is a serious and potentially destructive chronic Pulmonary disease, which characteristics include small artery vasospasm and hyperplasia in endometrium and medial, then vascular remodelling. The PAH’s main clinical symptoms include exertional dyspnea, shortness of breath, fatigue, chest pain, syncope et al. patients die eventually right heart failure. It is lack of effective treatment methods in currently. Just like malignant tumour, the prognosis so poor that the average survival time after diagnosis as PAH is2.8years.In recent years, although the biotechnology are developed very fast and made a series of research results about the pathogenesis of pulmonary hypertension, this pathogenesis is so complex that there are not yet a complete explaining. Until now, the main mechanism of PAH include as follow:One is the abnormal gene mutation related to type Ⅱ Bone Morphogenetic proteins receptor (BMPR2); Two is the impaired endothelial system and abnormal secretion; Three is abnormal potassium channels; Four is the action of inflammatory cells; Five is up regulated of the matrix metalloproteinases(MMPs). Tissue inhibitor of metalloproteinases(TIMPs), the MMP specific inhibitor, and Matrix metalloproteinases are dynamic equilibrium in cell matrix synthesis and degradation. In aortic and coronary artery, the effect of matrix metalloproteinases is hot research topic. In pulmonary arterial hypertension research, the earlier studies had shown that through the degradation of collagen and collagen denaturation of vascular basement membrane, which in turn antagonism other the degradation of matrix metalloproteinases, MMP-1and MMP-2promoted the proliferation and migration of pulmonary vascular smooth muscle and then affected the pulmonary vascular structural remodelling, so participated in the formation of pulmonary hypertension. The TIMP-1is specific inhibitor of the matrix metalloproteinases-1and-2which can specifically block the latter combined with the substrate, thus inhibiting the degradation of collagen, under normal circumstances, the balance maintain homeostasis. Brown who proved adequate supply of nitric oxide can decrease the secretion of inhibitor about matrix metalloproteinase. Animal studies have shown that nitric oxide can affect pulmonary vascular remodelling by reduced the accumulation of collagen through adjusting the TIMP-1/MMP-1balance and strengthened collagen degradation. It was found that inhibition of matrix metalloproteinase can significantly reduce pulmonary artery pressure in patients with pulmonary hypertension. The phenomenon will appear. For example the smooth muscle cell proliferation in pulmonary hypertension and the pulmonary no muscle arteries have muscle."Neointimal" appears between endothelial cells and internal elastic lamina while the severe pulmonary arterial hypertension, the neointimal are composed by the myofibroblasts and extracellular matrix. The proliferation and apoptosis appears loss of balance in the endothelial cells and smooth muscle cells and fibroblasts, then leading to pulmonary structural remodelling and ultimately causing the pulmonary arterial hypertension. In the process of pulmonary hypertension vascular remodelling, the first is to be activated adventitial fibroblasts to proliferate and synthesise matrix components. Then the matrix metalloproteinase, like MMP2and MMP9, are up regulated and mediated fibroblast migration from the outer membrane into the medial and intima of the blood vessels. Then the intimal and medial of pulmonary vascular will be hyperplasia and proliferation and formed vascular remodelling.A disinterring and metalloproteinase with thrombospondin motifs(ADAMTS) family is recently cloned who is a class of secreted metalloprotease family with Zn2+-dependent. The family and the family of matrix metalloproteinases and Depolymerization polyprotein-like metalloprotease belong metalloproteinase family. Because it is found just in the last ten years (in1997), we understand it is far less than the two families. The family same as the matrix metalloproteinase family have the ability to degrade the extracellular matrix. Compare to the other two family, the main difference of ADAMT family in structures is a repeat sequences containing at least one TSP (also known as TSP motifs) in carboxy-terminal. ADAMTS family are secreted proteases, which can be synthesized and secreted in vivo by a variety of synthetic and secretory cells, such as smooth muscle cells, endothelial cells, fibroblasts, macrophages. And ADAMTS is usually combined with the matrix. There were19family members. We are known some of their function, like the ADAMTS-1, ADAMTS-2, ADAMTS-4, ADAMTS-5, ADAMTS-9and ADAMTS-13.They degrade or regulate the extracellular matrix proteins and participate in a series of normal physiological functions, such as growth, angiogenesis, and coagulation et al. While their express abnormally and interact with the extracellular matrix, they will often lead to various diseases, such as cancer, arthritis, atherosclerosis, skin fragility-related diseases, coagulation disorders purpura and other diseases. The function of other family members remains unclear and let us to be studied.ADAMTS-7is the seventh of the members of the ADAMTS family, who is known gradually by people in-depth study due to the fine adjustment of blood vessel. Through its substrate cartilage oligomer matrix protein (COMP), ADAMTS-7maintains steady-state by fine regulation of blood vessels. ADAMTS-7is first reported by Hurskainen TL team in1999, whose structure is composed of a catalytic domain who have a zinc ions and several other non-catalytic domain structure, including:a disintegrin domain structure, a thrombospondin domain structure, a structure of cysteine-rich domain (CRD), a spacer-1structure domain, three thrombospondin motifs, a spacer-2domain structure, four thrombospondin motifs in the ends of the C. ADAMTS-7with the ADAMTS-12belong to one hypo type of ADAMTS family, who can be directly combined and degradation of the cartilage matrix proteins. The Professor Liu Chuan-Ju who are in New York University School of Medicine and Professor Wang Li’s research team whose are in the medical department of Peking University have done many researches over the past decade in internationally. Professor Liu does it in this field from arthritis and cartilage development. His research shows that the ADAMS-7who was found in patients with rheumatoid synovitis cartilage increased significantly through in vitro tests and who is overexpression associated with COMP degradation. In arthritis patients, they found COMP degradation fragments same as the COMP fragments through degrading by ADAMTS-7. In addition, through implicating the antibody of ADAMTS-7, it can significantly inhibit the COMP degradation by induced through tumour necrosis factor and interleukin-1β who are in cartilage cultured. Inhibition ADAMTS-7by siRNA silencing technology can also significantly prevent the degradation of COMP. The team of Wang Li study ADAMTS-7from blood vessels and also involve the COMP. They found that ADAMT-7could promote vascular smooth muscle cells calcification by regulating the degradation of COMP. they found that the microRNAs-29could regulate vascular calcification through intervened downstream reaction of ADAMTS-7. They also found ADAMT-7plays an important role in vascular smooth muscle cell proliferation and migration in balloon injury of rat carotid artery model. This research shows the ADAMTS-7may participate in the atherosclerosis in vascular matrix degradation and remodelling, which accelerated the process of atherosclerosis. Some research also reported that ADAMTS-7is involved in the intima hyperplasia after vascular injury. Through some related detected on the blood of non ST segment elevation myocardial infarction, some experts thought that the ADAMTS-7levels are closely associated with long-term prognosis of NSTEMI patients. The messenger RNA of ADAMTS-7is up regulate by TNF alpha in THP1in the monocyte or macrophage of people. The fine mechanism of adjustment is still unclear. Some research thought that its possible mechanism is through the transcription factor of the nuclear factor kappa B and AP-1to let the TNF alpha up regulate and stimulate ADAMTS-7induced rat smooth muscle proliferation.According to studies about detecting ADAMTS-7, it found that there are high expression levels in heart, pancreas, kidney, skeletal muscle and liver in the adult samples. It was found expressed in liver, testis, lung, ovary, kidney, fetal heart and thymus tissue in varying degrees. In skeletal muscle, meniscus and fat can also be detected lower levels. In the lung tissue of pulmonary hypertension patient, the expression of ADAMTS-7is not reported now.Based on the above information, we have known that the pathogenesis of pulmonary hypertension is very complex. The exact mechanism of PAH is still not very clear. There is one thing could make sure that the abnormal expression of matrix metalloproteinase is important in the pathogenesis of pulmonary hypertension. And the ADAMTS same as the family of matrix metalloproteinase, it also has the ability to degrade extracellular matrix. At the same time, the ADAMTS is widely distributed in mammalian organs. Previous research data had showed that the ADAMTS-7is closely associated with vascular development, in particular with circulation of atherosclerosis closely related to atherosclerosis. We hypothesized that ADAMTS-7and pulmonary circulation may also have close ties, as can be confirmed is expected to become a new target for the treatment of pulmonary hypertension. This paper intends to test the ADAMTS-7expression of lung tissue between adult pulmonary hypertension and normal pulmonary artery pressure in first, then could to clarify whether the ADAMTS-7expression in lung tissue, with or without expression in the lung tissue of patients with pulmonary hypertension. Second, cultured pulmonary artery smooth muscle cells, we test the expression of ADAMTS-7and some index of associated with proliferation and apoptosis and inflammation after intervened of the TNF-a. Finally, we verify the correlation detection of pulmonary hypertension in rats. In this study, there are three parts as follows.Part one. The expression of the ADAMTS-7in Patients lung tissue with mild-to-moderate pulmonary hypertensionObjective:To verify the expression of ADAMTS-7in lung tissue in patients with pulmonary hypertension. Methods:There was a case-control study. Selected40cases of patients randomly who want to do the open chest surgery in The Affiliated Hospital of Guangdong medical college,20cases showed mild-to-moderate pulmonary hypertension by tested pressure through right cardiac catheterization(as case group), the pulmonary artery pressure in other20cases was normal(as control group). Take a few lung tissue in the two groups of patients in the surgery, used the immune immunofluorescence histochemical method and RT-PCR method to detect the expression of ADAMTS-7in lung tissue of the two groups patients. Results: Through the immune histochemical methods, it showed that there is a little positive expression of ADAMTS-7in the lung tissue of the control group and numerous positive expressions of ADAMTS-7in the lung tissue of the case group. By the image analysis and statistical calculation software, the IOD value in case group was significantly higher than control group (P<0.05). Through immunofluorescence RT-PCR method to test the expression of ADAMTS-7in the lung tissue in the two groups, it showed that the two groups (control group and case group) compared with the blank group (rules its Folds value is1) were statistically significant (P<0.05). In the control group compared with the case group, which had a statistically significant difference (P<0.05). Conclusion:ADAMTS-7may be involved in the pathogenesis of pulmonary hypertension.Part two. The expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cellsObjective:to verify the expression and the possible mechanism of ADAMTS-7in pulmonary artery smooth muscle cells. Methods:Cultured the people primitive pulmonary artery smooth muscle cells, first we tested the expression of ADAMTS-7in pulmonary artery smooth muscle cells by immunofluorescence cytochemistry Then used the CCK8method to detect the TNF alpha intervene on cell proliferation, in order to make clear of the optimum time and concentration. Then divided into three groups:blank group, the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group. Cultured respectively for12hours,24hours,48hours,72hours and detected the relative content of ADAMTS-7, PCNA, NF-kappa B, PI3K, VEGF, MARKK4, ERK1, IL-1, IL-6by RT-PCR method in every groups. Chose the point of24hours in intervention group (the25ug/L TNF alpha intervention group, the ADAMTS-7siRNA silence group) with immune protein imprinting method (Western Blot) to detect the mRNA levels of ADAMTS-7, the Bcl-2and COMP. Results:(1) Through the cellular immune fluorescent method, there was numerous positive expression of ADAMTS-7in pulmonary smooth muscle cell cytoplasm.(2) Through CCK8method, the concentration of25ug/L TNF alpha and the cultured time of24hours were screen out as the optimal concentration and time for proliferation of pulmonary artery smooth muscle cell.(3) The RT-PCR detection showed that the expression levels of ADAMTS-7, PI3K, PCNA, VEGF, IL-1, IL-6in all time point in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The time of the highest express quantity of ADAMTS-7was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of PI3K, PCNA and IL-1was the48hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of VEGF, IL-6was the72hours after intervening by25ug/L TNF alpha. The RT-PCR detection showed that the expression levels of NF-kappa B, MARKK4in24hour and48hour and72hour in TNF alpha25ug/L intervention group were significantly higher than that of siRNA silent group (P<0.05). The two groups were no significant statistical significance (P>0.05) in the12hour time point. The time of the highest express quantity of NF-kappa B was the24hours after intervening by25ug/L TNF alpha. The time of the highest express quantity of MARKK4was the48hours after intervening by25ug/L TNF alpha. ERK1factor mRNA between two groups had no statistically significant difference in the various time points (P<0.05).(4) the western blot test results showed that the amount of ADAMTS-7express in TNF alpha intervention group was significant higher than that of ADAMTS-7siRNA silent group and control group(P<0.05), The amount of ADAMTS-7express in control group was significant higher than that of the ADAMTS-7siRNA silent group(P<0.05), While the COMP expression levels in TNF alpha intervention group was significance lower than that of ADAMTS-7siRNA silent group and control group(P<0.05), The COMP expression levels in the siRNA ADAMTS-7group was no statistical difference (P>0.05), the Bcl-2expression in three groups was no statistical difference (P>0.05). Conclusion:(1) The ADAMTS-7May regulate the pulmonary artery smooth muscle cell proliferation or apoptosis by the TNF alpha pathway, NF-kappa B pathway, JNK pathway, PI3K pathway.(2) The ADAMTS-7may not participate the apoptosis of pulmonary smooth muscle cells through ERK or Bcl-2pathway.(3) The ADAMTS-7may promote the migration of smooth muscle cells by promoting the degradation of COMP and the expression of the VEGF.Part three. The expression of ADAMTS-7in monocrotaline (MCT) induced pulmonary hypertension ratsObjective:By intervening the monocrotaline induced pulmonary hypertension rats, detecting the expression of ADAMTS-7and the proliferation and inflammation in pulmonary artery smooth muscle. Methods:30SD rats were randomly divided into three group:the normal control group (control group), the group of pulmonary hypertension induced by MCT (MCT), the doxycycline group. Measured the average pulmonary artery pressure (mPAP) and the right heart hypertrophy index (RVHI) after21days. And detect the ADAMTS-7by immunohistochemical and the TNF alpha, NF-kappa B, IL-1, IL-6, PI3K, MARKK4by RT-PCR in the lung tissue of rats. And detected the mRNA expression of ADAMTS-7, PCNA and Bcl-2by western blot method at each group. Results:(1) The mPAP and RVHI of rats in MCT group and doxycycline group were significantly higher than that of control group (P<0.05). The mPAP of rats in doxycycline group was lower than MCT group, but no statistical significance (P>0.05). The RVHI of rats in doxycycline group was less than the MCT group and has statistical significance (P<0.05).(2) Through immunohistochemical method for the lung tissue of rat, the positive of ADAMTS-7in MCT group was significantly higher than the control group and doxycycline group (P<0.05). While the doxycycline group compared with the control group, the former was higher than the latter and the difference was statistically significant (P<0.05).(3) By measuring used the RT-PCR method, the mRNA expression of the TNF alpha, the NF-kappa B, IL-1, IL-6, PI3K, MARKK4in MCT group and doxycycline group were significantly higher than that of control group (regulation control Folds value of1)(P<0.05). Each factor in MCT group obviously higher than that of doxycycline group, the difference was statistically significant (P<0.05).(4) Western blot analysis showed that the mRNA expression of ADAMTS-7, PCNA, Bcl-2in MCT group was significantly higher than that of the doxycycline group and the control group (P <0.05). The mRNA expression of Bcl-2and PCNA in doxycycline group were significantly higher that of the control group (P<0.05). The mRNA expression of ADAMTS-7in doxycycline group was equivalent that of the control group. It was no statistical significance (P>0.05). Conclusion:Doxycycline may adjust the pulmonary artery smooth muscle proliferation and inflammation by inhibiting ADAMTS-7. Doxycycline alone through the mechanism inhibiting ADAMTS-7and other metal loproteinase reduces pulmonary artery pressure is not obvious. |
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