Effects of MMP-1 and MMP-7 on Pulmonary Hypertension in Rats
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摘要:
目的 探究MMP-1和MMP-7在肺动脉高压(pulmonary arterial hypertension,PAH)发展中的作用。 方法 采用ELISA检测PAH患者血清中的MMPs(MMP-1、MMP-2、MMP-7、MMP-9、MMP-10、MMP-14)的含量并选择出2种MMPs进行动物体内实验。收集临床信息并比较PAH患者和健康人的体重指数(body mass index, BMI)、NYHA心功能等级、6 min步行距离(6 min walking distance,6MWD)、脑钠肽(brain natriuretic peptide,BNP)、平均肺动脉压(mean pulmonary arterial pressure,mPAP)、甘油三酯(triglyceride,TGs)、低密度脂蛋白(low-density lipoprotein,LDL)、总胆固醇(total cholesterol,TC)、血红蛋白(hemoglobin,HB)、心率(heart rate,HR)等指标的差异。采用野百合碱诱导PAH大鼠模型,然后使用过表达MMP-1质粒和shMMP-7质粒分别上调和下调PAH大鼠中MMP-1和MMP-7的表达。通过苏木精-伊红染色法 ( hematoxylin-eosin staining,HE)观察肺动脉病理变化,Masson染色观察肌纤维和胶原纤维的变化,免疫荧光检测α-SMA和VWF,对左心室、右心室和室间隔进行称重后计算右心室肥厚指数。 结果 与健康人相比,PAH患者血清中的MMP-1(P < 0.0001)、MMP-2(P < 0. 001)、MMP-7(P < 0.01)、MMP-9(P < 0.001)、MMP-10(P < 0.001)、MMP-14(P < 0.01)含量升高,6MWD降低(P < 0.05),BNP含量升高(P < 0.001),NYHA分级升高(P < 0.0001)。与对照组相比,PAH大鼠的MMP-1(P < 0.0001)和MMP-7(P < 0.0001)在肺组织中蛋白表达升高,右心室肥厚指数增加(P < 0.0001),肺动脉血管增厚(P < 0.05),胶原纤维沉积增多(P < 0.05),α-SMA(P < 0.05)和VWF(P < 0.05)在血管中表达增多。与PAH组相比,在PAH大鼠中过表达MMP-1促进PAH发展,而干扰MMP-7则延缓其发展。 结论 MMPs(MMP-1、MMP-2、MMP-7、MMP-9、MMP-10、MMP-14)在肺动脉高压患者血清中升高。MMP-1和MMP-7促进肺动脉高压大鼠模型的肺动脉血管重塑、肺纤维化和右心室功能不全。 Abstract:Objective To explore the effects of MMP-1 and MMP-7 on pulmonary arterial hypertension (PAH). Methods The contents of MMPs (MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, MMP-14) in the serum of PAH patients and healthy controls were detected and two MMPs were selected for in vivo experiments. Clinical information was collected and body mass index (BMI), NYHA cardiac function class, 6 min walking distance (6MWD), brain natriuretic peptide (BNP), mean pulmonary arterial pressure (mPAP), triglyceride (TGs), low-density lipoprotein (LDL), total cholesterol (TC), hemoglobin (HB), heart rate (HR) were compared between PAH patients and healthy controls. The PAH rat model was induced by monocrotaline, and then MMP-1 overexpression plasmid and shMMP-7 plasmid were used to up-regulate and down-regulate the expression of MMP-1 and MMP-7, respectively. Hematoxylin-eosin (HE) staining was used to observe the pathological of PAH, Masson staining was used to observe the changes of muscle fibers and collagen fibers, and immunofluorescence was used to detect the expression of α-SMA and VWF. The left ventricle, right ventricle and interventricular septum were weighed and the right ventricular hypertrophy index was calculated. Results The serum levels of MMP-1 (P < 0.0001), MMP-2 (P < 0.001), MMP-7 (P < 0.01), MMP-9 (P < 0.001), MMP-10 (P < 0.001) and MMP-14 (P < 0.01) in PAH patients were significantly higher than those in healthy controls. 6MWD was significantly decreased (P < 0.05), BNP level was increased (P < 0.001), NYHA class was increased (P < 0.0001). Compared with the control group, the protein expression of MMP-1 (P < 0.0001) and MMP-7 (P < 0.0001) in lung tissue, right ventricular hypertrophy index (P < 0.0001), pulmonary artery vascular thickness (P < 0.05), and collagen fiber deposition (P < 0.05) were increased in PAH rats. The expressions of α-SMA (P < 0.05) and VWF (P < 0.05) in blood vessels were increased in PAH rats than control rats. Compared with PAH group, overexpression of MMP-1 in PAH rats promoted the development of PAH, while interference with MMP-7 delayed its development. Conclusions The serum levels of MMPs (MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, MMP-14) are increased in patients with PAH. MMP-1 and MMP-7 promote pulmonary artery remodeling, pulmonary fibrosis and right ventricular dysfunction in PAH rats. -
Key words:
- MMP-1 /
- MMP-7 /
- Pulmonary arterial hypertension
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近年来,课题组对姜科姜花属植物萜类成分进行了系统研究[1-6],从滇姜花、圆瓣姜花和毛姜花中分离得到一系列对多种肿瘤细胞具有显著体外细胞毒活性的萜类化合物[7-9]。一些姜科植物中的二萜类成分具有抗菌、抗肿瘤等活性[10-11]。滇姜花(Hedychium yunnanense Gangep)中的呋喃二萜Coronarin E含量较高,该成分没有细胞毒活性和抗菌活性,将其通过光敏氧化反应制备具有生物活性的丁烯酸内酯结构的二萜衍生物[12],有产率较高、选择性高、绿色环保的特点。课题组对Coronarin E经二氧化硒氧化、酰化、光敏氧化三步反应,制备两个衍生物,对其抗菌活性(抑菌圈、MIC和联合用药)及体外抗肿瘤活性进行较为深入的研究,进一步验证了二萜衍生物的生物活性,为寻找较好的药物前体提供了理论基础和科学依据。
1. 材料与方法
1.1 仪器
78-1型磁力加热搅拌器(杭州仪器电机厂);分析天平(上海第二天平仪器厂);AM-500型核磁共振波谱仪((瑞士BRUKER公司);LED灯(上海一恒科学仪器有限公司);OSB-2100 旋转蒸发仪(上海爱朗仪器有限公司);ES-315 高压蒸汽灭菌锅,(TOMY 公司);恒温培养箱(上海一恒科学仪器有限公司);SW-CJ-2FD 超净工作台(AIRTECH公司);电热恒温鼓风干燥箱(上海一恒科技有限公司)。
1.2 试剂
Coronarin E由本课题组从姜科植物滇姜花中分离得到。所用有机试剂(化学纯)和化学试剂均购自昆明市医药公司化学试剂玻璃仪器采供站,柱色谱硅胶均为青岛海洋化工厂产品,培养基配料均购自雅云生物科技有限公司。
1.3 实验方法
1.3.1 Coronarin E的SeO2氧化反应
取400 mg coronarin E、168 mg SeO2溶于5 mL干燥的二氯甲烷中,加入368 mg过氧叔丁醇,在常温下搅拌反应2 h,TLC检测原料反应完全。反应液经200~300目硅胶柱色谱分离纯化,石油醚-乙酸乙酯(80∶1~40∶1)洗脱,得到化合物1(无色油状物,见图1)300 mg,产率为71%。
1.3.2 化合物1的酰化反应
取87 mg 1-萘甲酸、0.12 mL N,N-二异丙基碳二亚胺(DIC),0.5 mg对二甲氨基吡啶(DMAP),溶于5 mL干燥过的二氯甲烷,常温搅拌10 min后,加入79 mg化合物1,搅拌反应2 h后,TLC检测反应完全,加入0.2 mL水及50 mL石油醚继续搅拌10 min,超声30 min。产物用150 mL石油醚与100 mL 70%甲醇分配,后者再用100 mL石油醚萃取,合并两次萃取的石油醚层,浓缩后经200-300目硅胶柱色谱分离,石油醚-氯仿(10∶1~5∶1)洗脱。得到化合物2(白色固体,见图2)52.7 mg,产率为44.1%。
1.3.3 化合物2的光敏氧化反应
取52.7 mg化合物2溶于10 mL吡啶中,加入1.0 mg四苯基卟啉(TPP),通入氧气并搅拌,在LED灯照射下反应2 h,TCL检测原料反应完全。溶剂蒸干,经硅胶色谱分离纯化,石油醚-乙酸乙酯(4∶1~1∶1)洗脱,得到化合物3(白色固体)和4(白色固体),见图3,分别为12 mg 和8 mg,产率分别为21.3%和14.1%。
1.3.4 化合物的抗菌活性筛选
采用滤纸片扩散法测试化合物3和4的抑菌圈直径[13-14];采用微量倍比稀释法[15-16]测定化合物3和4的最小抑菌浓度(MIC);化合物4的联合用药测试采用棋盘法,测试该成分分别与万古霉素、氨苄西林、卡那霉素3种抗生素联合用药的最小抑菌浓度[17]。
1.3.5 体外细胞毒活性测试
采用MTT法[18],测定化合物3和4对五个肿瘤细胞株的体外细胞毒活性(阳性对照采用顺铂)。
2. 结果
2.1 化合物3和4的NMR波谱数据
化合物3:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.98(3H,s,H-19),1.19(3H,s,H-20),3.05(1H,d,J = 10.5 Hz,H-9),5.87(1H,t,J = 3.0 Hz,H-7),4.95(1H,br.s,H-17a),5.34(1H,br.s,H-17b),6.67(1H,dd,J = 16.5,10.5 Hz,H-11),5.94(1H,s,H-14),6.45(1H,d,J = 16.5 Hz,H-12),6.44(1H,s,H-16),8.98(1H,m,H-9′),7.80(1H,m,H-8′),7.36(1H,br.s,H-4′),8.51(1H,d,J = 8.1 Hz,H-3′),8.26(1H,d,J = 8.2 Hz,H-7′),8.23(1H,d,J = 8.2 Hz,H-5′),7.88(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.12(t,C-1),19.38(t,C-2),42.54(t,C-3),33.90(s,C-4),48.99(d,C-5),28.87(t,C-6),76.54(d,C-7),146.57(s,C-8),58.83(d,C-9),40.05(s,C-10),141.53(d,C-11),124.80(d,C-12),162.25(s,C-13),116.77(d,C-14),171.25(s,C-15),98.50(d,C-16),115.57(t,C-17),33.47(q,C-18),21.79(q,C-19),14.56(q,C-20),166.82(s,C-1′),128.88(s,C-2′),130.68(d,C-3′),125.68(d,C-4′),134.30(d,C-5′),132.00(s,C-6′),129.52(d,C-7′),126.39(d,C-8′),128.41(d,C-9′),127.15(d,C-10′),134.86(s,C-11′)。
化合物4:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.96(3H,s,H-19),1.19(3H,s,H-20),2.97(1H,d,J = 10.0 Hz,H-9),5.81(1H,t,J = 2.5 Hz,H-7),4.89(1H,br.s,H-17a),5.32(1H,br.s,H-17b),6.97/6.99(1H,dd,J = 15.5,10.0 Hz,H-11),6.23(1H,d,J = 15.5 Hz,H-12),7.15/7.16(1H,s,H-14),6.15/6.16(1H,s,H-15),8.98(1H,m,H-9′),7.16(1H,br.s,H-4′),7.64(1H,m,H-8′),8.01(1H,d,J = 8.2 Hz,H-3′),8.16(1H,d,J = 8.2 Hz,H-7′),8.22(1H,d,J = 8.2 Hz,H-5′),7.87(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.18(t,C-1),19.97(t,C-2),42.59(t,C-3),33.81(s,C-4),48.99(d,C-5),29.07(t,C-6),76.95(d,C-7),146.73(s,C-8),58.78(d,C-9),39.98(s,C-10),136.91(d,C-11),122.79(d,C-12),131.00(s,C-13),144.94(d,C-14),97.21(d,C-15),170.67(s,C-16),115.20(t,C-17),33.54(q,C-18),21.94(q,C-19),14.69(q,C-20),166.71(s,C-1′),128.98(s,C-2′),130.64(d,C-3′),125.66(d,C-4′),134.93(d,C-5′),131.97(s,C-6′),129.52(d,C-7′),126.35(d,C-8′),128.29(d,C-9′),127.16(d,C-10′),134.88(s,C-11′)。
2.2 化合物3和4的抗菌活性
化合物3对两种MRSA病原菌具有一定的抗菌活性,化合物4对多种革兰氏阳性菌、革兰氏阴性菌具有明显的抗菌活性,见表1。
表 1 化合物3和4对病原菌株抑菌活性筛选结果(抑菌圈直径:mm)Table 1. The result of antimicrobial activities against pathogens ( diameter of inhibition zone:mm)病原菌株 化合物3 化合物4 金黄色葡萄球菌29213 − 10.1 MRSA 1450 8.2 10.5 MRSA 1505 10.1 10.3 MRSA 2024 − 8.2 MRSA I-20 − 10.0 MRSA I-67 − 9.8 MRSA 1957 − 8.1 MRSA 28299 − 10.5 克雷伯氏菌13883 − 10.8 粪肠球菌29212 − 9.4 白色葡萄球菌1029 − 12.0 铜绿假单胞菌PA01 − 10.2 大肠杆菌25922 − 10.0 鼠伤寒沙门氏菌χ 8956 − 17.0 鲍曼不动杆菌19606 − 13.2 枯草芽孢杆菌6633 − − 注:表中抑菌圈直径为三次测量的平均值;“−”表示无抑菌圈。革兰氏阳性菌:金黄色葡萄球菌(Staphylococcus aureus ATCC 29213),7个耐甲氧西林金黄色葡萄球菌(MRSA 1450、1505、2024、1957、28299、I-20、I-67),白色葡萄球菌(Staphylococcus albus 1029);革兰氏阴性菌:鼠伤寒沙门氏菌(Salmonella typhimurium χ 8956),铜绿假单胞菌(Pseudomonas aeruginosa PA01),大肠杆菌(Escherichia coil ATCC 25922),枯草芽孢杆菌(Bacillus subtilis ATCC 6633),鲍曼不动杆菌(Acinetobacter baumanii ATCC 19606),肺炎克雷伯氏菌(Klebsiella pneumonia ATCC 13883),粪肠球菌(Enterococcus faecalis ATCC 29212)。 2.3 化合物与抗生素的联合用药
化合物4与三种抗生素联合使用时,对MRSA病原菌株抑制活性有不同程度的协同或相加作用,见表2。
表 2 化合物4与三种抗生素的联合用药测试结果Table 2. Combination test of compound 4 with three antibiotics菌株 药物 MIC(μg/ml) 最佳抑菌点(化合物4∶抗生素) FICI 作用方式 鼠伤寒沙门氏菌χ8956 化合物4 0.25 万古霉素 0.25 0.125∶0.0625 0.75 + 氨苄西林 2 0.125∶1 1 + 卡那霉素 2 0.0625∶1 0.75 + 鲍曼不动杆菌19606 化合物4 0.5 万古霉素 0.25 0.125∶0.125 0.75 + 氨苄西林 8 0.25∶8 1.5 − 卡那霉素 4 0.25∶4 1.5 − 白色葡萄球菌1029 化合物4 0.5 万古霉素 0.125 0.125∶0.03125 0.5 ++ 氨苄西林 0.5 0.25∶0.125 0.75 + 卡那霉素 4 0.125∶1 0.5 ++ 注:1、FICI = 甲药MIC联合/甲药MIC单用 + 乙药MIC联合/乙药MIC单用,其中甲药代表化合物4,乙药代表抗生素。FICI > 1,表示两药有无关作用;0.5 < FICI≤1,表示两药有相加作用;FICI≤0.5,表示两药有协同作用。2、以“++”表示协同作用,“+”表示相加作用,“−”表示无关。 2.4 化合物3和4对5种肿瘤细胞株的细胞毒活性
化合物3对5种人类肿瘤细胞株具有显著的体外细胞毒活性;化合物4具有较弱的体外肿瘤生长抑制活性,见表3。
表 3 产物对五种肿瘤细胞株的半数生长抑制浓度IC50(μM)Table 3. The IC50 value of 3 and 4 against five tumor cell lines (μM)化合物编号 白血病HL-60 肝癌SMMC-7721 肺癌A-549 乳腺癌MCF-7 结肠癌SW480 3 2.55 2.77 1.17 2.49 1.37 4 15.71 15.62 26.49 25.13 22.87 顺铂 5.00 4.33 2.17 9.18 13.19 评价标准:无效IC50 > 40 μM;有效IC50 < 40 μM;标示下划线的为活性高于阳性对照顺铂。 3. 讨论
二萜coronarin E经三步衍生化反应,制备具有丁烯酸内酯结构单元的二萜衍生物3和4。对3和4的生物活性测试表明:化合物4对多种革兰氏阳性菌、革兰氏阴性菌有明显的抗菌活性,化合物3对两种MRSA具有一定的抗菌活性。化合物4对鼠伤寒沙门氏菌(Salmonella typhimurium χ8956)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、白色葡萄球菌(Staphylococcus albus 1029)的抗菌效果显著。化合物4对鼠伤寒沙门氏菌的抗菌活性接近万古霉素,高于氨苄西林、卡那霉素;对鲍曼不动杆菌的抗菌活性高于氨苄西林、卡那霉素;对白色葡萄球菌的抗菌活性接近氨苄西林,高于卡那霉素。化合物4与三种抗生素联用时,对鼠伤寒沙门氏菌抑制活性均具有相加作用。化合物4与万古霉素、卡那霉素联用时对白色葡萄球菌抑制活性具有协同作用,与氨苄西林联用时具有相加作用。化合物4与万古霉素联用时对鲍曼不动杆菌抑制活性具有相加作用。
化合物3对5种人类肿瘤细胞株(白血病细胞株HL-60、肝癌细胞株SMMC-7721、肺癌细胞株A-549、乳腺癌细胞株MCF-7和结肠癌细胞株SW-480)均具有显著的体外细胞毒活性,超过阳性对照顺铂;化合物4具有较弱的体外肿瘤生长抑制活性。
由此可见,以姜科二萜为原料,经结构改造制备丁烯酸内酯结构单元的二萜衍生物,并从中寻找有苗头的抗菌、抗癌活性成分或先导化合物,可作为未来抗菌、抗肿瘤药物研究与开发的一个方向。
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图 1 健康人和PAH患者临床指标的差异
A:比较健康人与PAH组的6MWD差异;B:比较健康人与PAH组的BMI差异;C:比较健康人与PAH组的BNP差异;D:比较健康人与PAH组的NYHA分级差异;E:比较健康人与PAH组的LDH差异;F:比较健康人与PAH组的TGs差异;G:比较健康人与PAH组的mPAP差异;H:比较健康人与PAH组的HR差异;I:比较健康人与PAH组的HB差异;J:比较健康人与PAH组的TC差异。*P < 0.05,***P < 0.001,****P < 0.0001。
Figure 1. Differences of clinical indicators between healthy people and patients with PAH
图 2 Ctrl组与PAH患者血清中MMPs的水平
A:ELISA试剂盒检测健康人和PAH患者MMP-1水平;B:ELISA试剂盒检测健康人和PAH患者MMP-2水平;C:ELISA试剂盒检测健康人和PAH患者MMP-7水平;D:ELISA试剂盒检测健康人和PAH患者MMP-9水平;E:ELISA试剂盒检测健康人和PAH患者MMP-10水平;F:ELISA试剂盒检测健康人和PAH患者MMP-14水平。**P < 0.01,***P < 0.001,****P < 0.0001。
Figure 2. Concentration levels of MMPs in serum of patients with Ctrl group and PAH
图 3 筛选3个shMMP-7干扰质粒对MMP-7蛋白水平和mRNA水平的干扰效率
A和B:western blot检测比较3个shMMP-7干扰质粒对MMP-7蛋白表达的干扰效率; C:qRT-PCR检测比较3个shMMP-7干扰质粒对MMP-7 mRNA的干扰效率。*P < 0.05,**P < 0.01,***P < 0.001,****P < 0.0001。
Figure 3. Interference efficiency of three shMMP-7 interfering plasmids screened on MMP-7 protein level and mRNA level
图 5 MMP-1过表达质粒和MMP-7干扰质粒对PAH的蛋白表达水平和病理作用
A-C:western blot检测过表达MMP-1质粒和干扰MMP-7质粒的转染效率; D:HE染色观察MMP-1和MMP-7对PAH的形态学影响(40×);E:Masson染色观察MMP-1和MMP-7对PAH肌纤维和胶原纤维的影响(40×)。****P < 0.0001。
Figure 5. Protein expression levels and pathological effects of MMP-1 overexpression plasmid and MMP-7 interference plasmid on PAH
表 1 临床PAH患者和健康人的基线资料表[
$ \bar x \pm s $ /n(%)]Table 1. Baseline data table of clinical PAH patients and healthy individuals [
$ \bar x \pm s $ /n(%)]项目 健康人(n = 16) PAH患者(n = 15) 年龄(岁) 31.69 ± 11.45 69.07 ± 11.11 性别(女性) 10(62.50) 4(26.67) BMI(cm/kg2) 22.13 ± 2.68 23.60 ± 5.49 NYHA心功能等级 0 16(100.00) 0(0.00) 1 0(0.00) 6(40.00) 2 0(0.00) 9(60.00) 6MWD(m) 593.00 ± 69.19 508.67 ± 102.102 BNP(pg/mL) 55.19 ± 15.71 139.13 ± 89.37 mPAP(mmHg) 19.62 ± 1.20 19.86.25 ± 3.46 HR(次/min) ≤80 11(68.75) 10(66.67) > 80 5(31.25) 5(33.33) TGs(mmol/L) 1.74 ± 0.50 1.45 ± 0.99 LDL(mmol/L) 2.71 ± 0.55 2.63 ± 0.70 TC(mmol/L) 3.87 ± 0.55 4.41 ± 0.94 HB(g/L) 135.56 ± 11.37 141.47 ± 20.43 -
[1] Deng L,Blanco F J,Stevens H,et al. Microrna-143 activation regulates smooth muscle and endothelial cell crosstalk in pulmonary arterial hypertension[J]. Circ Res,2015,117(10):870-883. doi: 10.1161/CIRCRESAHA.115.306806 [2] Boutet K,Montani D,Jaïs X,et al. Therapeutic advances in pulmonary arterial hypertension[J]. Ther Adv Respir Dis,2008,2(4):249-265. doi: 10.1177/1753465808094762 [3] Liu Y,Zhang H,Yan L,et al. Mmp-2 and mmp-9 contribute to the angiogenic effect produced by hypoxia/15-hete in pulmonary endothelial cells[J]. J Mol Cell Cardiol,2018,121:36-50. doi: 10.1016/j.yjmcc.2018.06.006 [4] Tuder R M,Stacher E,Robinson J,et al. Pathology of pulmonary hypertension[J]. Clin Chest Med,2013,34(4):639-650. doi: 10.1016/j.ccm.2013.08.009 [5] George J,Sun J,D'armiento J. Transgenic expression of human matrix metalloproteinase-1 attenuates pulmonary arterial hypertension in mice[J]. Clin Sci (Lond),2012,122(2):83-92. doi: 10.1042/CS20110295 [6] Takahashi J,Orcholski M,Yuan K,et al. Pdgf-dependent β-catenin activation is associated with abnormal pulmonary artery smooth muscle cell proliferation in pulmonary arterial hypertension[J]. FEBS Lett,2016,590(1):101-109. doi: 10.1002/1873-3468.12038 [7] Safdar Z,Tamez E,Chan W,et al. Circulating collagen biomarkers as indicators of disease severity in pulmonary arterial hypertension[J]. JACC Heart Fail,2014,2(4):412-421. doi: 10.1016/j.jchf.2014.03.013 [8] Pittayapruek P,Meephansan J,Prapapan O,et al. Role of matrix metalloproteinases in photoaging and photocarcinogenesis[J]. Int J Mol Sci,2016,17(6):868. doi: 10.3390/ijms17060868 [9] Yao J,Xiong M,Tang B,et al. Simvastatin attenuates pulmonary vascular remodelling by down-regulating matrix metalloproteinase-1 and -9 expression in a carotid artery-jugular vein shunt pulmonary hypertension model in rats[J]. Eur J Cardiothorac Surg,2012,42(5):e121-127. doi: 10.1093/ejcts/ezs445 [10] Lee E,Grodzinsky A J,Libby P,et al. Human vascular smooth muscle cell-monocyte interactions and metalloproteinase secretion in culture[J]. Arterioscler Thromb Vasc Biol,1995,15(12):2284-2289. doi: 10.1161/01.ATV.15.12.2284 [11] Lepetit H,Eddahibi S,Fadel E,et al. Smooth muscle cell matrix metalloproteinases in idiopathic pulmonary arterial hypertension[J]. Eur Respir J,2005,25(5):834-842. doi: 10.1183/09031936.05.00072504 [12] Chi P L,Cheng C C,Hung C C,et al. Mmp-10 from m1 macrophages promotes pulmonary vascular remodeling and pulmonary arterial hypertension[J]. Int J Biol Sci,2022,18(1):331-348. doi: 10.7150/ijbs.66472 [13] Galiè N,Hoeper M M,Humbert M,et al. Guidelines for the diagnosis and treatment of pulmonary hypertension[J]. Eur Respir J,2009,34(6):1219-1263. doi: 10.1183/09031936.00139009 [14] Yancy C W,Jessup M,Bozkurt B,et al. 2013 accf/aha guideline for the management of heart failure: Executive summary: A report of the american college of cardiology foundation/american heart association task force on practice guidelines[J]. Circulation,2013,128(16):1810-1852. doi: 10.1161/CIR.0b013e31829e8807 [15] Condon D F,Agarwal S,Chakraborty A,et al. Novel mechanisms targeted by drug trials in pulmonary arterial hypertension[J]. Chest,2022,161(4):1060-1072. doi: 10.1016/j.chest.2021.10.010 [16] Benza R L,Miller D P,Gomberg-Maitland M,et al. Predicting survival in pulmonary arterial hypertension: Insights from the registry to evaluate early and long-term pulmonary arterial hypertension disease management (reveal)[J]. Circulation,2010,122(2):164-172. doi: 10.1161/CIRCULATIONAHA.109.898122 [17] Voelkel N F,Gomez-Arroyo J,Abbate A,et al. Pathobiology of pulmonary arterial hypertension and right ventricular failure[J]. Eur Respir J,2012,40(6):1555-1565. doi: 10.1183/09031936.00046612 [18] Harbaum L,Rhodes C J,Wharton J,et al. Mining the plasma proteome for insights into the molecular pathology of pulmonary arterial hypertension[J]. Am J Respir Crit Care Med,2022,205(12):1449-1460. doi: 10.1164/rccm.202109-2106OC [19] Overall C M,López-Otín C. Strategies for mmp inhibition in cancer: Innovations for the post-trial era[J]. Nat Rev Cancer,2002,2(9):657-672. doi: 10.1038/nrc884 [20] Drwal E,Rak A,Tworzydło W,et al. "Real life" polycyclic aromatic hydrocarbon (pah) mixtures modulate hcg,hpl and hplgf levels and disrupt the physiological ratio of mmp-2 to mmp-9 and vegf expression in human placenta cell lines[J]. Reprod Toxicol,2020,95:1-10. doi: 10.1016/j.reprotox.2020.02.006 [21] Schäfer M,Ivy D D,Nguyen K,et al. Metalloproteinases and their inhibitors are associated with pulmonary arterial stiffness and ventricular function in pediatric pulmonary hypertension[J]. Am J Physiol Heart Circ Physiol,2021,321(1):H242-h252. doi: 10.1152/ajpheart.00750.2020 [22] Thenappan T,Chan S Y,Weir E K. Role of extracellular matrix in the pathogenesis of pulmonary arterial hypertension[J]. Am J Physiol Heart Circ Physiol,2018,315(5):H1322-h1331. doi: 10.1152/ajpheart.00136.2018 [23] Frisdal E,Gest V,Vieillard-Baron A,et al. Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension[J]. Eur Respir J,2001,18(5):838-845. doi: 10.1183/09031936.01.00084601 [24] Uzui H,Lee J D,Shimizu H,et al. The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells[J]. Atherosclerosis,2000,149(1):51-59. doi: 10.1016/S0021-9150(99)00295-6 [25] George S J,Johnson J L,Angelini G D,et al. Adenovirus-mediated gene transfer of the human timp-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein[J]. Hum Gene Ther,1998,9(6):867-877. doi: 10.1089/hum.1998.9.6-867 [26] Botney M D,Liptay M J,Kaiser L R,et al. Active collagen synthesis by pulmonary arteries in human primary pulmonary hypertension[J]. Am J Pathol,1993,143(1):121-129. [27] Wei B,Du J,Li J,et al. The modulating effect of l-arginine on collagen metabolism of pulmonary artery in pulmonary hypertension induced by a left-to-right shunt[J]. Zhonghua Yi Xue Za Zhi,2002,82(18):1273-1275. [28] Dieffenbach P B,Mallarino Haeger C,Rehman R,et al. A novel protective role for matrix metalloproteinase-8 in the pulmonary vasculature[J]. Am J Respir Crit Care Med,2021,204(12):1433-1451. doi: 10.1164/rccm.202108-1863OC [29] Wright J L,Tai H,Wang R,et al. Cigarette smoke upregulates pulmonary vascular matrix metalloproteinases via tnf-alpha signaling[J]. Am J Physiol Lung Cell Mol Physiol,2007,292(1):L125-133. doi: 10.1152/ajplung.00539.2005 [30] Saunders W B, Bayless K J, Davis G E. Mmp-1 activation by serine proteases and mmp-10 induces human capillary tubular network collapse and regression in 3d collagen matrices[J]. J Cell Sci, 2005, 118(Pt 10): 2325-2340. [31] Cui N,Hu M,Khalil R A. Biochemical and biological attributes of matrix metalloproteinases[J]. Prog Mol Biol Transl Sci,2017,147:1-73. [32] Arvidsson M,Ahmed A,Bouzina H,et al. Matrix metalloproteinase 7 in diagnosis and differentiation of pulmonary arterial hypertension[J]. Pulm Circ,2019,9(4):1-8. [33] Rosanò L,Spinella F,Di Castro V,et al. Endothelin receptor blockade inhibits molecular effectors of kaposi's sarcoma cell invasion and tumor growth in vivo[J]. Am J Pathol,2003,163(2):753-762. doi: 10.1016/S0002-9440(10)63702-9 [34] Stenmark K R,Fagan K A,Frid M G. Hypoxia-induced pulmonary vascular remodeling: Cellular and molecular mechanisms[J]. Circ Res,2006,99(7):675-691. doi: 10.1161/01.RES.0000243584.45145.3f -