卡托普利对内皮细胞活化与损伤的保护作用及其在急性肺损伤中治疗作用的研究
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摘要
卡托普利对内皮细胞活化与损伤的保护作用及其在急性肺损伤中治疗作用的研究
前言
急性呼吸窘迫综合征(acute respiratory distress syndrome，ARDS)系由多种原因，如严重创伤和感染等肺内、肺外因素所致的急性缺氧性呼吸衰竭，其本质为急性肺损伤(acute lung injury，ALI)，以微血管渗透性增加，间质、肺泡水肿和低氧血症为特征，目前仍是引起高死亡率的疾病之一(40-60％)，并还缺乏有效的治疗办法。目前发病机制尚未明确，临床上缺乏敏感的早期诊断和病情监测指标及有效的治疗药物。因此，寻找特异而有效的药物成为该领域的研究热点，而利用传统己知的药物则成为理想的选择。
在所有的组织器官中，肺是富含内皮细胞的器官，在ALI的发展过程中总是伴随着内皮细胞(endothelial cell，EC)的激活和损伤。研究显示，ALI中肺血管EC是受损的主要靶细胞，更是活跃的炎症细胞和效应细胞，在调节凝血、纤溶和血管紧张度方面发挥作用。循环内皮细胞(circulatingendothelial cell，CEC)是循环血中的血管EC，导致EC损伤的因素均可使CEC增多，CEC可直接而特异的反映活体内EC损伤情况，且主要反映了EC的脱落性损伤。近来研究显示，CEC的改变与器官功能障碍及血管损伤明显相关。新近的研究也发现，凝血异常与炎症反应密切相关，并直接影响预后。有作者认为内皮细胞的活化和损伤是ARDS的重要标志，在疾病复杂的病理机制中起重要作用，并且其程度与疾病的严重程度及预后密切相关。因此，怎样保护及逆转内皮细胞功能障碍成为ARDS治疗中一个新的发展趋势。
卡托普利是一种血管紧张素转换酶抑制剂(angiotension convertingenzyme inhibitot，ACEI)，主要用于心血管疾病的降压治疗，新近的研究认为其还具有其他良好的药理作用，除清除氧自由基、抗氧化损伤以外，还具有内皮细胞保护作用，而其机制除与抑制血管紧张素转换酶(angiotensionconverting enzyme，ACE)有关外，是否还存在其他机制目前尚未明了。
我们通过体外培养人脐静脉内皮细胞(HUVEC)，观察细菌脂多糖诱导HUVEC活化及损伤的状态下及在体油酸诱导大鼠急性肺损伤(ALI)过程中，在血管内皮活化及损伤状态下，探讨卡托普利的保护作用及其可能的机制。
材料与方法
一、实验材料
(一)人脐静脉内皮细胞原代培养材料：
健康新生儿脐带25～30cm，由沈阳市铁西区妇婴医院提供。
(二)动物实验部分：
Wistar大鼠72只，体重220～260g，中国医科大学实验动物中心提供。
(三)临床实验对象：
ICU、RICU内危重病患者21例，根据临床表现、体征、X线胸片、血气分析结果确诊为ALI或ARDS。ICU内对照者10例，健康对照者15例。
二、实验步骤
(一)细胞培养部分：
1人脐静脉内皮细胞的培养：
采用改良Jaffe等法。用0.25％的胰蛋白酶灌注消化脐静脉后，离心收集内皮细胞，接种于75cm~2培养瓶，置于37℃、5％CO_2饱和湿度的孵箱中培养培养液为含20％新生牛血清的DMEM。待细胞长满2／3瓶底后进行传代。Ⅷ因子相关抗原免疫组化染色阳性，证实为内皮细胞。
2实验分组：
实验分为3组：对照组：常规无血清DMEM培养；LPS组：1μg／ml LPS；Cap+LPS组：根据Cap浓度的不同(10~(-7)mol／L、10~(-5)mol／L及10~(-3)mol／L)分为3个亚组，在每个亚组中LPS(1μg／m1)和Cap同时加入。
3细胞培养上清血管假性血友病因子(vWF)的测定：
用酶联免疫吸附试验(ELISA)检测各组培养上清中vWF的抗原含量(％)。抗体为兔抗人vWF的单克隆抗体，方法按试剂盒说明书进行。
4间接免疫荧光方法检测HUVEC的细胞间粘附分子-1(ICAM-1)蛋白表达：
各组HUVEC以PBS洗涤2次，0.25％胰蛋白酶消化，收集各组细胞，加入兔抗人ICAM-1多克隆抗体100μ1，37℃孵育1h，冷PBS洗涤3次，1000r／min，5min，离心去上清液，加入羊抗兔IgG-FITC 20μ1，室温避光反应90min，流式细胞仪测定每组细胞的平均荧光强度(MFI)，以Cell Quest软件分析细胞膜表面ICAM-1蛋白表达。
5原位杂交方法检测HUVEC的肿瘤坏死因子-α(TNFα)mRNA表达：
按照TNFαmRNA原位杂交试剂盒操作方法进行。培养于盖玻片的各组内皮细胞用4％多聚甲醛室温固定20min。胃蛋白酶于37℃消化60s。37℃预杂交液4h，37℃杂交过夜。以2×SSC，0.5×SSC及0.2×SSC洗片，封闭液37℃封闭30min，生物素化鼠抗地高辛37℃孵育1h，SABC孵育20min，DAB显色20min-30min，苏木素轻度复染，封片。阴性对照用预杂交液替代探针工作液。镜下观察并采用MetaMorth／DP10／BX41型细胞图像分析仪进行TNFαmRNA的半定量分析。
(二)动物实验部分：
1分组：
健康雄性Wistar大鼠按随机数字表随机分为对照组(C组)、ALI组(0A组)及Cap组(OA+Cap组)。OA组经颈静脉缓慢注射油酸(0.10 ml／kg)；OA+Cap组在注入油酸后立即腹腔注入Cap(1.25 mg／kg)，C组立即腹腔注射等量10％葡萄糖溶液。
2肺损伤的评价：
急性肺损伤通过动脉血气分析，肺组织学，肺干／湿比及肺泡灌洗液中细胞数量和蛋白含量测定来评价。
3肺组织细胞间粘附分子-1(ICAM-1)及核因子(NF)-κB蛋白表达的测定：
肺组织石蜡切片ICAM-1及NF-KB免疫组化染色采用链亲和素-生物素复合物(SABC)法免疫组化试剂盒检测(武汉博士德公司)。ICAM-1阳性反应定。位于胞浆(呈棕黄色颗粒)；NF-κB核阳性细胞为胞核呈棕黄色。每张ICAM-1免疫组化切片取5个不重复高倍视野，用MetaMorth／DP10／BX41型图象分析仪测其吸光度(A)值，取其平均值反映ICAM-1表达情况。每张NF-κB免疫组化片取5个不重复高倍视野，计算出NF-κB核阳性率(核阳性率=核阳性细胞数／总细胞数)，以反映活化程度。
4循环内皮细胞(circulating endothelial cell，CEC)的分离和计数：
CEC的检测采用Percoll密度梯度离心法。按Mbithe Mutunga法并稍做调整。每组动物观测时间结束前留取枸橼酸钠抗凝静脉血3mL，加入3mL生理盐水后轻轻倒置混匀，向试管中加入1.6mL 100％Percoll悬液，室温下1000r／min，离心10min，取中层液1mL计数用。其上清取出到另一试管中，3000r／min，离心20min后弃上清，加入0.5mL生理盐水，强烈震荡5min，取上层提取液和中层各1小滴分别加入血细胞计数池内，在光镜下计数全部9个大方格中的CEC数，以每0.9μL的细胞数表示，显微镜下观察细胞形态后照相。CEC的鉴定用Ⅷ因子相关抗原抗体进行免疫组化染色得到证实。
5血浆纤溶酶原激活物(tPA)及纤溶酶原激活物抑制物(PAI-1)活性的检测：
血标本的留取同步骤4。tPA及PAI-1活性的检测采用发色底物法测定，结果分别以IU／L及AU／L为单位表示
(三)临床实验部分：
1标本采集：
所有血标本均在确诊后24 h内通过静脉留置针留取并及时检测。
2血CEC检测：
方法同动物实验部分所用方法，Nikon UFX-Ⅱ型显微镜观察细胞形态后照相。
3血浆凝血酶原时间(prothrombin time，PT)、部分凝血活酶时间(activated partial thromboplastin time，APTT)、纤维蛋白原(fibrinogen，FG)、纤维蛋白原降解产物(fibrin degradationproducts，FDP)及D-二聚体的检测：
PT及APTT测定采用凝固法；FG测定采用Clouse法；FDP测定采用胶乳凝集法；D-二聚体测定采用ELISA法。
4血气分析的检测：
动脉血2mL，美国AVL-OMNI V型血气分析仪测定pH值、动脉血氧分压(PaO_2)、动脉血二氧化碳分压(PaCO_2)。
结果
1、ELISA及间接免疫荧光法检测的结果提示暴露于1μg／ml LPS后，HUVECs的vWF及ICAM-1表达明显强于对照组，加入Cap后，随Cap浓度增加明显下调LPS升高的HUVECs的vWF及ICAM-1表达，至Cap为10~(-3)mol／L时，其vWF与ICAM-1表达与LPS组有明显差别(P＜0.05)。Cap抑制LPS升高的HUVECs的vWF及ICAM-1表达呈一定的浓度依赖方式。
2、原位杂交显示Cap10~(-5) mol／L及10~(-3) mol／L时表现明显下调HUVECs的TNFαmRNA表达，与LPS组比较差异明显(P＜0.05，P＜0.01)。
3、与对照组比较，给予油酸2h后各时相ALI组氧合指数出现明显下降，CECs数量明显增加；Cap干预后，各时相Cap组氧合指数明显增加，CECs数量明显减少，氧合指数的变化与CECs数量的变化明显负相关(r=－0.7602，P＜0.05=。
4、给予油酸后，OA、OA+Cap两组的tPA较对照组明显下降，而PAI-1明显升高；Cap干预后，2h后PAI-1出现明显下降，4h后tPA出现明显升高，与对照组比较差异不明显(P＞0.05)。
5、OA+Cap组肺组织NF-KB核阳性率及ICAM-1表达显著高于对照组，但明显低于OA组(P＜0.05，)。肺组织ICAM-1表达与NF-KB核阳性率呈明显正相关(r=0.7861，P＜0.05)。
6、ALI和ARDS组患者CEC数量明显高于健康对照组和ICU内对照组(P＜0.05)，且ALI及ARDS组CEC数量与其APACHEII评分明显相关(r=0.55，P＜0.05及r=0.62，P＜0.05)，与LIS明显相关(r=0.60，P＜0.05及r=0.53，P＜0.05)；并且CEC数量与PaO_2明显负相关(r=－0.49，P＜0.05及r=－0.64，P＜0.05)。
7、ALI和ARDS组患者FDP和D-二聚体较健康对照组及ICU内对照组明显增加(P＜0.05)，ARDS组患者FG较健康对照组及ICU内对照组明显增加(P＜0.05)，ALI组患者FG较健康对照组明显增加(P＜0.05)。
结论
1、卡托普利对脂多糖诱导的HUVEC的活化及损伤有拮抗作用，其机制可能与卡托普利发挥直接的抗炎作用有关。
2、卡托普利对油酸所致的ALI大鼠血管内皮细胞损伤有一定的保护作用，其机制可能与卡托普利改善凝血系统的失衡及发挥直接的抗炎作用均有关系。
3、ARDS患者存在的血管内皮损伤可能在其复杂的发病机制中起重要作用。循环内皮细胞及凝血、纤溶指标可作为评价ARDS患者疾病严重程度及肺损伤程度的监测指标，指导临床治疗。
Objective
Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), remains one of the major challenges in intensive care medicine. Although tremendous efforts have been made, there is no specific therapy available yet in preventing and/or treating of ALI/ARDS. Searching for effective and specific therapies is still urgently needed, ideally with the conventional pharmaceutical drugs.
The features of ALI/ARDS, including diffuse interstitial and pulmonary edema, inflammatory cell infiltration, and protein-rich fluid in the alveolar space, indicate a significant endothelial injury with an increased vascular permeability, which has been considered as one of the initiating steps in this disorder. Pulmonary endothelium is one of the most vulnerable targets of a various insults. Endothelial injury and/or dysfunction have been found among patients and model animals of sepsis and ARDS. The activated/damaged endothelial cells are in turn involved in acute inflammatory responses by interacting and activating inflammatory cells, and affecting the coagulation/fibrinolysis system. We and others have reported that endothelial cells activation/damage and the related coagulation changes were closely correlated with the severity of injury and the clinical outcome in the ALI/ARDS patients. Protection of endothelial cells may offer a potential therapeutic strategy for ALI/ARDS.
Circulating endothelial cells (CECs) are vascular endothelial cells in circulation. All factors which may damage the endothelium can increase the number of CECs. CECs directly and specifically reflect the damage of endothelial cell in vivo, especially the exfoliative damage. Furthermore, because endothelial cells cover the surface of blood vessles, they are in tight contact with solid organs; thus, endothelial cell activation and damage are closely related to organ dysfunction. The objective of this investigation was to measure the changes of CECs, blood coagulation and fibrinolysis indexes in patients with critical illness, in order to study their clinical significance during the progress of ARDS.
The rennin-angiotensin system is crucial for maintaining blood pressure homeostasis, as well as fluid and salt balance. Angiotensin-converting enzyme (ACE) plays a crucial role by converting angiotensin I to angiotensin II, the main effecter of the system, as well as by degrading bradykinin and kallidin, the potent vasodilators. ACE inhibitors, such as captopril (Cap) and peridopril, have been widely used clinically for treatment of hypertension. Evidence showed that they also exert various beneficial effects on vascular structure and function by protecting endothelial cells. Treating endothelial cells in vitro with captopril showed an anti-apoptotic effect by up-regulating pro-survival genes. Administration of perindopril improved endotoxin-induced endothelial dysfunction in rabbits through a NO-dependent mechanism. Rosei and colleagues demonstrated that either inhibiting ACE or blocking angiotensin receptor could reduce the circulating levels of vascular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), and coagulation factors, such as von Willebrand factor (vWF), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1) in diabetes patients with hypertension. Recently, Imai et al. reported that rennin-angiotensin system may play an important role in the pathogenesis of ALI. Using transgenic animals and molecular biological approaches, they demonstrated that ACE, angiotensin II and its receptor type Ia may promote lung injury, while ACE2 and type II receptor of angiotensin II may protect the mice from severe ALI.
However, it is unknown whether inhibition of ACE with a pharmaceutical inhibitor like captopril could provide beneficial effects on ALI/ARDS in vivo. To investigate the effects of captopril on endothelial damage, we have chosen the oleic acid (OA)-induced acute lung injury in rats or on the cultured HUVEC caused by bacterial lipopolysaccharide as the model. Since ARDS has diversified etiology, this model only represents limited clinical situations. However, captopril prevented OA-induced severe endothelial damage and lung injury, implying that using of ACE inhibitors may be one of useful novel options for ALI/ARDS managing.
Methods
Experimental Materials
Part one: 25-30cm cords of healthy neonates were supplied by Woman's hospital of Tiexi district, Shenyang city.
Part two: Adult male Sprague-Dawley rats (Experimental Animal Centre,China Medical University, Shenyang, China) with body weights around 220-260 g were used, in compliance with the protocol approved by the Institutional Animal Care and Use Committee.
Part three: Twenty-one patients with critical illness admitted to the intensive care units of Emergency department and of medicine,the first affiliated hospital, China Medical University; and Shenyang Central Hospital affiliated to Shenyang Medical College between May 2002 and Apr 2003 were eligible for inclusion in the study. Ten patients with severe infection, 6 patients with severe trauma, 3 patients with large area burn and 2 patients with tumor were included. These patients with ALI or ARDS were diagnosed according to the criteria formulated by Respiratory Disease Division of Chinese Medical Association in 2000. Among them there are 14 patients with ALI, their mean age is (50±17)years; 7 patients with ARDS, their mean age is (49±15)years. Ten cases were ICU controls, they were other patients with critical illness recruited from among patients who did not meet the ARDS, ALI,sepsis or systemic inflammatory response syndrome(SIRS) criteria, their mean age is (51±19)years old. 15 cases were healthy volunteers, they were recruited from among members of physical examination, their mean age is (49±13)years old. Clinical information on each patient were recorded, including diagnosis, the Acute Physiology and Chronic Health Evaluation(APACHE) II, data set on ICU admission and Lung Injury Score (LIS).
Experimental Procedure
Part One: Cell Culture Experiment
Primary culture of human umblical veinendothelial cells: Improved Jaffe method was adopted.25-30cm cords of healthy neonates were used and 0.125% trypsin-0.02% EDTA (or 0.25% trypsin) were poured for 15-20 minutes at room temperature after cords were washed by PBS, then cell suspension was collected. After centrifugation, the number of endothelial cell s were adjusted to 3×10~8/Lby using DMEM culture media containing 20% inactivated neonate bovine serum, then cultivated on 24 pore culture boards with coverslips placed in advance and 6 pore culture boards. Culture media were changed every two days. Indirected immunofluorescence method was used to evaluate endothelial cells. Endothelial cell were cultured with serum-free medium for 24h before endothelial cells were used.
The content of vWF in the cell culture suspension was measured with ELISA kit according to the manufacture's introduction.
The expression of ICAM-1 protein in HUVECs was detected by indirected immunofluorescence method. HUVECs were washed by PBS, and digested with 0.25% tyrosine, cells were collected, and then washed by PBS, discard the supernatants, and PBS containing 10% serum was added for 30min at 4℃, centrifugated at 1000 r/min for 5min, discard the supernatants, then 100μL of rabbit anti human ICAM-1 polyclonal antibody(1:400) was added for 1h at 37℃. After washed by cold PBS, 20μL secondary antibody (goat anti rabbit IgG-FITC)(1:40) was added for 90min at room temperature without light. Then mean fluorescent intensity was evaluated by flow cytometry.
The expression of TNFamRNA was evaluated by in situ hybridization method. Endothelial cells were fixed for 20min in 4% paraformaldehyde at room temperature (containing 1/1 000 DEPC). Cells were digested by pepsin at 37℃for 60seconds. After addition of pre-hybridization solution, the fixed cells were incubated at 37℃for 4h. The pre-hybridization solution was discarded, then 20μL hybridization solution was added. The sections were put into a wet box and incubated overnight at 37℃. After being washed with 2×SSC, 0.5×SSC and 0.2×SSC, the sections were stained with DAB for 20min. Ralative quantative analysis of TNFamRNA was carried out by using Metamorph/DP10/BX41 image analyzer. Probe was replaced by pre-hybridization solution in the negative control group.
Part Two: Animal Experiment
Total 72 rats were randomly divided into control, oleic acid (OA) and OA plus Cap (OA+Cap) treated groups. The control group was treated with saline. Acute lung injury was induced by injection of OA (0.1 ml/kg) (Sigma, St. Louis, MO) via jugular vein over a 1-min period. The captopril (Changzhou Pharmaceutical, Changzhou, China) treatment was given immediately after the OA injection by intraperitoneal administration at a dose of 1.25 mg/kg in 10% glucose. The dose of captopril was chosen according to previous reports (10, 11) and our pilot study, in which we found that a higher dose of captopril (3 mg/kg) led to 75% mortality after OA challenge. The rats in OA alone and control groups received an equal volume of 10% glucose. At each of designated time points (1, 2, 4 and 6 hours after OA challenge), 6 rats from each group were sacrificed by exsanguinations.
Acute lung injury was assessed with blood gas, lung tissue histology and wet/dry lung weight ratio, as well as the cell number and albumin concentration in the bronchoalveolar lavage fluid (BALF). Partial pressure of oxygen (PaO_2) in the blood samples taken from the carotid artery was measured with a Blood Gas Analyzer (Omni 5, AVL Diagnostics, Switzerland). The rat lungs were removed en bloc after sacrifice of the animals. The upper right lung from each rat was fixed by inflating with 4% paraformaldehyde at 20 mmH_2O for hematoxylin and eosin (H&E) staining. The lung injury score was assessed by a pathologist in a blinded fashion using the method described by Nishina et al. Briefly, lung injury was accessed for alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in the airspace or vessel wall, and thickness of alveolar wall/hyaline membrane. The severity of lung injury was scored as: 0=minimum, 1=mild, 2=moderate, 3=severe and 4=maximum damage. For each animal, 6 high magnification fields were randomly selected and graded for the average lung injury score (LIS). The rest of the right lung was weighed immediately and after dried at 60℃for 7 days to calculate the wet/dry weight ratio. BAL was conducted on the left lung by infusing 2 ml saline for four times. The fluid recovery was similar among the groups, approximately 80-90%. The cell counts and albumin content in the BALF were determined with a hemocytometer and the Biuret assay, respectively.
ICAM-1 expression and NF-κB activation in the lung tissues were determined using immunohistochemistry staining kits (Boster Biotechnology, Wuhan, China). Lung tissue sections (4μm) were incubated with either an anti-ICAM-1 (1:200 dilution, Boster Biotechnology) or anti-p65 antibody (1:400 dilution, Boster Biotechnology, Wuhan, China) at 4℃overnight in a humidified chamber, and then incubated with biotinylated mouse anti-rabbit antibody (1:200) followed by peroxidase-conjugated avidin. The positive staining was revealed with diaminobenzidine (DAB). Total 6 fields (400x) per animal were randomly chosen under a microscope by a blinded investigator. A computer-assisted color image analyzer (LUZEX-F, NIRECO/Nikon, Tokyo, Japan) was used to determine the staining intensity (absorbance value) of ICAM-1 positive cells and to detect the cells with NF-κB p65 nuclear staining, respectively. The average intensity of ICAM-1 staining and the percentage of NF-κB nuclear positive cells in each group were calculated.
CECs were isolated by isopycnic centrifugation. Total 3 ml of whole blood were drawn into a tube containing 0.4 ml of sodium citrate and mixed with 3 ml saline, and then with 1.6 ml Percoll (specific gravity=1.130 g/ml; Sigma). The mixture was centrifuged at 1,000 g for 10 min and the top layer cells were further centrifuged at 3,000 g for 20 min to pellet the cells. The cells were resuspended in 0.5 ml of saline and counted with a hemocytometer. The isolated CECs were defined by their size (20-50μM in diameter) and morphology. For confirmation, 100μl cell suspension was used to prepare cytospin smears, which were incubated with a polyclone anti-vWF antibody (Boster Biotechnology) at 1:200 dilution and 37℃for 1 h, followed by a biotinylated mouse anti-rabbit antibody (1:200) and peroxidase-conjugated avidin.
Tissue plasminogen activator (tPA) activity and PAI-1 activity in the plasma were determined by a chromogenic method (Sun Biosource of American Diagnostica Inc., Shanghai, China) according to the manufacturer's instructions. Briefly, blood (1 ml) was drawn through right jugular vein to collect plasma. For tPA activity measurement, plasma was immediately acidified with acetate buffer (pH 3.9, 1:1 dilution). For PAI-1 activity measurement, a fixed amount of tPA provided in the kit was added in excess to plasma prior to acidification. The amount of plasmin formed is proportional to the tPA activity and inversely proportional to the PAI-1 activity in the plasma. The tPA activity is measured by adding samples (100μl) to a mixture (100μl) of Gluplasminogen and a chromogenic substrate S-2251 at neutral pH in a 96-well plate (Nunc, Roskilde, Denmark) for 2.5 h at 37℃. The tPA in the sample catalyzes the conversion of plasminogen to plasmin, which in turn hydrolyzes the chromogenic substrate. The optical density readings at 405 nm were proportional to the tPA activity in the samples, which was calculated against a standard curve. The tPA activities are expressed as international units/liter (IU/L). Since PAI-1 activities are indirectly measure, they are expressed as arbitrary units/liter (AU/L).
Part Three: Clinical Experiment
Blood sampling. Blood samples were taken from freshly placed flushed venous cannulae. The initial 1-2 mL blood sample drawn was discarded to minimize EC contamination from the puncture wound of the vascular wall. Samples were collected from patients within 24h of the initial diagnosis of ARDS or ALI. Blood samples were analyzed without access to clinical information.
EC isolation method is the same as what we described in Part two.
Laboratory Methods. Plasma prothrombin time (PT) and activated partial thromboplastin time(APTT) were detected by coagulation method, fibrinogen(FG) was detected by Clouse Method, fibrin degradation products(FDP) was detected by latex agglutination method. Using enzyme-linked immunosorbent assay (ELISA) method to detect D-dimer.
Arterial blood gas analysis. An arterial blood gas analysis was carried out within 5 minutes of obtaining the venous blood, and pH, PaO_2, PaCO_2 were recorded.
Results
1. The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1μg/mL led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P <0.05 = , whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10~(-3) mol/L) plus LPS group, and there was a significancant difference when compared with LPS group (P <0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner.
2. In situ hybridization revealed that the expression of TNFαmRNA in HUVECs was inhibited by Cap both in a concentration of 10~(-3)mol/L, and in a lower concentration of 10~(-5)mol/L, when compared with LPS group.
3. Typical lung injury induced by OA challenge is featured with thickening of the alveolar septa, alveolar hemorrhage and infiltration of inflammatory cells due to the direct damage of the pulmonary endothelium. All these features were observed in the lung tissue from OA-treated animals as early as 1 hour after the challenge. The lung injury score was significantly increased in the OA group. Treatment of captopril dramatically reduced OA-induced lung injury with less interstitial edema, hemorrhage, and cellular infiltration and a significantly lower lung injury score. Captopril treatment also significantly reduced the enhancements of albumin content and cell counts in the BALF as well as the increased wet/dry lung weight ratio induced by OA injection.
4. Elevated number of circulating endothelial cells reflects a compromise in the endothelium integrity and is an indicator of endothelial damage in a variety of disorders. One of the major effects of captopril observed on the OA-induced lung injury seems to protect the endothelial cells and reduce the vascular permeability. We therefore isolated and counted CECs from the blood collected for direct evidence. OA intravenous injection almost doubled the number of the CECs, which was significantly lower in the animals treated with captopril after 2 h. A highly negative correlation was also found between CEC counts and PaO_2/FiO_2, implicating that the integrity of endothelium is critical for blood gas exchange.
5. Increased expression of ICAM-1 reflects EC activation and is one of the major manifestations of acute inflammatory responses. ICAM-1 expression in lung tissues was barely detectable in the control group by immunohistochemistry staining, and markedly increased as early as 1 h after OA challenge. The elevated expression of ICAM-1 remained for the whole study period of 6 h. Captopril treatment significantly reduced ICAM-1 expression induced by OA. Activation of transcription factor NF-κB is the major signal transduction pathway that regulates the expression of multiple early response genes related to inflammation. We examined the NF-κB activation in the lung tissues by immunohistochemistry staining for NF-κB p65 subunit. Compared with the control group, OA-treatment induced a dramatic and consistent increase of nuclear staining of NF-κB in the lung tissues, which was blocked by captopril treatment significantly. It was known that NF-κB activation is involved in ICAM-1 expression in endothelial cells. Our data also showed a positive correlation between the NF-κB activation and ICAM-1 expression in the lung tissues.
6. Aggrandized coagulation activity is one of the hallmarks in ALI/ARDS, as a consequence of EC activation/damage. Disturbance of tPA and PAI-1 was observed in ALI animal models as well as in ARDS patients. The levels of tPA activity were decreased, while the activities of PAI-1 were increased significantly in OA treated groups. In captopril treated animals, the decreased tPA activities at 4 h were partially reversed. The OA-induced increase in PAI-1 activities was reduced by captopril after 2 h.
7. The number of CECs in ALI group patients (10.4±2.3) and ARDS group patients (16.1±2.7) was higher than that in healthy control group (1.9±0.5) and ICU control group (2.6±0.6)(P<0.05). Both in ALI and in ARDS patients,the number of CECs was correlated with APACHE II (r=0.55, P<0.05 and r=0.62, P<0.05, respectively) and LIS (r=0.60, P<0.05 and r=0.53, P<0.05, respectively) . The number of CECs was negatively correlated with PaO_2 in ALI and in ARDS (r=-0.49, P<0.05 and r=-0.64, P<0.05, respectively) .
8. The level of FDP and D-dimer were higher in ALI patients and ARDS patients than that in ICU control group and in healthy control group (P<0.05). The level of FG in ARDS group was significantly higher than in ICU control group and in healthy control group (P<0.05) , but in ALI group, the level of FG was significantly higher than that in healthy control group only (P<0.05) .
Conclusions
1. Captopril antagonized the activation and injury of HUVECs induced by LPS, which may be concerned with the decrease in TNFαmRNA expression.
2. With the OA-induced ALI model, our data demonstrated that ACE plays an important role in pathogenesis of ALI/ARDS by involving activation/damage of endothelial cells. A pharmaceutical approach with captopril prevents animals from severe lung injury induced by oleic acid. Captopril and other ACE inhibitors may offer a practical option for ALI/ARDS treatment, since they have been widely used in clinic for a variety of diseases.
3. Endothelial damage occurs in ARDS patients, which may play a major role in the pathophysiology of ARDS. Changes of the marker of endothelial cell activation and damage,such as CECs, plasma coagulation and fibrinolysis indexes ,to some extent, reflect the severity of the illness and the lung injury in ARDS.
前言
急性呼吸窘迫综合征(acute respiratory distress syndrome，ARDS)系由多种原因，如严重创伤和感染等肺内、肺外因素所致的急性缺氧性呼吸衰竭，其本质为急性肺损伤(acute lung injury，ALI)，以微血管渗透性增加，间质、肺泡水肿和低氧血症为特征，目前仍是引起高死亡率的疾病之一(40-60％)，并还缺乏有效的治疗办法。目前发病机制尚未明确，临床上缺乏敏感的早期诊断和病情监测指标及有效的治疗药物。因此，寻找特异而有效的药物成为该领域的研究热点，而利用传统己知的药物则成为理想的选择。
在所有的组织器官中，肺是富含内皮细胞的器官，在ALI的发展过程中总是伴随着内皮细胞(endothelial cell，EC)的激活和损伤。研究显示，ALI中肺血管EC是受损的主要靶细胞，更是活跃的炎症细胞和效应细胞，在调节凝血、纤溶和血管紧张度方面发挥作用。循环内皮细胞(circulatingendothelial cell，CEC)是循环血中的血管EC，导致EC损伤的因素均可使CEC增多，CEC可直接而特异的反映活体内EC损伤情况，且主要反映了EC的脱落性损伤。近来研究显示，CEC的改变与器官功能障碍及血管损伤明显相关。新近的研究也发现，凝血异常与炎症反应密切相关，并直接影响预后。有作者认为内皮细胞的活化和损伤是ARDS的重要标志，在疾病复杂的病理机制中起重要作用，并且其程度与疾病的严重程度及预后密切相关。因此，怎样保护及逆转内皮细胞功能障碍成为ARDS治疗中一个新的发展趋势。
卡托普利是一种血管紧张素转换酶抑制剂(angiotension convertingenzyme inhibitot，ACEI)，主要用于心血管疾病的降压治疗，新近的研究认为其还具有其他良好的药理作用，除清除氧自由基、抗氧化损伤以外，还具有内皮细胞保护作用，而其机制除与抑制血管紧张素转换酶(angiotensionconverting enzyme，ACE)有关外，是否还存在其他机制目前尚未明了。
我们通过体外培养人脐静脉内皮细胞(HUVEC)，观察细菌脂多糖诱导HUVEC活化及损伤的状态下及在体油酸诱导大鼠急性肺损伤(ALI)过程中，在血管内皮活化及损伤状态下，探讨卡托普利的保护作用及其可能的机制。
材料与方法
一、实验材料
(一)人脐静脉内皮细胞原代培养材料：
健康新生儿脐带25～30cm，由沈阳市铁西区妇婴医院提供。
(二)动物实验部分：
Wistar大鼠72只，体重220～260g，中国医科大学实验动物中心提供。
(三)临床实验对象：
ICU、RICU内危重病患者21例，根据临床表现、体征、X线胸片、血气分析结果确诊为ALI或ARDS。ICU内对照者10例，健康对照者15例。
二、实验步骤
(一)细胞培养部分：
1人脐静脉内皮细胞的培养：
采用改良Jaffe等法。用0.25％的胰蛋白酶灌注消化脐静脉后，离心收集内皮细胞，接种于75cm~2培养瓶，置于37℃、5％CO_2饱和湿度的孵箱中培养培养液为含20％新生牛血清的DMEM。待细胞长满2／3瓶底后进行传代。Ⅷ因子相关抗原免疫组化染色阳性，证实为内皮细胞。
2实验分组：
实验分为3组：对照组：常规无血清DMEM培养；LPS组：1μg／ml LPS；Cap+LPS组：根据Cap浓度的不同(10~(-7)mol／L、10~(-5)mol／L及10~(-3)mol／L)分为3个亚组，在每个亚组中LPS(1μg／m1)和Cap同时加入。
3细胞培养上清血管假性血友病因子(vWF)的测定：
用酶联免疫吸附试验(ELISA)检测各组培养上清中vWF的抗原含量(％)。抗体为兔抗人vWF的单克隆抗体，方法按试剂盒说明书进行。
4间接免疫荧光方法检测HUVEC的细胞间粘附分子-1(ICAM-1)蛋白表达：
各组HUVEC以PBS洗涤2次，0.25％胰蛋白酶消化，收集各组细胞，加入兔抗人ICAM-1多克隆抗体100μ1，37℃孵育1h，冷PBS洗涤3次，1000r／min，5min，离心去上清液，加入羊抗兔IgG-FITC 20μ1，室温避光反应90min，流式细胞仪测定每组细胞的平均荧光强度(MFI)，以Cell Quest软件分析细胞膜表面ICAM-1蛋白表达。
5原位杂交方法检测HUVEC的肿瘤坏死因子-α(TNFα)mRNA表达：
按照TNFαmRNA原位杂交试剂盒操作方法进行。培养于盖玻片的各组内皮细胞用4％多聚甲醛室温固定20min。胃蛋白酶于37℃消化60s。37℃预杂交液4h，37℃杂交过夜。以2×SSC，0.5×SSC及0.2×SSC洗片，封闭液37℃封闭30min，生物素化鼠抗地高辛37℃孵育1h，SABC孵育20min，DAB显色20min-30min，苏木素轻度复染，封片。阴性对照用预杂交液替代探针工作液。镜下观察并采用MetaMorth／DP10／BX41型细胞图像分析仪进行TNFαmRNA的半定量分析。
(二)动物实验部分：
1分组：
健康雄性Wistar大鼠按随机数字表随机分为对照组(C组)、ALI组(0A组)及Cap组(OA+Cap组)。OA组经颈静脉缓慢注射油酸(0.10 ml／kg)；OA+Cap组在注入油酸后立即腹腔注入Cap(1.25 mg／kg)，C组立即腹腔注射等量10％葡萄糖溶液。
2肺损伤的评价：
急性肺损伤通过动脉血气分析，肺组织学，肺干／湿比及肺泡灌洗液中细胞数量和蛋白含量测定来评价。
3肺组织细胞间粘附分子-1(ICAM-1)及核因子(NF)-κB蛋白表达的测定：
肺组织石蜡切片ICAM-1及NF-KB免疫组化染色采用链亲和素-生物素复合物(SABC)法免疫组化试剂盒检测(武汉博士德公司)。ICAM-1阳性反应定。位于胞浆(呈棕黄色颗粒)；NF-κB核阳性细胞为胞核呈棕黄色。每张ICAM-1免疫组化切片取5个不重复高倍视野，用MetaMorth／DP10／BX41型图象分析仪测其吸光度(A)值，取其平均值反映ICAM-1表达情况。每张NF-κB免疫组化片取5个不重复高倍视野，计算出NF-κB核阳性率(核阳性率=核阳性细胞数／总细胞数)，以反映活化程度。
4循环内皮细胞(circulating endothelial cell，CEC)的分离和计数：
CEC的检测采用Percoll密度梯度离心法。按Mbithe Mutunga法并稍做调整。每组动物观测时间结束前留取枸橼酸钠抗凝静脉血3mL，加入3mL生理盐水后轻轻倒置混匀，向试管中加入1.6mL 100％Percoll悬液，室温下1000r／min，离心10min，取中层液1mL计数用。其上清取出到另一试管中，3000r／min，离心20min后弃上清，加入0.5mL生理盐水，强烈震荡5min，取上层提取液和中层各1小滴分别加入血细胞计数池内，在光镜下计数全部9个大方格中的CEC数，以每0.9μL的细胞数表示，显微镜下观察细胞形态后照相。CEC的鉴定用Ⅷ因子相关抗原抗体进行免疫组化染色得到证实。
5血浆纤溶酶原激活物(tPA)及纤溶酶原激活物抑制物(PAI-1)活性的检测：
血标本的留取同步骤4。tPA及PAI-1活性的检测采用发色底物法测定，结果分别以IU／L及AU／L为单位表示
(三)临床实验部分：
1标本采集：
所有血标本均在确诊后24 h内通过静脉留置针留取并及时检测。
2血CEC检测：
方法同动物实验部分所用方法，Nikon UFX-Ⅱ型显微镜观察细胞形态后照相。
3血浆凝血酶原时间(prothrombin time，PT)、部分凝血活酶时间(activated partial thromboplastin time，APTT)、纤维蛋白原(fibrinogen，FG)、纤维蛋白原降解产物(fibrin degradationproducts，FDP)及D-二聚体的检测：
PT及APTT测定采用凝固法；FG测定采用Clouse法；FDP测定采用胶乳凝集法；D-二聚体测定采用ELISA法。
4血气分析的检测：
动脉血2mL，美国AVL-OMNI V型血气分析仪测定pH值、动脉血氧分压(PaO_2)、动脉血二氧化碳分压(PaCO_2)。
结果
1、ELISA及间接免疫荧光法检测的结果提示暴露于1μg／ml LPS后，HUVECs的vWF及ICAM-1表达明显强于对照组，加入Cap后，随Cap浓度增加明显下调LPS升高的HUVECs的vWF及ICAM-1表达，至Cap为10~(-3)mol／L时，其vWF与ICAM-1表达与LPS组有明显差别(P＜0.05)。Cap抑制LPS升高的HUVECs的vWF及ICAM-1表达呈一定的浓度依赖方式。
2、原位杂交显示Cap10~(-5) mol／L及10~(-3) mol／L时表现明显下调HUVECs的TNFαmRNA表达，与LPS组比较差异明显(P＜0.05，P＜0.01)。
3、与对照组比较，给予油酸2h后各时相ALI组氧合指数出现明显下降，CECs数量明显增加；Cap干预后，各时相Cap组氧合指数明显增加，CECs数量明显减少，氧合指数的变化与CECs数量的变化明显负相关(r=－0.7602，P＜0.05=。
4、给予油酸后，OA、OA+Cap两组的tPA较对照组明显下降，而PAI-1明显升高；Cap干预后，2h后PAI-1出现明显下降，4h后tPA出现明显升高，与对照组比较差异不明显(P＞0.05)。
5、OA+Cap组肺组织NF-KB核阳性率及ICAM-1表达显著高于对照组，但明显低于OA组(P＜0.05，)。肺组织ICAM-1表达与NF-KB核阳性率呈明显正相关(r=0.7861，P＜0.05)。
6、ALI和ARDS组患者CEC数量明显高于健康对照组和ICU内对照组(P＜0.05)，且ALI及ARDS组CEC数量与其APACHEII评分明显相关(r=0.55，P＜0.05及r=0.62，P＜0.05)，与LIS明显相关(r=0.60，P＜0.05及r=0.53，P＜0.05)；并且CEC数量与PaO_2明显负相关(r=－0.49，P＜0.05及r=－0.64，P＜0.05)。
7、ALI和ARDS组患者FDP和D-二聚体较健康对照组及ICU内对照组明显增加(P＜0.05)，ARDS组患者FG较健康对照组及ICU内对照组明显增加(P＜0.05)，ALI组患者FG较健康对照组明显增加(P＜0.05)。
结论
1、卡托普利对脂多糖诱导的HUVEC的活化及损伤有拮抗作用，其机制可能与卡托普利发挥直接的抗炎作用有关。
2、卡托普利对油酸所致的ALI大鼠血管内皮细胞损伤有一定的保护作用，其机制可能与卡托普利改善凝血系统的失衡及发挥直接的抗炎作用均有关系。
3、ARDS患者存在的血管内皮损伤可能在其复杂的发病机制中起重要作用。循环内皮细胞及凝血、纤溶指标可作为评价ARDS患者疾病严重程度及肺损伤程度的监测指标，指导临床治疗。
Objective
Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), remains one of the major challenges in intensive care medicine. Although tremendous efforts have been made, there is no specific therapy available yet in preventing and/or treating of ALI/ARDS. Searching for effective and specific therapies is still urgently needed, ideally with the conventional pharmaceutical drugs.
The features of ALI/ARDS, including diffuse interstitial and pulmonary edema, inflammatory cell infiltration, and protein-rich fluid in the alveolar space, indicate a significant endothelial injury with an increased vascular permeability, which has been considered as one of the initiating steps in this disorder. Pulmonary endothelium is one of the most vulnerable targets of a various insults. Endothelial injury and/or dysfunction have been found among patients and model animals of sepsis and ARDS. The activated/damaged endothelial cells are in turn involved in acute inflammatory responses by interacting and activating inflammatory cells, and affecting the coagulation/fibrinolysis system. We and others have reported that endothelial cells activation/damage and the related coagulation changes were closely correlated with the severity of injury and the clinical outcome in the ALI/ARDS patients. Protection of endothelial cells may offer a potential therapeutic strategy for ALI/ARDS.
Circulating endothelial cells (CECs) are vascular endothelial cells in circulation. All factors which may damage the endothelium can increase the number of CECs. CECs directly and specifically reflect the damage of endothelial cell in vivo, especially the exfoliative damage. Furthermore, because endothelial cells cover the surface of blood vessles, they are in tight contact with solid organs; thus, endothelial cell activation and damage are closely related to organ dysfunction. The objective of this investigation was to measure the changes of CECs, blood coagulation and fibrinolysis indexes in patients with critical illness, in order to study their clinical significance during the progress of ARDS.
The rennin-angiotensin system is crucial for maintaining blood pressure homeostasis, as well as fluid and salt balance. Angiotensin-converting enzyme (ACE) plays a crucial role by converting angiotensin I to angiotensin II, the main effecter of the system, as well as by degrading bradykinin and kallidin, the potent vasodilators. ACE inhibitors, such as captopril (Cap) and peridopril, have been widely used clinically for treatment of hypertension. Evidence showed that they also exert various beneficial effects on vascular structure and function by protecting endothelial cells. Treating endothelial cells in vitro with captopril showed an anti-apoptotic effect by up-regulating pro-survival genes. Administration of perindopril improved endotoxin-induced endothelial dysfunction in rabbits through a NO-dependent mechanism. Rosei and colleagues demonstrated that either inhibiting ACE or blocking angiotensin receptor could reduce the circulating levels of vascular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), and coagulation factors, such as von Willebrand factor (vWF), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1) in diabetes patients with hypertension. Recently, Imai et al. reported that rennin-angiotensin system may play an important role in the pathogenesis of ALI. Using transgenic animals and molecular biological approaches, they demonstrated that ACE, angiotensin II and its receptor type Ia may promote lung injury, while ACE2 and type II receptor of angiotensin II may protect the mice from severe ALI.
However, it is unknown whether inhibition of ACE with a pharmaceutical inhibitor like captopril could provide beneficial effects on ALI/ARDS in vivo. To investigate the effects of captopril on endothelial damage, we have chosen the oleic acid (OA)-induced acute lung injury in rats or on the cultured HUVEC caused by bacterial lipopolysaccharide as the model. Since ARDS has diversified etiology, this model only represents limited clinical situations. However, captopril prevented OA-induced severe endothelial damage and lung injury, implying that using of ACE inhibitors may be one of useful novel options for ALI/ARDS managing.
Methods
Experimental Materials
Part one: 25-30cm cords of healthy neonates were supplied by Woman's hospital of Tiexi district, Shenyang city.
Part two: Adult male Sprague-Dawley rats (Experimental Animal Centre,China Medical University, Shenyang, China) with body weights around 220-260 g were used, in compliance with the protocol approved by the Institutional Animal Care and Use Committee.
Part three: Twenty-one patients with critical illness admitted to the intensive care units of Emergency department and of medicine,the first affiliated hospital, China Medical University; and Shenyang Central Hospital affiliated to Shenyang Medical College between May 2002 and Apr 2003 were eligible for inclusion in the study. Ten patients with severe infection, 6 patients with severe trauma, 3 patients with large area burn and 2 patients with tumor were included. These patients with ALI or ARDS were diagnosed according to the criteria formulated by Respiratory Disease Division of Chinese Medical Association in 2000. Among them there are 14 patients with ALI, their mean age is (50±17)years; 7 patients with ARDS, their mean age is (49±15)years. Ten cases were ICU controls, they were other patients with critical illness recruited from among patients who did not meet the ARDS, ALI,sepsis or systemic inflammatory response syndrome(SIRS) criteria, their mean age is (51±19)years old. 15 cases were healthy volunteers, they were recruited from among members of physical examination, their mean age is (49±13)years old. Clinical information on each patient were recorded, including diagnosis, the Acute Physiology and Chronic Health Evaluation(APACHE) II, data set on ICU admission and Lung Injury Score (LIS).
Experimental Procedure
Part One: Cell Culture Experiment
Primary culture of human umblical veinendothelial cells: Improved Jaffe method was adopted.25-30cm cords of healthy neonates were used and 0.125% trypsin-0.02% EDTA (or 0.25% trypsin) were poured for 15-20 minutes at room temperature after cords were washed by PBS, then cell suspension was collected. After centrifugation, the number of endothelial cell s were adjusted to 3×10~8/Lby using DMEM culture media containing 20% inactivated neonate bovine serum, then cultivated on 24 pore culture boards with coverslips placed in advance and 6 pore culture boards. Culture media were changed every two days. Indirected immunofluorescence method was used to evaluate endothelial cells. Endothelial cell were cultured with serum-free medium for 24h before endothelial cells were used.
The content of vWF in the cell culture suspension was measured with ELISA kit according to the manufacture's introduction.
The expression of ICAM-1 protein in HUVECs was detected by indirected immunofluorescence method. HUVECs were washed by PBS, and digested with 0.25% tyrosine, cells were collected, and then washed by PBS, discard the supernatants, and PBS containing 10% serum was added for 30min at 4℃, centrifugated at 1000 r/min for 5min, discard the supernatants, then 100μL of rabbit anti human ICAM-1 polyclonal antibody(1:400) was added for 1h at 37℃. After washed by cold PBS, 20μL secondary antibody (goat anti rabbit IgG-FITC)(1:40) was added for 90min at room temperature without light. Then mean fluorescent intensity was evaluated by flow cytometry.
The expression of TNFamRNA was evaluated by in situ hybridization method. Endothelial cells were fixed for 20min in 4% paraformaldehyde at room temperature (containing 1/1 000 DEPC). Cells were digested by pepsin at 37℃for 60seconds. After addition of pre-hybridization solution, the fixed cells were incubated at 37℃for 4h. The pre-hybridization solution was discarded, then 20μL hybridization solution was added. The sections were put into a wet box and incubated overnight at 37℃. After being washed with 2×SSC, 0.5×SSC and 0.2×SSC, the sections were stained with DAB for 20min. Ralative quantative analysis of TNFamRNA was carried out by using Metamorph/DP10/BX41 image analyzer. Probe was replaced by pre-hybridization solution in the negative control group.
Part Two: Animal Experiment
Total 72 rats were randomly divided into control, oleic acid (OA) and OA plus Cap (OA+Cap) treated groups. The control group was treated with saline. Acute lung injury was induced by injection of OA (0.1 ml/kg) (Sigma, St. Louis, MO) via jugular vein over a 1-min period. The captopril (Changzhou Pharmaceutical, Changzhou, China) treatment was given immediately after the OA injection by intraperitoneal administration at a dose of 1.25 mg/kg in 10% glucose. The dose of captopril was chosen according to previous reports (10, 11) and our pilot study, in which we found that a higher dose of captopril (3 mg/kg) led to 75% mortality after OA challenge. The rats in OA alone and control groups received an equal volume of 10% glucose. At each of designated time points (1, 2, 4 and 6 hours after OA challenge), 6 rats from each group were sacrificed by exsanguinations.
Acute lung injury was assessed with blood gas, lung tissue histology and wet/dry lung weight ratio, as well as the cell number and albumin concentration in the bronchoalveolar lavage fluid (BALF). Partial pressure of oxygen (PaO_2) in the blood samples taken from the carotid artery was measured with a Blood Gas Analyzer (Omni 5, AVL Diagnostics, Switzerland). The rat lungs were removed en bloc after sacrifice of the animals. The upper right lung from each rat was fixed by inflating with 4% paraformaldehyde at 20 mmH_2O for hematoxylin and eosin (H&E) staining. The lung injury score was assessed by a pathologist in a blinded fashion using the method described by Nishina et al. Briefly, lung injury was accessed for alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in the airspace or vessel wall, and thickness of alveolar wall/hyaline membrane. The severity of lung injury was scored as: 0=minimum, 1=mild, 2=moderate, 3=severe and 4=maximum damage. For each animal, 6 high magnification fields were randomly selected and graded for the average lung injury score (LIS). The rest of the right lung was weighed immediately and after dried at 60℃for 7 days to calculate the wet/dry weight ratio. BAL was conducted on the left lung by infusing 2 ml saline for four times. The fluid recovery was similar among the groups, approximately 80-90%. The cell counts and albumin content in the BALF were determined with a hemocytometer and the Biuret assay, respectively.
ICAM-1 expression and NF-κB activation in the lung tissues were determined using immunohistochemistry staining kits (Boster Biotechnology, Wuhan, China). Lung tissue sections (4μm) were incubated with either an anti-ICAM-1 (1:200 dilution, Boster Biotechnology) or anti-p65 antibody (1:400 dilution, Boster Biotechnology, Wuhan, China) at 4℃overnight in a humidified chamber, and then incubated with biotinylated mouse anti-rabbit antibody (1:200) followed by peroxidase-conjugated avidin. The positive staining was revealed with diaminobenzidine (DAB). Total 6 fields (400x) per animal were randomly chosen under a microscope by a blinded investigator. A computer-assisted color image analyzer (LUZEX-F, NIRECO/Nikon, Tokyo, Japan) was used to determine the staining intensity (absorbance value) of ICAM-1 positive cells and to detect the cells with NF-κB p65 nuclear staining, respectively. The average intensity of ICAM-1 staining and the percentage of NF-κB nuclear positive cells in each group were calculated.
CECs were isolated by isopycnic centrifugation. Total 3 ml of whole blood were drawn into a tube containing 0.4 ml of sodium citrate and mixed with 3 ml saline, and then with 1.6 ml Percoll (specific gravity=1.130 g/ml; Sigma). The mixture was centrifuged at 1,000 g for 10 min and the top layer cells were further centrifuged at 3,000 g for 20 min to pellet the cells. The cells were resuspended in 0.5 ml of saline and counted with a hemocytometer. The isolated CECs were defined by their size (20-50μM in diameter) and morphology. For confirmation, 100μl cell suspension was used to prepare cytospin smears, which were incubated with a polyclone anti-vWF antibody (Boster Biotechnology) at 1:200 dilution and 37℃for 1 h, followed by a biotinylated mouse anti-rabbit antibody (1:200) and peroxidase-conjugated avidin.
Tissue plasminogen activator (tPA) activity and PAI-1 activity in the plasma were determined by a chromogenic method (Sun Biosource of American Diagnostica Inc., Shanghai, China) according to the manufacturer's instructions. Briefly, blood (1 ml) was drawn through right jugular vein to collect plasma. For tPA activity measurement, plasma was immediately acidified with acetate buffer (pH 3.9, 1:1 dilution). For PAI-1 activity measurement, a fixed amount of tPA provided in the kit was added in excess to plasma prior to acidification. The amount of plasmin formed is proportional to the tPA activity and inversely proportional to the PAI-1 activity in the plasma. The tPA activity is measured by adding samples (100μl) to a mixture (100μl) of Gluplasminogen and a chromogenic substrate S-2251 at neutral pH in a 96-well plate (Nunc, Roskilde, Denmark) for 2.5 h at 37℃. The tPA in the sample catalyzes the conversion of plasminogen to plasmin, which in turn hydrolyzes the chromogenic substrate. The optical density readings at 405 nm were proportional to the tPA activity in the samples, which was calculated against a standard curve. The tPA activities are expressed as international units/liter (IU/L). Since PAI-1 activities are indirectly measure, they are expressed as arbitrary units/liter (AU/L).
Part Three: Clinical Experiment
Blood sampling. Blood samples were taken from freshly placed flushed venous cannulae. The initial 1-2 mL blood sample drawn was discarded to minimize EC contamination from the puncture wound of the vascular wall. Samples were collected from patients within 24h of the initial diagnosis of ARDS or ALI. Blood samples were analyzed without access to clinical information.
EC isolation method is the same as what we described in Part two.
Laboratory Methods. Plasma prothrombin time (PT) and activated partial thromboplastin time(APTT) were detected by coagulation method, fibrinogen(FG) was detected by Clouse Method, fibrin degradation products(FDP) was detected by latex agglutination method. Using enzyme-linked immunosorbent assay (ELISA) method to detect D-dimer.
Arterial blood gas analysis. An arterial blood gas analysis was carried out within 5 minutes of obtaining the venous blood, and pH, PaO_2, PaCO_2 were recorded.
Results
1. The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1μg/mL led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P <0.05 = , whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10~(-3) mol/L) plus LPS group, and there was a significancant difference when compared with LPS group (P <0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner.
2. In situ hybridization revealed that the expression of TNFαmRNA in HUVECs was inhibited by Cap both in a concentration of 10~(-3)mol/L, and in a lower concentration of 10~(-5)mol/L, when compared with LPS group.
3. Typical lung injury induced by OA challenge is featured with thickening of the alveolar septa, alveolar hemorrhage and infiltration of inflammatory cells due to the direct damage of the pulmonary endothelium. All these features were observed in the lung tissue from OA-treated animals as early as 1 hour after the challenge. The lung injury score was significantly increased in the OA group. Treatment of captopril dramatically reduced OA-induced lung injury with less interstitial edema, hemorrhage, and cellular infiltration and a significantly lower lung injury score. Captopril treatment also significantly reduced the enhancements of albumin content and cell counts in the BALF as well as the increased wet/dry lung weight ratio induced by OA injection.
4. Elevated number of circulating endothelial cells reflects a compromise in the endothelium integrity and is an indicator of endothelial damage in a variety of disorders. One of the major effects of captopril observed on the OA-induced lung injury seems to protect the endothelial cells and reduce the vascular permeability. We therefore isolated and counted CECs from the blood collected for direct evidence. OA intravenous injection almost doubled the number of the CECs, which was significantly lower in the animals treated with captopril after 2 h. A highly negative correlation was also found between CEC counts and PaO_2/FiO_2, implicating that the integrity of endothelium is critical for blood gas exchange.
5. Increased expression of ICAM-1 reflects EC activation and is one of the major manifestations of acute inflammatory responses. ICAM-1 expression in lung tissues was barely detectable in the control group by immunohistochemistry staining, and markedly increased as early as 1 h after OA challenge. The elevated expression of ICAM-1 remained for the whole study period of 6 h. Captopril treatment significantly reduced ICAM-1 expression induced by OA. Activation of transcription factor NF-κB is the major signal transduction pathway that regulates the expression of multiple early response genes related to inflammation. We examined the NF-κB activation in the lung tissues by immunohistochemistry staining for NF-κB p65 subunit. Compared with the control group, OA-treatment induced a dramatic and consistent increase of nuclear staining of NF-κB in the lung tissues, which was blocked by captopril treatment significantly. It was known that NF-κB activation is involved in ICAM-1 expression in endothelial cells. Our data also showed a positive correlation between the NF-κB activation and ICAM-1 expression in the lung tissues.
6. Aggrandized coagulation activity is one of the hallmarks in ALI/ARDS, as a consequence of EC activation/damage. Disturbance of tPA and PAI-1 was observed in ALI animal models as well as in ARDS patients. The levels of tPA activity were decreased, while the activities of PAI-1 were increased significantly in OA treated groups. In captopril treated animals, the decreased tPA activities at 4 h were partially reversed. The OA-induced increase in PAI-1 activities was reduced by captopril after 2 h.
7. The number of CECs in ALI group patients (10.4±2.3) and ARDS group patients (16.1±2.7) was higher than that in healthy control group (1.9±0.5) and ICU control group (2.6±0.6)(P<0.05). Both in ALI and in ARDS patients,the number of CECs was correlated with APACHE II (r=0.55, P<0.05 and r=0.62, P<0.05, respectively) and LIS (r=0.60, P<0.05 and r=0.53, P<0.05, respectively) . The number of CECs was negatively correlated with PaO_2 in ALI and in ARDS (r=-0.49, P<0.05 and r=-0.64, P<0.05, respectively) .
8. The level of FDP and D-dimer were higher in ALI patients and ARDS patients than that in ICU control group and in healthy control group (P<0.05). The level of FG in ARDS group was significantly higher than in ICU control group and in healthy control group (P<0.05) , but in ALI group, the level of FG was significantly higher than that in healthy control group only (P<0.05) .
Conclusions
1. Captopril antagonized the activation and injury of HUVECs induced by LPS, which may be concerned with the decrease in TNFαmRNA expression.
2. With the OA-induced ALI model, our data demonstrated that ACE plays an important role in pathogenesis of ALI/ARDS by involving activation/damage of endothelial cells. A pharmaceutical approach with captopril prevents animals from severe lung injury induced by oleic acid. Captopril and other ACE inhibitors may offer a practical option for ALI/ARDS treatment, since they have been widely used in clinic for a variety of diseases.
3. Endothelial damage occurs in ARDS patients, which may play a major role in the pathophysiology of ARDS. Changes of the marker of endothelial cell activation and damage,such as CECs, plasma coagulation and fibrinolysis indexes ,to some extent, reflect the severity of the illness and the lung injury in ARDS.
引文
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26 Boldt J, Papsdorf M, Rothe A, et al. Changes of the hemostatic network in critically ill patients-Is there a difference between sepsis, trauma, and neurosurgery patients? Crit Care Med, 2000, 28:445-450
27 Yan SB, Helterbrand JD, Hartman DL, et al. Low levels of protein C are associated with poor outcome in severe sepsis. Chest, 2001, 120:915-922
28 Hite RD, Morris PE. Acute respiratory distress syndrome: pharmacological treatment options in development. Drugs, 2001, 61:897-907
29 Masters RM, Helterbrand J, Utterback BG, et al. Factor VIIa and antithrombinlll activity during severe sepsis and septic shock in neutropenic patients. Blood, 1996, 88:881-886
30 DschietzigT, AlexiouK, Laule M, et al. Stimulation of pulmonary and endothelin-1 by antithrombinlll and endothelin antagonists in sepsis-related acute respiratory distress syndrome? Crit Care Med, 2000, 28:2445-9