Activation And Damage Of Endothelium And Potential Therapeutic Effect Of Captopril In Acute Lung Injury

ObjectiveAcute 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.MethodsExperimental MaterialsPart 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 ProcedurePart One: Cell Culture ExperimentPrimary 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 3×10⁸/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 ExperimentTotal 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 (PaO₂) 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 mmH₂O 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 ExperimentBlood 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, PaO₂, PaCO₂ were recorded.Results1. 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^-3mol/L, and in a lower concentration of 10^-5mol/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 PaO₂/FiO₂, 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 PaO₂ 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) .Conclusions1. 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.