A New Treatment Strategy For ALI/ARDS: Renin-angiotensin System

OBJECTIVE:The lack of specific and efficient therapies is a major cause for the high mortality rate observed in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recently, much research interest was directed towards the role renin-angiotensin system (RAS) played in the pathogenesis of ALI/ARDS. Therefore, this study was undertaken to clarify the relation of ALI/ARDS and RAS by using a mouse cecal ligation and puncture (CLP) model. We also investigated the impacts of RAS drugs on ALI/ARDS and its'mechanisms. In addition, to further elucidate the mechanisms, of losartan prevent lung injury, we looked at the ultrastructure of vascular endothelial cell in ALI/ARDS model, and investigated the impacts of losartan on HUVEC apoptosis in vitro. We hope that these findings can aid in the development of a new therapeutic strategy for ALI/ARDS in future studies.METHODS:Part 1. The changes of RAS on ALI/ARDS modelsALI/ARDS animal models were induced by the CLP operaten. The acute lung injury of these models was assessed by measuring blood gas, wet/dry lung weight rate (W/D), and lung tissue histology 18 h after operaten. After the building of the ALI/ARDS models is successful, immunohistochemistry, Western blotting and radioimmunity were used to investigate the changes of several key enzymes of RAS, such as ACE, ACE2 and Ang II.Part 2. The impacts of RAS drugs on ALI/ARDSThe mice were than divided into three group. Each group received a separate intraperitoneal injection of angiotensin-converting enzyme (ACE) inhibitor captopril, or recombinant mouse ACE2 (rmACE2), or AT1 inhibitor losartan after CLP. These treatments would help in illustrating the effects these drugs have on the ALI/ARDS models, especially losartan.Part 3. The possible mechanisms of losartan prevented lung injuryLung tissue samples were collected from sacrificed samples of the SHAM group, CLP group and losartan group. Then, nuclear protein and cytosol protein extraction were performed. Then the extraction is subjected to Western blotting or EMSA (Electrophoretic Mobility Shift Assay) which NF-ΚB activations, IκB-degradations, phosphorylations of p38 MAPK, ERK1/2, and JNK expressions are evaluated.Part 4. The impacts of losartan on HUVEC apoptosisTo further elucidate the injure pervention mechanisms of losartan in the lung, we observed the ultrastructure of lung vascular endothelial cell in ALI/ARDS model. Moreover, to investigate the impacts of losartan on HUVEC (Human umbilical vein endothelial cell), we conducted the apoptosis in vitro experiments on the models. HUVEC apoptosis rate was measured by the flow cytometry at 24 hours after AngII treatment. Losartan was then added to the Ang II treated HUVEC. Then it was measured again for the apoptosis rate as described previously.RESULTS:Part 1. The changes of RAS on ALI/ARDS models1. The common lung injuries induced by CLP challenge typically features the thickening of the alveolar septa, alveolar hemorrhage, and infiltration of the inflammatory cells. All these features can be observed in the lung tissue from CLP-treated animals 18 h after operation. Furthermore, the CLP-induced ALI/ARDS led to an increase in the wet/dry weight rate (W/D:6.08±0.64 vs 4.38±0.93, P<0.01. vs sham group) of the lung tissues, and a decrease in the PaO2/FiO2( 194.3±23.9 vs 346.7±20.5. P<0.01, vs sham group).2. Immunohistochemistry and Western blotting tests of the lung tissues from CLP-treated animals showed a decrease in the ACE2 protein level. However, in both the CLP and sham mice there is a very high level of ACE protein present, and there is no significant differences between the two groups.3. CLP markedly increased Ang II level in lungs and plasma of mice, and several RAS drugs significantly impacted the Ang II levels of mice. Compared with the CLP group, captopril or rmACE2 led to decreasing of the Ang II level in mice (Lung,1.58±0.16, 1.65±0.21 vs 2.38±0.41; Plasma,178.04±17.87, 153.74±10.24 vs 213.38±25.44).Part 2. The impacts of RAS drugs on ALI/ARDS1. Compared with the CLP group, W/D rate of lung tissue decreased significantly in captopril group (5.35±0.25 vs 6.06±0.22, P<0.05). But the W/D rate of rmACE2 group was no significant different from controls, whereas a decreased tendency was observed (5.61±0.59 vs 6.06±0.22, P=0.096). The losartan administration had led to a more significant decrease in W/D rate of the models when compared with the captopril group and rmACE2 group.2. The effectiviness of losartan in the prevention of the ALI/ARDS is partialy depended on the concentrate of losartan administered. The index of lung injury measured shown a persistent improving trend in the mice that received intraperitoneal injections of 5 mg/kg -15 mg/kg losartan. However, a decreasing trend is also observed in mice that received a dose of over 15 mg/kg. The maximum improvement was observed at 15 mg/kg losartan injection.3. Losartan treatment significantly attenuated TNF-α, IL-6, and IL-1β6 h after CLP, prevented histopathologic appearance of ALI/ARDS after CLP, and significantly improved 7-day survival in mice.Part 3. The possible mechanisms of losartan prevented lung injury1. Western blot analysis showed that CLP mice lung cytosolic extracts exhibited a degradation in the amount of sham IκB-αlevels in the cytosol , whereas similar degradation was not observed in the CLP+losartan lung tissues. Accordingly, CLP-induced activation of NF-κB signaling was inhibited significantly by losartan treatment 6 h after CLP in an EMSA experiment.2. Losartan given post - CLP led to the inhibition of the phosphorylation of p38MAPK, ERK1/2, and JNK pathways, which are critical for cytokine release in ALI/ARDS models.Part 4. The impacts of losartan on HUVEC apoptosis1. Cell edema or dissolved, separated widened perinuclear space, widened intercellular junctions of cells was detected in the lung vascular endothelial cell of CLP-induced ALI/ARDS mice.2. 24 hours after, the Ang II stimulation of the HUVEC cell apoptosis was dose-dependent. The cell apoptosis rate of three AngII groups (10-7mol/l, 10-6mol/l,10-5mol/l) were 7.85 %, 9.96% and 10.4 % respectively. After adding 10-6mol/l losartan, HUVEC apoptosis rate was decreased to 7.95%, 8.62%, 8.67% accordingly.CONCLUSIONS :1. RAS activation is one of the casusative agent of CLP-induced ALI/ARDS in mice models. And they are manifested as an increased secretion of AngⅡ.2. ACE and ACE2 in RAS have a different role in the regulation of the synthesis AngⅡ. While ACE has a positive effect in generating AngⅡ, ACE2 shows a negative effect. At the same time, captopril (ACE inhibitor) and rmACE2 can also partly reduce the secretion of AngⅡ, and thus prevent lung injuries of CLP-induced ALI/ARDS.3. The AT1 inhibitor, Losartan, can prevent the manifestation of blood gas and histopathologic in ALI/ARDS after CLP, and significantly improved the 7-day survival rate.4. The possible mechanisms of the prevention lung injury by Losartan are: the intervention of NF-κB and MAPK signaling pathway that reduced the ALI/ARDS systemic inflammatory responses.5. Since vascular endothelial cell injuries are observed in CLP-induced ALI/ARDS mice, Losartan's ability to decrease vascular endothelial cell apoptosis and reduces vascular permeability, can potiential become an effective treatment for ALI / ARDS. 6. RAS may be a new therapeutic strategy for ALI/ARDS.