The Role Of Lipocalin 2 In Kawasaki Disease-related Myocardial Injury And Pulmonary Vascular Remodeling

Kawasaki Disease(KD)is an acute fever and rash disease with systemic vascular inflammation as the main pathological change.Kawasaki disease mainly occurs in children,and the incidence rate has increased year by year.It has replaced rheumatic fever as one of the most important causes of childhood heart disease.Although Kawasaki disease has received increasing attention,the etiology and pathogenesis of Kawasaki disease have not yet been elucidated.Exploration of the mechanism of Kawasaki disease induced organ damage,and to find therapeutic strategies is still an important issue to be solved.We gradually noticed in clinical observation that in addition to the clinical manifestations of acute fever and major criteria for rash,important organ damage and decreased function caused by Kawasaki disease should not be overlooked.Cardiopulmonary function is vital to life.Kawasaki disease can affect lung function and heart function.However,the biological mechanism of heart and lung injury caused by Kawasaki disease is not very clear.Previous studies have shown that abnormal inflammatory responses play an important role in the pathogenesis of Kawasaki disease.Therefore,the role and mechanism of inflammatory factors in Kawasaki disease should be the focus of attention.Lipocalin-2(Lcn 2)is a cytokine mainly secreted by adipose tissue and secreted by neutrophils,macrophages,endothelial cells,etc.It can be used as a trigger factor for inflammation.It has recently been found that in some malignancies,changes in Lcn 2 levels have an important effect on cell survival and correlate with the extent of heart and lung organ damage.More importantly,Lcn 2 has different biological effects that promote or inhibit apoptosis in different types of cells.At present,the role and specific mechanism of Lcn 2 in Kawasaki disease-associated cardiopulmonary function impairment is unknown.In order to further explore the biological mechanism of Kawasaki disease-associated cardiopulmonary impairment,KD rat model was used in this study to analyze whether Lcn 2 is involved in the pathophysiological processes of myocardial damage and pulmonary vascular remodeling in Kawasaki disease and to elucidate the underlying signal mechanism from in vivo and in vitro experiments.This research includes of two parts.Part I Kawasaki disease induced pulmonary vascular remodelingRecently,clinical cases have been reported that Kawasaki disease patients have pulmonary hypertension and have clinical manifestations such as pulmonary artery dilatation and increased right ventricular pressure.Some patients with Kawasaki disease who died of sudden coronary artery disease were found to be involved extensively in autopsy.Pulmonary arteries also have pathological structural changes such as stenosis and hypertrophy,suggesting that Kawasaki disease may involve the pulmonary artery and cause changes in the structure and function of the pulmonary artery.Objective: To investigate the role and potential mechanism of Lcn 2 in Kawasaki disease-induced changes in pulmonary artery structure and function.Methods: Kawasaki disease rat model was established by intraperitoneal injection of Lactobacillus casei cell wall extract(LCWE);pathological changes of lung tissue were detected by histomorphology;circulating levels and Lcn 2 content in lung homogenate were measured by ELISA;hemodynamics The mean left common carotid artery pressure(m CAP)and peak right ventricular systolic pressure(RVSP)were measured.In vitro,Lcn 2 levels were measured by primary cultured rat pulmonary arterial smooth muscle cells(PASMCs)and neutrophils(PMNs)with ELISA methods,and further analyzed by Western blotting and RT-PCR analysis of related protein and m RNA levels.Results:1.Kawasaki disease rats have elevated right ventricular pressure and pulmonary arterioles remodeling.After SD rats(body weight 80-100g)were given intraperitoneal injection of LCWE 0.5 ml(1 mg/ml)for 14 days,edema and localized spotting and flaky hemorrhage occurred in the lung tissue.A large number of neutrophils were mainly associated with a small amount of lymphocytes and monocytes infiltration.Although there was no significant change in m CAP in all groups,RVSP increased and right ventricular hypertrophy index [RV/(LV+S)]% increased significantly.After 28 days,focal atelectasis and emphysema were found,and pulmonary arterial endothelial cells were swollen.Microscopic observation of pulmonary arteriolar thickening,luminal stenosis,suggested pulmonary vascular smooth muscle hyperplasia.The results showed that animals with Kawasaki disease have elevated pulmonary vascular resistance, structural changes of the right heart,and pulmonary vascular inflammatory cell infiltration,smooth muscle hyperplasia,vascular stenosis and other pulmonary vascular remodeling performance.2.Kawasaki disease rat pulmonary vascular tissue to promote proliferative endoplasmic reticulum stress(ER stress).Endoplasmic reticulum stress is closely related to pulmonary hypertension and smooth muscle proliferation.Our experiments showed that the ER stress-associated proliferative protein ATF6 cleavage(60 k D)in the pulmonary vascular tissue of Kawasaki disease increased compared with the control group,significantly increased ATF6 nuclear translocation,and then increased the m RNA and protein levels of GRP78 and NOGO.On the other hand,ER stress-related pro-apoptotic protein CHOP expression and nuclear translocation did not change significantly,and the expression of pro-apoptotic protein caspase 12 did not increase.3.Lcn 2 levels were significantly increased in plasma and lung tissues of Kawasaki disease rats.Compared with the control group,the Lcn 2 content in plasma and lung homogenates of Kawasaki disease rats peaked at 14 days after LCWE injection,and maintained at a high level.Through in vitro experiments,it was found that the increased Lcn 2 after LCWE stimulation may originate from PMNs in the circulation or lung tissue.4.Lcn 2 promotes proliferation of pulmonary artery smooth muscle cells.The above results are clear: Lung vascular tissue hyperplasia of Kawasaki disease is accompanied by elevation of Lcn 2.We further examined whether Lcn 2 causes pulmonary artery smooth muscle cell proliferation.Cytological experiments found that Lcn 2 treatment of PASMCs can directly lead to ATF6 cleavage and nuclear translocation,significant increase of GRP78 and NOGO;otherwise,pro-apoptotic CHOP and Caspase 12 levels did not increase.The detection of cell proliferation rate showed that Lcn 2 treatment significantly promoted the proliferation of PASMCs.The above results indicate that Lcn 2 plays an important role in the proliferation of pulmonary artery smooth muscle cells.5.Inhibition of proliferative endoplasmic reticulum stress improves pulmonary vascular remodeling in Kawasaki disease rats.In order to find specific preventive measures,we used PBA,a specific inhibitor of endoplasmic reticulum stress,in rats with Kawasaki disease.Cytological experiments revealed that 1 m M PBA treatment significantly inhibited the proliferation of PASMCs induced by Lcn 2.The whole animal experiment confirmed that the treatment of PBA(500 mg/kg)for 14 days can effectively reduce RVSP and right heart hypertrophy,and effectively inhibit the structural changes of the small pulmonary blood vessels.Summary: Kawasaki disease can induce pulmonary vascular remodeling and pulmonary artery smooth muscle cell proliferation accompanied by a significant increase in Lcn 2.Lcn 2-induced proliferative endoplasmic reticulum stress plays an important role in the proliferation of pulmonary artery smooth muscle cells.Targeted inhibition of ER stress is an effective measure to prevent and treat pulmonary vascular injury associated with Kawasaki disease.Part II Kawasaki disease induced myocardial injuryIn addition to vascular inflammatory lesions,does Kawasaki disease cause direct myocardial damage? It is still not clear.Studies have shown that Lcn 2,as an important inflammatory molecule,is closely related to cardiomyocyte and vascular endothelial cell damage and apoptosis,and participates in the development of cardiovascular diseases.In the first part of our research,we found that the plasma Lcn 2 level was significantly increased in the Kawasaki disease state,which prompted us to further explore Kawasaki disease-induced myocardial injury,and to analyze the role of Lcn 2 in this process.Objective: To explore the role and potential mechanism of Lcn 2 in Kawasaki disease induced injury.Methods: Kawasaki disease rat model was established(the method was the same as the first part).ELISA was used to detect the plasma Lcn 2 levels in plasma and cardiac tissue homogenates of Kawasaki disease rats and patients with Kawasaki disease;cardiac ultrasound was used to detect systolic and diastolic function in rats.Masson staining to evaluate the degree of myocardial fibrosis;the expression of IL-6,CD3,CD45,and Collagen I m RNA in myocardial tissue was detected by real-time fluorescence quantitative PCR;Caspase 3,CHOP,ATF6,p-PERK、p-el F2α and Caspase 12 were detected by Western blotting.Results:1.Kawasaki disease induces systolic diastolic dysfunction and inflammatory reactions.The use of echocardiography confirmed that the left ventricular end-diastolic dimension(LVIDd)and left ventricular ejection fraction(LVEF)were decreased in Kawasaki disease rats compared with the control group(P<0.05).This result suggests that the heart is dilated and there is a decrease in pumping capacity during Kawasaki disease.Masson staining showed a certain degree of myocardial fibrosis and collagen deposition in Kawasaki disease rats.The m RNA expression levels of inflammation-related molecules such as IL-6,CD3,and CD45 in the myocardium were significantly elevated(P<0.05).2.The cardiac tissue and circulation levels of Lcn 2 are elevated in Kawasaki disease state.We further found that Lcn 2 levels in rat plasma gradually increased during Kawasaki disease,and there was a statistically significant difference at 7 days of modeling.Peak values were reached by 14 days and remained high for more than 28 days.After 28 days of modeling,myocardial tissue homogenate detection also confirmed that Lcn 2 levels in myocardial tissue of Kawasaki disease rats were significantly higher than those in the control group(P<0.05).More importantly,clinical plasma samples also confirmed that circulating levels of Lcn 2 in patients with Kawasaki disease were higher than those in normal controls(n=6,P<0.05).3.Lcn 2 promotes collagen formation.To determine whether Lcn 2 is involved in myocardial collagen fibril deposition.We used cultured cardiac fibroblasts and treated Lcn 2(10 ng/ml)for 48 hours.The results showed that the m RNA and protein expression levels of Collagen I were significantly increased(P < 0.05).It was also found that Lcn 2 treatment significantly up-regulated the phosphorylation of NF-кB and IκB in cardiac fibroblasts.Treatment with IκB inhibitor BAY 11-7082(2.5 μmol/L)effectively inhibited Lcn 2 up-regulation of Collagen I protein.The above results indicate that Lcn 2 may promote the formation of collagen by activating the NF-кB signal.4.Lcn 2 induces apoptotic endoplasmic reticulum stress(ER stress)in cardiomyocytes.In addition to fibroblasts,we further explored the direct effect of Lcn 2 on cardiomyocytes.We used primary cultured cardiomyocytes and treated Lcn 2(500 ng/ml)for 48 hours.It was found that Lcn 2 induces apoptosis in cardiomyocytes.The ER stress markers ATF6,CHOP,p-PERK,and p-el F2α all increased,resulting in a significant increase in the apoptotic signals Caspase 12 and cleave-Caspase 3 levels.Quantitative detection of Caspase 3 activity in cardiomyocytes was significantly increased(P<0.05).The above results show that unlike fibroblasts,the direct effect of Lcn 2 on cardiomyocytes is to induce endoplasmic reticulum stress-induced myocardial apoptosis.5.Inhibition of endoplasmic reticulum stress relieves cardiomyocyte apoptosis and improves cardiac function in Kawasaki disease rats.After clarifying the role of Lcn2-induced pro-apoptotic endoplasmic reticulum stress in cardiomyocytes,we used endoplasmic reticulum stress inhibitor 4-phenylbutyrate(4-PBA)(500 mg/kg,14 days).The results showed that PBA treatment effectively inhibited the degree of myocardial endoplasmic reticulum stress in Kawasaki disease,reduced the levels of ATF6,CHOP,p-PERK,and p-el F2α and decreased the levels of Caspase 12 and cleave-Caspase 3(P<0.05).TUNEL results confirmed that inhibition of endoplasmic reticulum stress can effectively reduce cardiomyocyte apoptosis in Kawasaki disease rats and ultimately improve diastolic function in Kawasaki disease rats.Summary: Our study confirmed that Kawasaki disease can induce increased collagen synthesis in cardiac fibroblasts and inflammatory responses accompanied by a significant increase in Lcn 2.We found that the direct effect of Lcn 2 on cardiomyocytes was pro-apoptotic endoplasmic reticulum stress.Inhibiting myocardial endoplasmic reticulum stress under Kawasaki disease can inhibit myocardial apoptosis and improve cardiac function.Conclusion: This series of studies clearly revealed that Lcn 2 plays an important role in Kawasaki disease-related cardiopulmonary impairment.For different types of cells(proliferative pulmonary vascular smooth muscle cells or non-proliferating cardiomyocytes),Lcn 2 has different biological effects that promote proliferation or promote apoptosis.Lcn 2 triggered endoplasmic reticulum stress is the main cellular mechanism of the above biological phenomena.These mechanisms together results exist in the coexistence of myocardial injury and pulmonary arterial remodeling in the Kawasaki disease state.Since there are no mature and feasible Lcn 2 interventions(eg,neutralizing antibodies),inhibition of endoplasmic reticulum stress in Kawasaki disease may be a feasible approach to prevent Kawasaki disease-related cardiopulmonary impairment.