The Effect Of A Novel Nitric Oxide Donor (CDDO-NO) On Monocrotaline–Induced Pulmonary Arterial Hypertension In Rats

Background:Pulmonary arterial hypertension(PAH)is a rapidly progressive and severe disease with elevated pulmonary artery pressure and increased pulmonary vascular resistance,leading to right heart failure and death.At present,at least three major pathways,including nitric oxide(NO),endothelin and prostacyclin are well-known contributors for the development of PAH.The strategies targeting these pathways have well been established and adopted in clinical practice,such as inhaled NO,bosentan,eloprostol etc.However,mortality in patients with PAH remains dismal.Pulmonary vascular remolding is a pathological feature of PAH,which shares close relationship with prognosis of patients with PAH.Existing strategies do not effectively inhibit pulmonary vascular remolding.Therefore,it is essential to explore a new strategy to curatively treatment of PAH.NO is an endothelium derived factor,endogenously produced from L-arginine catalytically by nitric oxide synthase(NOS).NO is able to activate soluble guanylyl cyclase(s GC)to convert guanosine 5’-triphosphate(GTP)into cyclic guanosine monophosphate(c GMP),which in turn blocks calcium influx,resulting in vascular dilation.Inhaled NO(i NO)is found to be an efficient approach for the treatment of patients with severe PAH.However,the therapeutic window of i NO is narrow(5-20 ppm),and excess i NO could generate methemoglobinemia and toxic nitrogen dioxide(NO2).Interestingly,it has been found that inhaled NO-donor compounds can be controlled to release NO in a slow and sustained manner,thereby reducing pulmonary vascular pressure with few side effects,providing a potentially promising approach for PAH treatment.CDDO-Me(also as bardoxolone methyl),a semi-synthetic derivative of oleanolic acid(OA),has been found to protect endothelial cell from injury and inhibit blood vessel hyperplasia by activating nuclear factor-erythroid 2 related factor 2(Nrf2)and inhibiting nuclear factor kappa(Nf-k B).Moreover,CDDO-Me directly improves cellular mitochondrial function and energy production by reducing oxidative stress and inflammation,which are beneficial for reversing the mitochondrial dysfunction in PAH.A phase II clinical trial has demonstrated that CDDO-Me was able to improve exercise capacity in PAH patients.Thus,CDDO-Me is a promising agent for the treatment of PAH.In view of the pathological complexity of PAH,it is believed that combination therapy simultaneously targeting multiple pathologic targets could generate improved efficacy relative to monotherapy.In this study,we investigated the biological effect and pharmacokinetics of a novel hybrid(CDDO-NO)from CDDO-Me and NO donor isosorbide 5’-mononitrate(ISMN)and dual effects of CDDO-NO on pulmonary vasodilation and vascular remodeling on PAH rats.Part I Biological Effect of CDDO-NO on Pulmonary Vascular Cells and its PharmacokineticsObjective: To investigate the biological effect of CDDO-NO on human PAECs or PASMCs,and the pharmacokinetics characteristics of CDDO-NO in rats.Methods: 1.The effect of CDDO-NO or CDDO-Me on the growth of PAECs or PASMCs was assessed by CCK-8 assay.Ed U assay was applied to detect cells proliferations.Transell migration assay was used to evaluate the migrations of PAECs or PASMCs stimulated by CDDO-NO.The translocation of Nrf2 and the expression of VE-cadherin in PAECs were determined using Immunofluorescence analysis.DCFH-DA probe was used to detect ROS production in PAECs stimualted by CDDO-NO or CDDO-Me.2.For pharmacokinetics experiment,the lung tissues and plasma were collected from rats,and then analyzed by LC-MS-MS to determine the concentrations of CDDO-Me and CDDO-NO in lungs and plasma after trachea injections of two agents.Results: 1.In vitro study,CDDO-NO was less toxic in PAECs and PASMCs compared to CDDO-Me.CDDO-NO selectivly inhibited proliferation and migration of PASMCs.In comparison to CDDO-Me,CDDO-NO had less effect on VE-cadherin expression of PAECs.CDDO-NO could activate Nrf2,and then reduce ROS production in PAECs under hypoxic conditon.2.In pharmacokinetics study,the concentrations of CDDO-NO in lungs increased gradually within initial 60 min and reached the peak of 243 ng/m L,but decreased rapidly and almost under detection limit at 180 min.After injection,CDDO-NO was gradually decomposed to CDDO-Me in lungs,and the highest concentration appeared at 90 min was 78.5 ng/m L.Even at 12 h,15.9 ng/m L of CDDO-Me can be detected.CDDO-Me reached the peak concentration at 60 min,and then decreased rapidly.However,CDDO-Me were undetected at 360 min.Conclusions: 1.CDDO-NO is less toxic to pulmonary vascular cells compared to CDDO-Me.Moreover,CDDO-NO has more cytotoxicity to PASMCs and protects PAECs from oxidative stress injury.2.CDDO-NO administrated to airway slowly releases CDDO-NO and NO gas,which is a novel NO donor.Part II The Effect of CDDO-NO inhalation on MCT-Induced PAH in ratsObjective: To investigate the effect of CDDO-NO on haemodynamics and pulmonary vascular remolding in PAH rats.Methods: 1.A rat model of PAH was established by subcutaneously injected with a single dose of monocrotaline(MCT).CDDO-NO,CDDO-Me,ISMN or CDDO-Me and ISMN was inhaled daily at the beginning of MCT administration by nose-only exposure system.At the end of 28 days,RVSP and m PAP of rats was detected by a polygraph system.Meanwhile,lung and heart tissues were isolated for determing the ratio of RV/LV+S.2.Modified Griess reaction method was used to measure the plasma nitrate and nitrite.ET-1 in plasma was assayed by ELISA kits.3.Histological stainings(Hematoxylin-eosin,masson and picro-sirius red)and immunohistochemical assay were used to reveal the effect of CDDO-NO on pulmonary vascular remodeling and cardiomyocyte hypertrophy in PAH rats.Results: 1.After treatment for 28 days,the m PAP,RVSP and RV/LV+S were significantly improved in PAH rats receiving CDDO-NO treatment.However,inhalation of CDDO-Me,ISMN or CDDO-Me and ISMN did not alter haemodynamics of PAH rats.2.CDDO-NO reduced ET-1 production and increased NO production in PAH rats.3.CDDO-NO reduced PAMT,inhibited pulmonary vascular muscularization and decreased Collagen-I and TGF-β production in PAH rats.Additionally,CDDO-NO effectively reduced PCNA postive cells and CD68 positive cells infiltrations and inhibited oxidative stress in perivasular tissues of PAH rats.4.CDDO-NO decreased cardiomyocyte hypertrophy and fibrosis in PAH rats.Conclusions: 1.CDDO-NO significantly reduces m PAP and RVSP in PAH rats.2.CDDO-NO effectively improves pulmonary vascular remolding in PAH rats.3.CDDO-NO ameliorates myocardial remolding in PAH rats.4.CDDO-NO may provide a new strategy for the treatement of PAH.