Construction Of Lung-targeted ACE2 Plasmid Delivery System And Its Therapeutic Effect On Hypoxic Pulmonary Hypertension In Rats

Background:Hypoxic pulmonary hypertension(HPH)is a progressive malignant pulmonary vascular disease,and the three-year survival rate of patients is less than 70%.At present,there is still a lack of effective drugs for the treatment of HPH-induced pulmonary vascular remodeling and pulmonary inflammatory microenvironment.HPH activates the angiotensin-converting enzyme(ACE)/angiotensin Ⅱ(Ang Ⅱ)/AT1 R axis of the renin angiotensin system(RAS)in the lung.This subsequently causes persistent contraction of pulmonary vessels,progressive remodeling of pulmonary vessels and pulmonary inflammation.The angiotensin-converting enzyme 2(ACE2)/angiotensin-(1-7)(Ang-(1-7))/Mas(ACE2/Ang-(1-7)/Mas)axis of RAS can play the role of vasodilation,anti-pulmonary smooth muscle cell proliferation,anti-pulmonary vascular remodeling and anti-inflammatory to fight against the classical ACE/AngⅡ/AT1 R axis.Herein,the hypoxia and vascular endothelial responsive plasmid of ACE2 was constructed by gene recombination technology.The tyrosine kinase receptor Tie2 was selected as the promoter.Consequently,ACE2 was highly expressed in endothelial cells.Furthermore,ACE2 is expressed at the site where HIF-1α is increased by designing hypoxia-regulated plasmids that specifically bind to hypoxia-inducible factor(HIF-1α).Thus,ACE2 plasmid is controlled to be highly expressed only in hypoxic endothelial cells.However,ACE2 plasmid has some shortcomings such as poor stability,low transfection efficiency and lack of targeting in vivo.Therefore,it is necessary to construct a suitable ACE2 plasmid delivery system to solve above problem.Protamine(PRT)can compress the plasmid and improve its ability to resist violent forces.Protamine also has the "proton sponge effect",which can make the plasmid escape from the lysosome and improve the transfection efficiency of the plasmid.Therefore,the stability and transfection efficiency of ACE2 plasmid can be improved by using PRT.In addition,von Willebrand factor(v WF)is highly expressed in hypoxia injured vascular endothelial cell.GPIbα on the platelet membrane(PM)can bind to the A1 domain of von Willebrand factor(v WF),allowing platelets to adhere to the surface of damaged blood vessels.Wrapping PM on the surface of ACE2-containing nanoparticles can deliver ACE2 plasmid to the hypoxia-injured pulmonary artery endothelium of HPH,thereby improving the targeting of ACE2 plasmid for the treatment of HPH.Objective:In order to explore a new method for the treatment of HPH,a lung-targeted ACE2 plasmid delivery system was constructed to make ACE2 highly expressed in HPH injured pulmonary artery endothelium.The hypoxia-responsive ACE2 plasmid delivery system is able to reverse HPH by inhibiting Ang II/AT1 R axis,activating ACE2/Ang-(1-7)/Mas axis,reducing pulmonary artery pressure,inhibiting pulmonary vascular remodeling and improving pulmonary inflammatory microenvironment.Methods:1.Hypoxia and vascular endothelial responsive plasmid p UC57-HRE-Tie2-Fc-r ACE2(ACE2 plasmid)was constructed by using Tie2 as promoter and 6 × HREs as enhancer.ACE2 plasmid was identified by double digestion and sequence analysis.2.ACE2 plasmid,CS and PRT complex(ACE2-CS-PRT)was prepared by electrostatic adsorption,and the optimal mass ratio of ACE2 plasmid,CS and PRT was screened by agarose gel electrophoresis.Next,the platelet membrane(PM)was extracted,and PM was coated on the surface of ACE2-CS-PRT complex by ultrasound and extrusion method to obtain the PM-coated ACE2 plasmid delivery system(ACE2-CS-PRT@PM).Finally,the particle size,zeta potential,stability,morphology and PM coating of ACE2-CS-PRT@PM were characterized by dynamic laser particle size analyzer,coomassie brilliant blue staining,transmission electron microscopy(TEM)and laser scanning confocal microscope(LSCM).3.Primary pulmonary microvascular endothelial cells(PMVECs)and primary pulmonary artery smooth muscle cells(PASMCs)were cultured and identified by immunofluorescence staining.4.The hypoxia and vascular endothelial responsiveness of ACE2-CS-PRT@PM was investigated by Western blot.The transfection activity of ACE2-CS-PRT@PM was evaluated by immunofluorescence staining,western blot and ELISA.PMVECs transfected with ACE2-CS-PRT@PM were co-cultured with pulmonary artery smooth muscle cells(PASMCs)to investigate the effect of ACE2-CS-PRT@PM on the proliferation PASMCs by MTT assay and Ki67 immunofluorescence staining.5.Flow cytometer and LSCM were used to investigate the uptake mechanism of ACE2-CS-PRT@PM by PMVECs under normoxic or hypoxic conditions.The intracellular traffic of ACE2 plasmid in PMVECs was observed under hypoxic conditions.6.The HPH rat model was established,and Cy5 was used to label ACE2-CS-PRT@PM.After tail vein administration,the in vivo distribution of ACE2-CS-PRT@PM was investigated by in vivo imaging,immunofluorescence staining and ELISA.7.The effects of ACE2-CS-PRT@PM on pulmonary artery pressure and right ventricular hypertrophy in HPH rats were evaluated by measuring mean left carotid artery pressure(m CAP),right ventricular peak systolic pressure(RVSP)and right ventricular hypertrophy index RV/(LV+S)%.Hematoxylin-eosin(HE)staining and immunofluorescence staining were used to observe the effect of ACE2-CS-PRT@PM on pulmonary artery remodeling in HPH rats.8.Western blot was used to detect the protein expression of AT1 R,ACE2 and Mas in the lung tissue in HPH rats.ELISA was used to measure the levels of Ang II and Ang-(1-7)in the lung tissue to investigate the effect of ACE2-CS-PRT@PM on Ang II/AT1 R axis and ACE2/Ang-(1-7)/Mas axis in the lung tissue in HPH rats.9.HE staining was used to evaluate the effect of ACE2-CS-PRT@PM on the inflammatory microenvironment in the lung tissue in HPH rats,and ELISA was used to detect the contents of TNF-α and IL-6 in the lung tissues in HPH rats.10.HE staining was used to observe the effect of ACE2-CS-PRT@PM on the morphology of normal rat brain,heart,liver,spleen,lung,and kidney tissues.The effect of ACE2-CS-PRT@PM on liver and kidney function in normal rats was detected by automatic biochemical analyzer.The coagulation analyzer was used to investigate the effect of ACE2-CS-PRT@PM on the coagulation function in normal rats.Results:1.After double enzyme digestion,the target plasmid p UC57-HRE-Tie2-Fc-r ACE2 with a gene band of 852 bp+4787 bp was obtained by agarose-gel electrophoresis.The sequencing results were completely consistent with the original sequence.This indicated that the target plasmid p UC57-HRE-Tie2-Fc-r ACE2 was successfully constructed.2.ACE2 plasmid was completely adsorbed when the ratio of ACE2:CS:PRT(w/w/w)was 1:4:4.The results of SDS-PAGE gel electrophoresis,TEM and LSCM showed that PM was coated on the surface of ACE2-CS-PRT.The particle size and zeta potential of ACE2-CS-PRT@PM was 194.5±2.16 nm and-27.9±1.1 m V,respectively.ACE2-CS-PRT@PM remained stable in PBS for 6 days.The hemolysis rate of ACE2-CS-PRT@PM at the concentration of 2 mg/m L was less than 5%.3.More than 95% of the PMVECs expressed CD31 protein,which proved that more than 95% of the extracted cells were PMVECs.More than 95% of the PASMCs expressedα-SMA protein,which indicated that more than 95% of the extracted cells were PASMCs,the primary PMVECs and PASMCs could be used in subsequent experiments.4.ACE2-CS-PRT@PM exhibited hypoxia and vascular endothelial responsiveness,and ACE2 was overexpressed only in hypoxic PMVECs.ACE2 protein levels in PMVECs and cell culture medium were significantly increased after the transfection of ACE2-CS-PRT@PM under hypoxic condition.Transfection of ACE2-CS-PRT@PM into PMVECs could inhibit the proliferation of PASMCs significantly when PMVECs were co-cultured with PASMCs.5.The uptake of ACE2-CS-PRT@PM by PMVECs exhibited a time-dependent manner,and the uptake was mainly through large pinocytosis and caveolin mediated endocytosis.In addition,ACE2 plasmids were able to escape from lysosomes and enter the nucleus of PMVECs.6.ACE2-CS-PRT@PM selectively distributed in the lungs,pulmonary artery vessels and pulmonary artery endothelium in HPH rats.7.ACE2-CS-PRT@PM significantly reduced the pulmonary artery pressure and right ventricular hypertrophy index in HPH rats.ACE2-CS-PRT@PM also inhibited the proliferation of PASMCs and the pulmonary vascular remodeling,subsequently reversed HPH in rats.8.ACE2-CS-PRT@PM significantly reduced the expression levels of Ang Ⅱ and AT1 R proteins and increased the expression levels of ACE2,Ang-(1-7)and Mas proteins in the lung tissues in HPH rats,which resulted in the inhibition of Ang II/AT1 R axis and the activation of ACE2/Ang-(1-7)/Mas axis.9.ACE2-CS-PRT@PM significantly reduced pulmonary edema,neutrophil infiltration,hemorrhage and bronchial epithelial desquamation in HPH rats.ACE2-CS-PRT@PM also significantly reduced the levels of pro-inflammatory factors IL-6 and TNF-α in lung tissues in HPH rats,indicating ACE2-CS-PRT@PM could improve the pulmonary inflammatory microenvironment in HPH rats.10.After ACE2-CS-PRT@PM was administrated to normal rats,no obvious abnormal morphological changes were observed in brain,heart,liver,spleen,lung,and kidney tissue,and the levels of ALT,AST,BUN and CREA were all within the normal range.There was no significant difference in coagulation function indexes between ACE2-CS-PRT@PM treated rats and normal saline treated rats.Conclusions:ACE2-CS-PRT@PM improved the transfection efficiency of ACE2 plasmid.ACE2-CS-PRT@PM showed a pulmonary hypoxic vascular endothelial responsive characteristic and caused ACE2 to be overexpressed in hypoxic-injured pulmonary artery endothelial cells.ACE2-CS-PRT@PM could accumulate in the lung tissues in HPH rats,and subsequently targeted to hypoxia-injured pulmonary artery endothelial cells.ACE2-CS-PRT@PM could inhibit pulmonary vascular remodeling and improve pulmonary inflammatory microenvironment by inhibiting Ang II/AT1 R axis and activating ACE2/Ang-(1-7)/Mas axis in HPH rats,which subsequently reversed HPH in rats.ACE2-CS-PRT@PM has a potential in the treatment of HPH.