Mitochondrial Heterogeneity And Hypoxic Pulmonary Hypertension

Pulmonary hypertension is a serious disease which harm to human physical and mentalwell-being, its pathogenesis is second only to hypertension and coronary heart disease,ranking third in cardiovascular disease, characterized by pulmonary vasoconstriction andpulmonary vascular remodeling, and eventually lead to the right heart failure and death.Pulmonary hypertension caused by many reasons, with the development of society,hypoxia and respiratory diseases caused by pulmonary hypertension incidence rate isincreasing year by year. Hypoxia-induced pulmonary vasoconstriction is an importantevent in the development of pulmonary hypertension and pulmonary heart disease.However, hypoxic vasodilation in the circulation system. Pulmonary vasoconstriction andare closely related with intracellular calcium concentration. Vadula found that hypoxiacan cause increased production of mitochondria-derived reactive oxygen species in theliver cells1, and then the role of mitochondria in HPV is of particular concern. A largenumber of studies have confirmed that the core mechanism of HPV depends onhypoxia-inducible mitochondrial reactive oxygen species, thereby the voltage-gated K⁺channels is inhibited in pulmonary vascular smooth muscle cells, membranedepolarization, extracellular calcium influx, leading to cell contraction ultimately2. Onthe contrary, vascular relaxation when exposed to hypoxia in the circulation system, inorder to increase blood flow, oxygen supply to ensure that the organization’s performancein hypoxia. Studies have shown that lack of oxygen makes the pulmonary circulationvascular K⁺channel inhibition, vasoconstriction, K⁺currents increase. Stimulated byhypoxia3, energy generation and utilization of increase in pulmonary artery smooth muscle,while decreased rapidly in femoral artery smooth muscle, thus the pulmonaryvasoconstriction and the femoral artery diastolic. Thus, the response of pulmonary arterialvascular smooth muscle to hypoxia differs from the ones in systemic circulation.Mitochondria are the powerhouse of cell, mainly engaged in energy production andoxidative phosphorylation, energy requirements are not the same in types of organization,thus the mitochondrial is different too. The differences is not only in the number ofmitochondria4, but also in many other aspects of the form5, structure6,7and function8.This difference is also exsist in the smooth muscle cells of the pulmonary vascular andsystemic vascular, neither the function or morphology vary.The mitochondria in thepulmonary artery smooth muscle display spindle-shaped or oval normally, and the basalrespiration rate is lower than the one of femoral artery smooth muscle cells9.while themitochondria in the femoral artery smooth muscle are much more elongated.Mitochondrial-derived reactive oxygen species play a key role in HPV, mitochondrialdiversity led to increasing attention in the different response of pulmonary and systemicvascular vascular to hypoxia, however, the underline mechanism is subject to the newtechnology roadmap and research strategy.In our pre-experimental studies,we found that mitochondria isolated from the tissue orcells can maintain the function and characteristics which are same with the one in vivo,and be able to integrate into to cultured cells, isolated vessels as well as in vivopulmonary circulation vascular smooth muscle cells. And then, this study will explore thedifferences of mitochondrial between pulmonary and circulatory systerm, and thesubsequent impaction on hypoxic pulmonary vasoconstriction and pulmonaryhypertension, for discovery of the new approach which is expected to provide for thetreatment of pulmonary hypertension mechanism and pulmonary hypertension. Part â… Heterogeneity of mitochondria onacute hypoxia response of vascular smooth muscle cellsObjectExplore the the dynamic characteristics of exogenous mitochondria into cells, andobserve the affect of homologous and heterologous mitochondria transplantion on thehypoxia response of PASMC and FASMC.MethodMarked smooth muscle cell mitochondria with DsRed-Mito and GFP-Mito, thefluorescently labeled mitochondria are extracted from the cells useing the gradientcentrifugation and then incubated with cells, observing the distribution of exogenousmitochondria in cells by confocal fluorescence microscope, and analyzing the dynamiccharacteristics of the endogenous mitochondrial,Inhibiting the endogenous mitochondrialDNA synthesis by EB,detecting of intracellular calcium ion by Fura-2/AM probe and cellmembrane potential by JC-1.Result1. There was no fluorescent in pulmonary artery smooth muscle cells of the controlgroup by live cell confocal imaging, while the pulmonary artery smooth muscle cellsincubated DsRed-maked exogenous mitochondria, showed red fluorescence intracellular,indicating that the exogenous mitochondria were successfully transplanted into the cells.Counted by two-dimensional fluorescence analysis, the exogenous mitochondria into thecells depended on the time and concentration of incubation. It was found that exogenouscells are mainly distributed in the area close to the cell membrane by the combination ofthree-dimensional reconstruction.2. Hypoxia increased the concentration of intracellular calcium in PASMC of thecontrol group, similar to the changes in pulmonary artery smooth muscle cells pretreatedwith Py and Ur. On the contrary, there was no increasing in calcium concentration in thePASMC pre-processing with EB when exposed to hypoxic,but after transplanting PASMC-mito,[Ca²⁺]iincreased in PASMC even pretreated with EB exposed to hypoxia.However, when transplanted with the FASMC-mito,[Ca²⁺]iin the PASMC pre-processingwith EB was reduced exposed to hypoxia. We replaced PASMC with FASMC, and gotthe same results. In the control group,[Ca²⁺]iin the FASMC reduced when exposed tohypoxia, similar to the ones pretreated with Py and Ur.While EB made the changedisappear. But after transplanting PASMC-mito, the [Ca²⁺]iof FASMC displayed similarto the PASMC’s.3. Hypoxia-induced membrane depolarization in PASMC of control group and of theone transplanted with PASMC-mito.While membrane hyperpolarization whentransplanted with FASMC-mito.We replaced PASMC with FASMC, and got the sameresults. Hypoxia-induced membrane hyperpolarization in FASMCs of control group andof the one transplanted with FASMC-mito, while membrane depolarization whentransplanted with PASMC-mito.Conclusion1. The number of exogenous mitochondria which transplanted into cells was dependedon time and concentration, and the spatial distribution is mainly located close tomembrane of the cells.2. FASMC-mito which transplanted into cells can reduced calcium concentration in thepulmonary artery smooth muscle cells to hypoxia, PASMC-mito which transplanted intocells increased concentration of calcium ions in the femoral artery smooth muscle cells tohypoxia.3. FASMC-mito which transplanted into cells can reduce membrane potential ofpulmonary artery smooth muscle cell to hypoxia, PASMC-mito which transplanted intocells increased membrane potential femoral artery smooth muscle cell to hypoxia. Part â…¡Heterogeneity of mitochondria on hypoxiapulmonary vasoconstriction and pulmonary hypertensionObjectExplore the affect of homologous and heterologous mitochondria transplantion onacute hypoxia-induced vascular responses in vitro and attenuation of HPV, HPR andHPH.MethodMitochondria were isolated by gradient centrifugation and incubated with vascular ringin vitro, and then detecting the tension and function of vascular ring. Exogenousmitochondria were transplanted into rats in vivo by tail vein injection.Establish the modelof chronic hypoxic pulmonary hypertension model, and detect pulmonary arterialpressure and systemic vascular pressure by four-derivative instruments, and observe theremodeling of pulmonary vascular by HE staining of paraffin.Result1. PA which removal of the endothelium contraction when exposed to hypoxia, the PAwhich transplanted with FASMC-mito contraction lower than the control group. Wereplaced PA with FA, and found that FA vasodilation due to hypoxia in control group andthe PA of being transplanted with FASM-mito. But what interesting was that after beingtransplanted with PASMC-mito, the FA contraction exposed to hypoxia.In some experiments, we kept the intact endothelium of the vascular ring, and then thevascular ring exposed to hypoxia for60min. The PA of the control group and the onebeen transplanted with PASMC-mito showed a typical biphasic contractile response,compared to the control group, the PA transplanted with FASMC-mito showed muchlower biphasic contractile response. We replaced PA with FA, and found that hypoxiainduced FA of the control group and the one transplanted with FASMC-mito steady andconstant vasodilation, however a biphasic contractile response in PA of beingtransplanted with PASMC-mito, which was similar to the PA of control group.2. Pulmonary artery pressure of the control group and the one transplanted with PASMC-mito rats rose higher, pulmonary artery pressure of the group transplanted withFASMC-mito rose too, but at a lower state compared with the control group. Thepulmonary resistance showed the same results. The pressure of systemic artery was notaffected by exogenous mitochondria at all.3. The Ppa, PVR and Fulton’s index were increase when transplanted with PBS andexogenous mitochondria, but they increased at a lower state contract with the group ofPBS and PASMC-mito. There was no change of the control group, and the pressure ofsystemic artery was not affected by exogenous mitochondria either. The vascular wallthickness showed the same results. Moreover, another model was been established, wetransplanted exogenous mitochondria in the process of chronic hypoxia, and came to thesame conclusion.Conclusion1. Exogenous mitochondria can be successfully transplanted into vascular ring in vitro,and FASMC-mito made a lower PA contraction to hypoxia, PASMC-mito made FAcontraction instead of relaxation to hypoxia.2. Exogenous mitochondria can be successfully transferred into the pulmonaryvascular smooth muscle cells by tail vein injection, and FASMC-mito can reducepulmonary artery pressure, PASMC-mito had no effect on pulmonary artery pressure,exogenous mitochondria has no effect on the systemic circulation pressure.3. FASMC-Mito had improvement in reducing pulmonary hypertension, pulmonaryvascular resistance and vascular remodeling induced by chronic hypoxic.