Role of ROS/Kv/HIF Axis in the Development of Hypoxia-Induced Pulmonary Hypertension

Hypoxic pulmonary hypertension (HPH) is a common complication in patients with chronic obstructive pulmonary disease (COPD), sleep-disordered breathing, or dwellers in high altitude. The exact mechanisms underlying the development of HPH still remain unclear. Reactive oxygen species (ROS),hypoxia inducible factors (HIF), and potassium channels (KV) are believed as the main factors during the development of HPH. We propose that the “ROS/Kv/HIF axis” may play an important initiating role in the development of HPH. Being formed under a hypoxic condition, ROS affects the expression and function of HIFs or KV, and consequently triggers multiple downstream signaling pathways and genes expression that participate in promoting pulmonary vasoconstriction and arterial remodeling. Thus, further study determining the initiating role of “ROS/Kv/HIF axis” in the development of HPH could provide theoretic evidences to better understand the underlying mechanisms of HPH, and help identify new potential targets in the treatment of HPH.

Effects of Endoplasmic Reticulum Stress on Pulmonary Hypertension in Rat Induced by Chronic Hypoxia and Hypercapnia

Objective: To observe the pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia, and to explore the role of endoplasmic reticulum stress in pulmonary hypertension. Methods: 1) 40 SD rats were randomly divided into four groups: normoxic control group (N), hypoxia hypercapnia group (HH), endoplasmic reticulum stress (ERS) inhibitor 4-phenyl butyric acid group (4-PBA), ERS pathway agonist tunicamycin group (TM). 2) The mean pulmonary arterial pressure (mPAP) and the right ventricular hypertrophy index (RV/(LV + S)) were measured in each group. 3) Identification of pulmonary artery smooth muscle cells (PASMCs) in each group by immunofluorescence α-SMA. 4) Morphological changes of lung tissue and pulmonary artery were observed by electron microscope. 5) The apoptotic index of PASMCs in each group was detected by TUNEL. 6) Reverse transcription polymerase chain reaction (RT-PCR) and Western Blot (WB) were used to detect the expression of ERS related protein and mRNA (GRP78, CHOP, JNK, Caspase-12) in each group. Results: 1) Compared with the N group, the mPAP, RV/(LV + S) and vascular wall area (WA)/total area (TA) value of HH group, 4-PBA group and TM group were increased (P < 0.01), and the vascular lumen area (LA)/TA values, PASMCs apoptosis index were significantly decreased. GRP78, CHOP, JNK, Caspase-12 expression were increased, and the differences were statistically significant. 2) Compared with the HH group, the mPAP, RV/(LV + S) and WA/TA of 4-PBA group were decreased (P < 0.01);the LA/TA value and PASMCs apoptosis index were increased (P < 0.05);and the mRNA and protein expression of CHOP, JNK, Caspase-12 and GRP78 had a significant decrease (P < 0.05). 3) Compared with the HH, the mPAP, RV/(LV + S) and WA/TA of TM group were increased (P P P < 0.01);and? PASMCs apoptotic index was increased (P < 0.01). Meanwhile, the mRNA expression of Caspase-12, CHOP, JNK and GRP78 was increased to varying degrees (P < 0.05), and the protein expression of Caspase-12, CHOP and JNK was also increased significantly (P Conclusion: Hypoxia and hypercapnia induced pulmonary vascular remodeling may be related to the proliferation of PASMCs, and ERS related factors (JNK, Caspase12 and CHOP) are involved in the regulation of hypoxic hypercapnia.

Inhibition of Expression of Hypoxia-inducible Factor-1α mRNA by Nitric Oxide in Hypoxic Pulmonary Hypertension Rats

In order to study the effect of nitric oxide (NO) on the expression of hypoxia inducible factor 1 alpha (HIF 1α) mRNA in hypoxic pulmonary hypertension (HPH) rats, 30 healthy male Wistar rats were randomly divided into normoxic control group, chronic hypoxic group and hypoxia plus L argine (L Arg) group. The animal model of HPH was developed. The mean pulmonary arterial pressure (mPAP) was measured by inserting a microcatheter into the pulmonary artery. The HIF 1α mRNA expression levels were detected by in situ hybridization (ISH) and semiquantitative RT PCR. It was found that after 14 days hypoxia, the mPAP in normoxic control group (17.6±2 7 mmHg,1 mmHg=0 133 kPa) was significantly lower than that in chronic hypoxic group(35.8±6.1 mmHg, t =0.2918, P <0.05) and mPAP in chronic hypoxic group was higher than that in hypoxia plus L argine group(24.4±3.8 mmHg, t =0.2563, P <0.05). ISH showed that the expression of HIF 1α mRNA in the intraacinar pulmonary arteriolae (IAPA) in normoxic control group (0.1076±0.0205) was markedly weaker than that in chronic hypoxic group (0.3317±0.0683, t =3.125, P <0.05) and that in chronic hypoxic group was stronger than that in hypoxia plus L argine group (0.1928±0.0381, t =2.844, P <0.05). RT PCR showed that the content of HIF 1α mRNA in chronic hypoxic group (2.5395±0.6449) was 2.16 times and 1.75 times higher than that in normoxic control group (1.1781±0.3628) and hypoxia plus L argine group (1.4511±0.3981), respectively. It is concluded that NO can reduce the mPAP by the inhibition of the expression of HIF 1α mRNA, which may be one of the mechanisms through which NO affects the pathogenesis of HPH.

Constant daily hypoxia leads to different degrees of pulmonary hypertension in two different rat strains

Pulmonary diseases associated with diurnal hypoxemia are known to be associated with pulmonary hypertension in some patients. In this study we examined the effects of daily hypoxia (10% oxygen;8h/day for 14 days) on two strains of rats to simulate sleep related hypoxia in pulmonary diseases expecting to find differences in vascular responses, the develop-ment of right ventricular hypertrophy and pulmonary hypertension according to genetic background. In response to daily hypoxia, Sprague Dawley rats developed right ventricular hypertrophy while Brown Norway rats did not. Both strains developed pulmonary hypertension (elevated right ventricular pressure) although the increase was significantly greater in the Sprague Dawley strain. Pulmonary artery (first branch) vasoconstrictive responses to potassium chloride were increased equally in both strains and the subsequent vasodilation with acetylcholine were reduced equally with daily hypoxia in both strains. Taken together, these findings suggest that the genetic makeup of the rats contributed significantly to the development of right ventricular hypertrophy and the degree of pulmonary hypertension. Moreover, this response is not secondary to differences in the intralobar pulmonary vascular reactivity. Genetic background could explain why certain patients do worse with hypoxia inducing pulmonary vascular diseases.

The Role of Endogenous Carbon Monoxide in the Hypoxic Vascular Remodeling of Rat Model of Hypoxic Pulmonary Hypertension

We investigated the expression of heme oxygenase 1 (HO 1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO 1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10 % O 2). Reverse transcriptase polymerase chain reaction (RT PCR) was performed to determine the level of HO 1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO 1 mRNA in normal rat lung tissue, but the level of HO 1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats ( P <0.01). The quantity of COHb was 2-3 times those of control group ( P <0.01 or P <0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO 1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO 1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.

CircPMS1 promotes proliferation of pulmonary artery smooth muscle cells,pulmonary microvascular endothelial cells,and pericytes under hypoxia

Background:Circular RNAs(circRNAs)have been recognized as significant regulators of pulmonary hypertension(PH);however,the differential expression and function of circRNAs in different vascular cells under hypoxia remain unknown.Here,we identified co-differentially expressed circRNAs and determined their putative roles in the proliferation of pulmonary artery smooth muscle cells(PASMCs),pulmonary microvascular endothelial cells(PMECs),and pericytes(PCs)under hypoxia.Methods:Whole transcriptome sequencing was performed to analyze the differential expression of circRNAs in three different vascular cell types.Bioinformatic analysis was used to predict their putative biological function.Quantitative real-time polymerase chain reaction,Cell Counting Kit-8,and EdU Cell Proliferation assays were carried out to determine the role of circular postmeiotic segregation 1(circPMS1)as well as its potential sponge mechanism in PASMCs,PMECs,and PCs.Results:PASMCs,PMECs,and PCs exhibited 16,99,and 31 differentially expressed circRNAs under hypoxia,respectively.CircPMS1 was upregulated in PASMCs,PMECs,and PCs under hypoxia and enhanced the proliferation of vascular cells.CircPMS1may upregulate DEP domain containing 1(DEPDC1)and RNA polymerase II subunit D expression by targeting microRNA-432-5p(miR-432-5p)in PASMCs,upregulate MAX interactor 1(MXI1)expression by targeting miR-433-3p in PMECs,and upregulate zinc finger AN1-type containing 5(ZFAND5)expression by targeting miR-3613-5p in PCs.Conclusions:Our results suggest that circPMS1 promotes cell proliferation through the miR-432-5p/DEPDC1 or miR-432-5p/POL2D axis in PASMCs,through the miR-433-3p/MXI1 axis in PMECs,and through the miR-3613-5p/ZFAND5 axis in PCs,which provides putative targets for the early diagnosis and treatment of PH.

Experimental animal models of pulmonary hypertension:Development and challenges

Pulmonary hypertension(PH) is clinically divided into 5 major types, characterized by elevation in pulmonary arterial pressure(PAP) and pulmonary vascular resistance(PVR), finally leading to right heart failure and death. The pathogenesis of this arteriopathy remains unclear, leaving it impossible to target pulmonary vascular remodeling and reverse the deterioration of right ventricular(RV) function. Different animal models have been designed to reflect the complex mechanistic origins and pathology of PH, roughly divided into 4 categories according to the modeling methods: noninvasive models in vivo, invasive models in vivo, gene editing models, and multi-means joint modeling. Though each model shares some molecular and pathological changes with different classes of human PH, in most cases the molecular etiology of human PH is poorly known. The appropriate use of classic and novel PH animal models is essential for the hunt of molecular targets to reverse severe phenotypes.

ROLE OF COLLAGEN METABOLISM CHANGES IN THE PATHOGENESIS OF PULMONARY HYPERTENSION IN RATS AND ITS REVERSIBILITY

Pulmonary arterial pressure (PAP) was increased obviously in rats after 3 days of normobaric hypoxic exposure and reached a maximum at 14 days of hypoxia. It remained at the same level during prolonged hypoxic exposure of up to 21 days. Right ventricular weight (RV/LV +S) and hydroxyproline (HP) content in the pulmonary artery began to increase at day 7. HP content had increased much faster than the relative rate of increase of PAP after 14 days, but HP content in the thoracic aorta showed no change. The relative proportion of type Ⅰto Ⅲ collagen increased singnificantly, and compliance of the pulmonary vessels obviously decreased. All parameters returned to the normal range within 14 days after recovery from hypoxia, except for HP content as expressed per vessel. 764-3 treatment obviously attenuated most of the changes caused by hypoxia, though it had no effect on compliance of the pulmonary vessels. It is suggested that collagen, especially type Ⅰcollagen, accumulation may play an important role in maintaining pulmonary hypertension. 764-3 has certain protective effects and may be useful in the treatment of early chronic obstructive pulmonary disease.

Novel Role of Calcium-Sensitive Receptors in Chronic Hypoxia-Induced Proliferation of Pulmonary Vein Smooth Muscle Cells

Objective:Vascular remodeling due to chronic hypoxia(CH)occurs not only in the pulmonary arteries but also in the pulmonary veins.Pulmonary vascular remodeling arises from the proliferation of pulmonary vascular myocytes.However,the mechanism by which CH induces the proliferation of pulmonary vein smooth muscle cells(PVSMCs)is unknown.This study aimed to investigate the mechanism by which CH affects the proliferation of PVSMCs.Methods:PVSMCs were isolated from rat distal pulmonary veins and exposed to CH(4%O2,60h),and the expression of the calcium-sensitive receptor(CaSR)was detected by Western blotting and immunofluorescence.MTT assay was used to detect the proliferation viability of the cells,and the changes in the intracellular calcium concentration were detected by laser confocal scanning technique.Results:CaSR expression was present in rat distal PVSMCs,and CaSR protein expression was upregulated under hypoxia.The positive regulator spermine not only enhanced CH-induced CaSR upregulation but also enhanced CH-induced increase in cell viability and calcium ion concentration.The negative CaSR regulator NPS2143 not only attenuated CH-induced CaSR upregulation but also inhibited CH-induced cell viability and calcium ion concentration.Conclusion:CaSR-mediated hyperproliferation is a novel pathogenic mechanism for the development of proliferation in distal PVSMCs under CH conditions.

Effect of safflower injection on endoplasmic reticulum stress-induced apoptosis in rats with hypoxic pulmonary hypertension

Objective To explore the effects of safflower injection on prevention and treatment of hypoxic pulmonary hypertension and clarify the function of the endoplasmic reticulum stress apoptosis pathway during the process.Methods Thirty male SD rats were randomly grouped as normal control group,hypoxiahypercapnia group and hypoxia+safflower group.The latter two groups were put in the cabin with oxygen concentration ranged from 9% to 11% and carbon dioxide concentration from 5% to 6%.The pulmonary artery pressure and the index of right ventricular hypertrophy were determined after hypoxia exposure(8 h/d×28 d).Changes in morphology of lung tissue were observed by electron microscopy.To explore the possible mechanisms,we also detected apoptosis and apoptosis-related genes/proteins in lung tissue by TUNEL reactivity and PCR and Western blot.Results Compared with the normal control group,pulmonary artery pressure and the index of right ventricular hypertrophy in hypoxia group were 45% and 33.4% higher,respectively.Tiny blood vessel wall of lungs was thickened and edema,and proliferation of collagen fibers was obvious under the electron microscope.TUNEL staining of apoptotic cells in lung tissues showed more high brightness green fluorescence(+～++),but less green fluorescence showed in the pulmonary vascular smooth muscle cell layer,and apoptosis index(AI) value was 150% higher;gene and protein expression levels of endoplasmic reticulum stress pathway were increased.Compared with hypoxia-hypercapnia group,pulmonary artery pressure and the index of right ventricular hypertrophy in the hypoxia+safflower group were 18% and 15.6% lower,respectively;collagen fibers were decreased,and smooth muscle cells and epithelial cells were got apoptotic-like changes under the electron microscope.TUNEL staining of apoptotic cells in lung tissues showed brighter green fluorescence(++～+++);the high brightness green fluorescence showed in pulmonary vascular smooth muscle cell layer,and apoptotic index(AI) value was 40% higher;gene and protein expressions of endoplasmic reticulum stress pathway were significantly upregulated.Conclusion Our findings demonstrate that safflower injection could activate endoplasmic reticulum stressinduced apoptosis and especially promote apoptosis in pulmonary vascular smooth muscle cells.

The effect of bone marrow mesenchymal stem cell transplantation on hypoxic pulmonary hypertension in rats

Objective:To study the influence of bone marrow mesenchymal stem cells(MSCs)transplantation on hypoxic pulmonary hypertension(HPH)in rats.Methods:MSCs in SD rats were separated,cultivated,identified in vitro,and labeled by the green fluorescence protein(GFP)adenovirus.Healthy male SD rats were randomly divided into four groups:normal control group(NC group)and HPH group,with eight rats in each group respectively;HPH+mesenchymal stem cell transplantation group(MSCs group)and HPH+vascular endothelial growth factor+mesenchymal stem cell transplantation group(VEGF+MSCs group),with twenty-four rats in each group respectively.In this experiment,intermittent normobaric hypoxia was employed to establish the pulmonary hypertension rat models,with stem cells transfected and transplanted.The mean pulmonary artery pressure(mPAP)was observed in rats to calculate the right ventricular hypertrophy index(RVHI);the morphological changes of pulmonary arterioles in each group of rats were observed under the microscope;the distribution and manifestation of MSCs fluorescently labeled by adenovirus transfection were observed in pulmonary arterioles under the fluorescence microscope at the set time points of 7 d,14 d and 28 d after the transplantation of stem cells.Results:For NC group,the mPAP(mmHg)was 15.5±1.5 at 28 d,while the mPAP in HPH,MSCs and VEGF+MSCs groups were 26.1±1.9,21.6±2.7 and 20.1±2.9 respectively which were apparently higher than that in NC group(p<.01).Compared with HPH group(p<.01),the mPAP was obviously decreased in MSCs and VEGF+MSCs groups.There was no significant difference between MSCs and VEGF+MSCs groups.At 28 d,RVHI for NC group was 0.28±0.02,while the RVHI in HPH,MSCs and VEGF+MSCs groups were 0.43±0.07,0.34±0.03 and 0.35±0.01 respectively which were apparently higher than that in NC group(p<.01).In comparison with HPH group,RVHI was significantly decreased in MSCs and VEGF+MSCs groups(p<.05).There was no significant difference between MSCs and VEGF+MSCs groups.For HPH group,at 28 d,pulmonary arterioles were apparently thickened,with luminal stenosis&obliteration and incomplete endothelial cells.Compared with HPH group,pulmonary arterioles in MSCs group became thinning,with the lumen unobstructed and the integrity of endothelial cells improved.The changes in the manifestation of MSCs and VEGF+MSCs groups were not significant.Conclusions:The transplantation of MSCs can improve the remodeling of pulmonary arterioles to partially reverse the progress of HPH;the combined transplantation of VEGF and MSCs doesn’t improve the effect of MSC transplantation.

Effects of Hypoxia on Calmodulin Levels of Lung Tissues and Small Pulmonary Arterial Walls in Young Pigs

The effects of chronic hypoxia on calmodulin levels of lung tissues and small pulmonary ar-terial walls were studied in young pigs, The tissue specimens of hypoxic animals were obtained underhypoxic conditions. The following results were collected:(1) The swine exposed to chronic intermittent hypoxia showed a significant pulmonary pressor re-sponse at a simulated high altitude of 4000 m.(2) A higher level of calmodulin was found in the lung tissues of chronic hypoxic animals. It maybe related to the increased release of some vaosactive substances from pulmonary non-muscularcalls.(3) No significant difference of calmodulin level of small arterial walls was demonstrated between theexperimental animals and the control.The findings suggest that pulmonary vasoconstriction due to hypoxia is not likely to be associatedwith obvious change in calmodulin level in the smooth muscle of blood vessels.

The Effect of Hypoxia on Expression of Basic Fibroblast Growth Factor in Pulmonary Vascular Pericytes

To examine whether hypoxia exerts effect on the expression of basic fibroblast growth ac- tor (bFGF) in pulmonary vascular pericytes (PC), cell culture, in .citu hybridization with probe of digoxigenin-11-dUTP-labled cDNA, immunocytochemistry and image analysis were employed in this study. The results showed that the expression amount of bFGF mRNA and protein in PC of hypoxia (H) group was 1.31 times (P<0. 01) and 1. 17 times (P<0. 01) that of normoxia (N) group re- spectively. It suggests that hypoxia can directly enhance the expression of bFGF mRNA and protein in PC. Increased expression of bFGF may play an important role in the process of PC proliferation and differentiation of PC into smooth muscle-like cells.

NR4A1 enhances glycolysis in hypoxia-exposed pulmonary artery smooth muscle cells by upregulating HIF-1αexpression

Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.

Quercetin,the key constituent of Astragali Radix,modulates ferroptosis in PASMCs and attenuates hypoxia pulmonary hypertension via the MAPK signaling pathway

This study delved into the mechanism by which the principal component of Astragali Radix regulated ferroptosis in the context of hypoxia-induced pulmonary hypertension,employing a combination of network pharmacology and experimental validation techniques.Active constituents of Astragali Radix and their corresponding targets were identified using the TCMSP database,while therapeutic targets associated with hypoxia-induced pulmonary hypertension were sourced from the GeneCards database.The Venn online tool facilitated the identification of overlapping targets between the active constituents of Astragali Radix and hypoxia-induced pulmonary hypertension.Interaction network diagrams depicting the relationship between Astragali Radix’s active constituents and their targets were constructed using Cytoscape software,with core targets and sub-networks identified using the CytoHubba plug-in.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted using the DAVID database.Additionally,the FerrDb database was consulted to analyze genes implicated in regulating ferroptosis.The investigation revealed 18 active constituents selected from Astragali Radix,with quercetin emerging as the key component.A total of 35 potential targets associated with Astragali Radix in regulating ferroptosis and addressing hypoxia-induced pulmonary hypertension were predicted.Experimental validation demonstrated that quercetin could inhibit the MAPK signaling pathway,resulting in reduced Fe2+and lipid peroxide levels,increased GPX4 expression,and the reversal of ferroptosis.In summary,this study elucidated the fundamental constituents and pivotal signaling pathways through which Astragali Radix modulated ferroptosis and mitigated hypoxia-induced pulmonary hypertension.Specifically,quercetin,a core constituent of Astragali Radix,was observed to inhibit ferroptosis in pulmonary arterial smooth muscle cells via the MAPK pathway and alleviate hypoxia-induced pulmonary hypertension.

BMP-7/Smads通路参与EndoMT在大鼠HHPH中的作用

目的:探讨骨形态发生蛋白(BMP)-7/Smads通路参与的内皮-间充质转化(EndoMT)在大鼠低氧高二氧化碳性肺动脉高压(HHPH)中的作用。方法:将大鼠肺动脉内皮细胞(RPAECs)分为常氧对照(Con)组、低氧高二氧化碳(HH)组、BMP受体激动剂rhBMP-7组、BMP受体抑制剂DMH-1组和溶剂DMSO组。各组给予相应的药物后,Con组置于常氧培养箱中培养,其余4组置于低氧高二氧化碳培养箱中培养。采用免疫荧光标记法观察细胞CD31和α-平滑肌肌动蛋白(α-SMA)的表达水平;RT-PCR和Western blot法分别检测各组细胞α-SMA、CD31和Smad1/5/8的mRNA和蛋白表达水平;CCK-8法检测细胞活力;Transwell小室法检测细胞的迁移水平。结果:与Con组相比,HH组α-SMA的mRNA和蛋白表达水平升高,而CD31的mRNA和蛋白表达水平、Smad1/5/8的mRNA和p-Smad1/5/8的蛋白表达水平降低,同时细胞活力降低而迁移增多(P<0.05)。与HH组相比,rhBMP-7组α-SMA的mRNA和蛋白表达水平降低,而CD31的mRNA和蛋白表达水平、Smad1/5/8的mRNA和p-Smad1/5/8的蛋白表达水平升高,同时细胞活力升高而迁移减少(P<0.05)。与rhBMP-7组相比,DMH-1组α-SMA的mRNA和蛋白表达水平升高,而CD31的mRNA和蛋白表达水平及Smad1/5/8的mRNA和p-Smad1/5/8的蛋白表达水平降低,同时细胞活力降低而迁移增多(P<0.05)。结论:低氧高二氧化碳可促进RPAECs发生EndoMT,从而促进HHPH的发展,其机制可能与抑制BMP-7/Smads通路有关。

TGF-β/Smads通路参与EndoMT在HHPH中的作用及益气温阳活血化痰方的干预效应

目的:探讨转化生长因子β(TGF-β)/Smads信号通路与低氧高二氧化碳性肺动脉高压(HHPH)中肺动脉内皮-间充质转化(Endo MT)的关系及益气温阳活血化痰方的调控作用。方法:取健康雄性清洁级SD大鼠为研究对象,随机分为5组:常氧对照(N)组、低氧高二氧化碳(HH)组及益气温阳活血化痰方高剂量(YH)组、中剂量(YM)组和低剂量(YL)组。N组置于常氧环境下饲养,其余4组置于低氧高二氧化碳(9%~11%O2和5%~6%CO2)环境下饲养4周。各中药组在放入氧舱前以相同体积不同浓度的益气温阳活血化痰方灌胃,YH、YM和YL组浓度分别为200 g/L、100 g/L和50 g/L。术中测平均肺动脉压和平均颈动脉压,术毕取右心室游离壁及左心室加心室间隔称重,以此计算右心室肥大指数。HE染色和免疫荧光法观察肺组织形态学的变化,采用RT-PCR和Western blot检测α-平滑肌肌动蛋白(α-SMA)、CD31、TGF-β1和Smad2/3的m RNA和蛋白表达以及p-Smad2/3的蛋白水平。结果:与N组比,其余4组肺动脉平均压、α-SMA、TGF-β1和Smad2/3的m RNA和蛋白表达以及pSmad2/3的蛋白水平上升,CD31 m RNA和蛋白表达水平降低,镜下观察到组织形态学损伤;与HH组比,YH、YM和YL组肺动脉平均压、α-SMA、TGF-β1和Smad2/3的m RNA和蛋白表达以及p-Smad2/3的蛋白水平下降,CD31m RNA和蛋白表达水平上升,肺组织形态学的损伤显著减轻。结论:在HHPH中,TGF-β/Smads通路可能参与了Endo MT;益气温阳活血化痰方可通过抑制TGF-β/Smads通路的表达降低肺动脉高压。

西地那非联合硫酸镁治疗HPH的临床观察

目的探讨西地那非联合硫酸镁治疗新生儿缺氧性肺动脉高压(HPH)的疗效及对患儿血清缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)、内皮素-1(ET-1)表达的影响。方法选择2018年1月~2020年2月我院收治的HPH患儿62例,采用随机数字表法分为观察组和对照组,每组各31例。对照组给予硫酸镁治疗,观察组给予西地那非联合硫酸镁治疗,比较两组血气分析指标,血清HIF-1α、VEGF、ET-1表达含量,不良反应及疗效。结果治疗后观察组mPAP、PaCO2均明显低于对照组,PaO2明显高于对照组(P<0.05);治疗后观察组血清HIF-1α、VEGF、ET-1表达含量均明显低于对照组(P<0.05);两组不良反应总发生率比较,差异无统计学意义(P>0.05);观察组总有效率为96.77%,明显高于对照组的77.42%(P<0.05)。结论西地那非联合硫酸镁治疗新生儿缺氧性肺动脉高压的疗效确切,可有效改善患儿血气分析指标,并下调血清HIF-1α、VEGF、ET-1表达含量,改善预后。

TDAG8、HMGB1及HIF-1在HPH炎症调控中的作用

肺动脉高压( Pulmonary Hypertension,PH)是多种累及心、肺的疾病进展到一定阶段所表现出来的病理生理状态。其中,低氧性肺动脉高压(Hypoxic Pulmonary Hypertension,HPH)是由于低氧状态引起的PH,可引起严重及可危及生命的并发症,死亡率和长期残疾的风险明显增加[1]。HPH治疗尚缺乏特异性的方法,目前的治疗多具有引起体循环压力改变且副作用较高方法,成为临床急需解决的严峻问题。HPH主要特点是肺血管阻力(PVR)和肺动脉压(PAP)持续性升高,导致右心衰竭,发生在慢性缺氧引起的各种形式的慢性肺疾病(如慢性阻塞性肺疾病、囊性纤维化、支气管肺的发育不良等)。

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development

Background:Pulmonary arterial hypertension(PAH)is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells(PASMCs),endothelial dysfunction,and extracellular matrix remodeling.G protein-coupled receptor kinase 2(GRK2)plays an important role in the maintenance of vascular tone and blood flow.However,the role of GRK2 in the pathogenesis of PAH is unknown.Methods:GRK2 levels were detected in lung tissues from healthy people and PAH patients.C57BL/6 mice,vascular smooth muscle cell-specific Grk2-knockout mice(Grk2^(∆SM22)),and littermate controls(Grk2^(flox/flox))were grouped into control and hypoxia mice(n=8).Pulmonary hypertension(PH)was induced by exposure to chronic hypoxia(10%)combined with injection of the SU5416(cHx/SU).The expression levels of GRK2 and Yes-associated protein(YAP)in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining.The mRNA expression levels of Grk2 and Yes-associated protein(YAP)in PASMCs were quantified with real-time polymerase chain reaction(RT-PCR).Wound-healing assay,3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT)assay,and 5-Ethynyl-2′-deoxyuridine(EdU)staining were performed to evaluate the proliferation and migration of PASMCs.Meanwhile,the interaction among proteins was detected by immunoprecipitation assays.Results:The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice.Moreover,cHx/SU-induced PH was attenuated in Grk2^(∆SM22) mice compared with littermate controls.The amelioration of PH in Grk2^(∆SM22) mice was accompanied by reduced pulmonary vascular remodeling.In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration,whereas this effect was severely intensified by overexpression of GRK2.We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs,resulting in excessive PASMCs proliferation and migration.Furthermore,GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions.Conclusion:Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH,via regulating YAP expression and nuclear translocation.Therefore,GRK2 serves as a novel therapeutic target for PAH treatment.