Research progress on the mechanism of phenotypic transformation of pulmonary artery smooth muscle cells induced by hypoxia / 浙江大学学报·医学版

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) is a key factor in pulmonary vascular remodeling. Inhibiting or reversing phenotypic transformation can inhibit pulmonary vascular remodeling and control the progression of hypoxic pulmonary hypertension. Recent studies have shown that hypoxia causes intracellular peroxide metabolism to induce oxidative stress, induces multi-pathway signal transduction, including those related to autophagy, endoplasmic reticulum stress and mitochondrial dysfunction, and also induces non-coding RNA regulation of cell marker protein expression, resulting in PASMCs phenotypic transformation. This article reviews recent research progress on mechanisms of hypoxia-induced phenotypic transformation of PASMCs, which may be helpful for finding targets to inhibit phenotypic transformation and to improve pulmonary vascular remodeling diseases such as hypoxia-induced pulmonary hypertension.

Role and diagnostic value of miRNA-205 on vascular calcification in patients with chronic kidney disease / 中华肾脏病杂志

Objective:To investigate the role and diagnostic value of miRNA-205 in chronic kidney disease (CKD) patients with vascular calcification.Methods:It was divided into in vitro cell experiment and retrospective cohort study. In vitro experiments were conducted by using rat thoracic aortic smooth muscle cells. Alizarin red staining and calcium content detection were used to detect the calcification of vascular smooth muscle cells (VSMCs). Alkaline phosphatase (ALP) test kit was used to measure ALP activity. Western blotting was used to detect the protein expression levels of osteogenic transcription factors runt-related transcription factor 2 (Runx2), α smooth muscle actin (α-SMA) and smooth muscle-22α (SM-22α) in VSMCs. qRT-PCR was used to detect miRNA-205 and Runx2 expression levels. The double luciferase reporter gene assay was used to verify the targeted relationship between miRNA-205 and Runx2. The non-dialysis patients with CKD 3-5 stage from June 2020 to January 2021 in the Department of Nephrology of Fourth Hospital, Hebei Medical University were selected. According to coronary artery calcium score (CACs), the patients were divided into non-calcification group (CACs=0), mild-moderate calcification group (0<CACs≤400), and severe calcification group (CACs > 400). Spearman correlation analysis was used to analyze the correlation between miRNA-205 and Runx2 and vascular calcification. Logistic regression model and receiver operating characteristic (ROC) curve analysis were used to analyze the ability of miRNA-205 to predict the vascular calcification in patients with CKD. Results:(1)Compared with the control group, calcium nodules were more, and the calcium content, ALP activity and Runx2 protein level were higher, and the expression levels of miRNA-205, α-SMA and SM-22α were significantly lower in high phosphorus group (all P<0.05). Overexpression of miRNA-205 significantly reduced the calcification of VSMCs and Runx2 protein level, and increased the protein levels of α-SMA and SM-22α (all P<0.05). miRNA-205-5p reduced the activity of luciferase in the wild-type Runx2-3'-end non-coding region plasmid. (2) Eighty CKD patients were enrolled, with age of (57.50±14.93) years old and 49 males (61.3%). The results of comparison of miRNA-205 and Runx2 expression levels in non-calcification group ( n=26), mild- moderate calcification group ( n=30) and severe calcification group ( n=24) showed that, the higher degree of calcification, the lower miRNA-205 expression level and the higher Runx2 mRNA expression level (all P<0.05). miRNA-205 was negatively correlated with CACs ( r=-0.50, P<0.01) and Runx2 was positively correlated with CACs ( r=0.55, P<0.01). Multivariate logistic regression analysis results suggested that miRNA-205 ( OR=0.451, 95% CI 0.122-0.873) was an independent influencing factor of vascular calcification in CKD patients. The area under the ROC curve of miRNA-205 and miRNA-205 combined with Runx2 for predicting vascular calcification were 0.796 (95% CI 0.697-0.859) and 0.924 (95% CI 0.866-0.982), respectively. Conclusions:miRNA-205 inhibits vascular calcification by targeting Runx2 to negatively regulate osteogenetic phenotype transformation of VSMCs and is expected to be an early diagnostic marker of vascular calcification in CKD patients.

Salidroside inhibits phenotypic transformation of rat pulmonary artery smooth muscle cells induced by hypoxia / 中国中药杂志

This study investigated the effect of salidroside on phenotypic transformation of rat pulmonary artery smooth muscle cells(PASMCs) induced by hypoxia. Rat pulmonary arteries were isolated by tissue digestion and PASMCs were cultured. The OD values of cells treated with salidroside at different concentrations for 48 hours were measured by cell counting kit-8(CCK-8) to determine the appropriate concentration range of salidroside. The cells were divided into a normal(normoxia) group, a model(hypoxia) group, and three hypoxia + salidroside groups(40, 60, and 80 μg·mL~(-1)). Quantitative real-time PCR(qRT-PCR) was used to detect the mRNA expression of cell contractile markers in each group, such as α-smooth muscle actin(α-SMA), smooth muscle 22(SM22), and calcium-binding protein(calponin), and synthetic marker vimentin. The expression levels of cell phenotypic markers and proliferating cell nuclear antigen(PCNA) were detected by Western blot. The proliferation of cells in each group was detected by the 5-ethynyl-2'-deoxyuridine(EdU) assay. Cell migration was measured by Transwell assay. As revealed by results, compared with the normal group, the model group showed decreased mRNA and protein expression of contractile phenotypic markers of PASMCs and increased mRNA and protein expression of synthetic markers. Compared with the conditions in the model group, salidroside could down-regulate the mRNA and protein expression of synthetic markers in PASMCs and up-regulated the mRNA and protein expression of contractile phenotypic markers. Compared with the normal group, the model group showed potentiated proliferation and migration. Compared with the model group, the hypoxia + salidroside groups showed blunted proliferation and migration of cells after phenotypic transformation. The results suggest that salidroside can inhibit the expression of synthetic markers in PASMCs and promote the expression of contractile markers to inhibit the hypoxia-induced phenotypic transformation of PASMCs. The mechanism of salidroside in inhibiting the proliferation and migration of PASMCs is related to the inhibition of the phenotypic transformation of PASMCs.

Effect and mechanism of phenotypic transformation of vascular smooth muscle cells regulated by TGF-β2 on the venous thrombus wall remodeling / 解放军医学杂志

Objective To investigate the role and mechanism of transforming growth factor-β2 (TGF-β2) on regulating the phenotype transformation of vascular smooth muscle cells (SMC) during the wall remodeling after venous thrombosis. Methods Human umbilical vein smooth muscle cells (HVSMCs) were treated with different concentration (0, 1, 10, 20 ng/ml) of TGF-β2 for 24 hours, or treated with 10 ng/ml TGF-β2 at different time points (0, 2, 6, 24 h). RT-PCR was performed to detect the mRNA expressions of phenotype markers (contractile type: α-SMA and Elastin; synthetic type: Col1A1 and Col1A2). SD rat models of inferior vena cava thrombosis (IVCT) were established. Rats were randomly divided into sham surgery group (n=5), thrombus group (n=9) and TGF-β2 experimental group (n=5). The successful establishment of rat model was confirmed by HE and Masson staining of vein wall tissues on 4 and 7 days after modelling. At the 14th day after modelling, IVCT rats were locally treated with hydrogel containing TGF-β2 10 ng per rat for 24 hours. RT-PCR was used to detect the expression of SMC phenotypic marker mRNA in the vein wall of each group. Results TGF-β2 up-regulated mRNA expressions of both contractile and synthetic markers in HVSMCs in a dose- and time-dependent manner (P<0.05). Notely, the mRNA expression of contractile markers was more up-regulated than that of synthetic markers. HE and Masson staining showed the acute thrombosis phase 4 days after modelling and the chronic organized thrombus and recanalization 7 days after modelling, confirming the successful establishment of IVCT models. At the 14th day after modelling, compared with the sham surgery group, the mRNA expression in SMC venous wall contractile markers of thrombus group was down-regulated obviously, and was up-regulated markedly in synthetic markers (P<0.05). When TGF-β2 was used to locally treat the thrombus, the expressions of contractile markers mRNA were more significantly up-regulated (P<0.05). Conclusions TGF-β2 may induce the expression of contractile and synthetic markers in HVSMCs, particularly up-regulating the contractile markers. In IVCT rats model, TGF-β2 can significantly up-regulate the mRNA expressions of SMC contraction markers, which is in favour of maintenance of SMC contractile phenotype in the thrombus vein wall.

High-salt exposure induces macrophage polarization to promote proliferation and phenotypic transformation of co-cultured renal fibroblasts / 南方医科大学学报

OBJECTIVE@#To investigate high-salt exposure-induced polarization of mononuclear macrophages and the changes in proliferation and phenotypic transformation of renal fibroblasts in a co-culture system.@*METHODS@#Cultured mononuclear macrophages were exposed to high salt (161 mmol/L Na +) for 2 h and the surface markers of M0, M1 and M2-type macrophages were detected with RT-qPCR. The culture medium of the macrophages in normal and high-salt groups was collected for detection of the mRNA and protein levels of IL-6 and TGF-β1 using RT-qPCR and ELISA. A co-culture system of high salt-exposed macrophages and renal fibroblasts (NRK-49F) was established using a Transwell chamber, and the changes in proliferation and migration of NRK-49F cells were examined using EdU assay and Transwell assay, respectively. Western blotting was performed to detect the expressions of collagen I, collagen III and collagen α-SMA in NRK-49F cells.@*RESULTS@#The high salt-exposed macrophages showed significantly increased mRNA levels of M2-type macrophage surface markers mannose receptor and arginase (@*CONCLUSIONS@#High-salt exposure induces polarization of mononuclear macrophages into M2-type macrophages and promotes secretion of IL-6 and TGF-β1 by the macrophages to induce the proliferation and phenotypic transformation of NRK-49F cells.

Effects of C-C motif chemokine receptor 2 on phenotypic transformation of cardiac fibroblasts after hypoxia / 解放军医学杂志

Objective To investigate the effect of C-C motif chemokine receptor 2 (CCR2) on phenotypic transformation of cardiac fibroblasts after hypoxia. Methods The mouse myocardium primary fibroblasts were extracted by collagenase digestion. Cells were divided into control, hypoxia-24h and hypoxia-48h, the mRNA and protein expression of CCR2 was examined by real-time PCR and Western blotting. CCR2 low expression cell (si-CCR2) was established using by small interfering RNA. Cells were divided into four groups including si-control, si-CCR2, si-control+hypoxia, si-CCR2+hypoxia. The mRNA and protein expressions of CCR2, α smooth muscle actin (α-SMA) and Collagen 1A (Col 1A) were detected by real-time PCR and Western blotting, cell proliferation was detected by Cell Counting Kit-8 (CCK8) and Bromodeoxyuridine (BrdU). Results Compared with control, the mRNA and protein levels of CCR2 significantly increased in hypoxia-24h and hypoxia-48h group (P<0.01), however, no significance was found in these two time points. Compared with si-control group, the mRNA and protein expressions of CCR2, α-SMA and Col 1A not significantly changed in si-CCR2 group. Compared with si-control group, the mRNA and protein levels of CCR2, α-SMA and Col 1A significantly increased in si-control+hypoxia group (P<0.01 or P<0.05), and cell proliferation increased (P<0.01). Compared with si-control+hypoxia group, the protein expression of α-SMA and Col 1A decreased in si-CCR2+hypoxia group (P<0.05), and cell proliferation also decreased in si-CCR2+hypoxia group (P<0.05). Conclusions CCR2 could affect the phenotypic transformation of cardiac fibroblasts after hypoxia.

Advances on epithelial mesenchymal transformation and airway remodeling of asthma / 中国药理学通报

Bronchial asthma is a respiratory system disease char-acterized by airway remodeling as a pathological basis. Repeated inflammatory infiltration and tissue damage repair can lead to airway remodeling. At present,the mechanism of airway remod-eling is not comprehensive. Studies have shown that epithelial-mesenchymal transition (EMT) plays an important role in the genesis and development of airway remodeling. Airway epithelial cells can be induced to mesenchymal transition through a variety of secretion factors and signaling pathways,leading to airway re-modeling in asthma. This review summarizes the study of EMT and airway remodeling in asthma,providing a reference for clini-cal follow-up treatment and research.

Effects of inhibition of KLF4 expression by miR-29a on transformation of phenotype of vascular smooth muscle cells / 解放军医学杂志

Objective To explore whether miR-29a can influence the phenotypic transformation of vascular smooth muscle cells (VSMCs) by inhibiting expression of Kruppel-like factor 4 (KLF4). Methods Rat VSMCs were cultured primarily. The luciferase reporter system was used to verify whether KLF4 is the target gene of miR-29a. VSMCs were divided into miR-29a-transfected expression plasmid group, transfection-negative expression plasmid group, and no-transfection group. The expression of KLF4 and VSMC contractile phenotype protein levels were determined by Western blotting. The proliferation of VSMCs was analyzed by3H thymidine-incorporation assay. Results The lucierase activity was significantly decreased in wild-type KLF4 luciferase report gene and miR-29a expression plasmid co-transfection group. The KLF4 protein expression level (0.36 ± 0.02) was significantly lower in miR-29a-transfected expression plasmid group than that in untransfected and transfection-negative expression plasmid group (1.52 ± 0.06, 1.55 ± 0.05, respectively, P<0.01). Meanwhile, compared with untransfected and transfection-negative expression plasmid groups, the contractile phenotype associated protein SM-MHC, SM-22α, calponin expression levels were increased, while the proliferation capability was decreased in miR-29a-transfected expression plasmid group. Conclusion MiR-29a targets KLF4 and inhibits its expression, thus maintains contractile phenotype of VSMCs, and reduces the cell proliferation ability.

Influence of pathologic factors on phenotypic transformation of renal tubular epithelial cells in vitro / 中华肾脏病杂志

Objective To study the effects of high albumin, high glucose and low bovine serum on the phenotypic transformation of renal tubular epithelial cells. Methods The normal human kidney proximal tubular eel] line (HKC) was cultured for 30 days in the presence of high albumin (1.5 g/L), high glucose(25 mmol/L) and low bovine serum(2% ) . Morphological changes were observed by electronic microscopy. Immunohistochemistry stain was used to examine the expression of cytokeratin, vimentin, a-SMA, collagen Ⅰ and TGF-pl protein. Western blot was applied to further detect the process of collagen I protein expression, and in situ hybridization was used to examined the expression of collagen Ⅰ gene. Results Renal tubular epithelial cells cultured in high albumin, high glucose and low bovine serum showed obvious morphologic changes, including elongated shape, decrease of microvilli and mitochondria, and increase of rough endoplasmic reticulum under electronic microscopy. Immunohistochemistry stain revealed the reduction of cytokeratin, and enhancement of vimentin, ?-SMA, TGF-?1 and collagen Ⅰ. Western blot demonstrated that the expression of collagen Ⅰincreased in a time-dependent manner, and in situ hybridization showed that collagen type Ⅰ mRNA increased as well. Conclusion High albumin, high glucose and low bovine serum induce phenotypic transformation of renal tubular epithelial cells into mesanchymal cells.