Urotensin II activates the ferroptosis pathway through circ0004372/miR-124/SERTAD4 to promote the activation of vascular adventitial fibroblasts.

Both vascular adventitial fibroblasts (VAFs) and urotensin II (UII) play important roles in vascular remodeling diseases, but the mechanism of UII in VAFs is still unclear. UII inhibited miR-124 expression through up-regulating circ0004372 expression, thereby promoting SERTAD4 expression. UII significantly promoted the generation of ROS, MDA and 4-HNE, reduced the activities of SOD, GST and GR, increased Fe2+ concentration and inhibited GPX4 expression through circ0004372/miR-124/SERTAD4. Both UII and ferroptosis inducer Erastin significantly promoted the expression of α-SMA, Collagen I and TGF-ß1 in VAFs, but circ0004372 siRNA, miR-124 mimics, SERTAD4 siRNA or Ferrostatin-1 significantly inhibited the effect of UII and Erastin on cell activation. When co-transfected with circ0004372 siRNA and miR-124 inhibitors or miR-124 mimics and SERTAD4 overexpression vector, UII still significantly increased the expression of α-SMA, Collagen I and TGF-ß1. After transfection with circ0004372 overexpression vector, miR-124 inhibitors or SERTAD4 overexpression vector and then treating with UII and Ferrostatin-1, the expression of α-SMA, Collagen I and TGF-ß1 was still significant; when the circ0004372 overexpression vector and miR-124 mimics or miR-124 inhibitors and SERTAD4 siRNA were co-transfected and then UII and Ferrostatin-1 were added, the expression of α-SMA, Collagen I and TGF-ß1 was not significantly increased. Therefore, these results indicate that UII promotes the activation of VAFs through the circ0004372/miR-124/SERTAD4/ferroptosis pathway.

Glutamine switches vascular smooth muscle cells to synthetic phenotype through inhibiting miR-143 expression and upregulating THY1 expression.

AIMS: Vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of many human cardiovascular diseases. They modulate their phenotype from "contractile" to "synthetic" in response to changes in local environmental cues. How glutamine regulates the differentiation of VSMCs and the underlying mechanisms remain largely unknown. MAIN METHODS: Here, we explored the effects of various doses of glutamine (0 mM, 1 mM, 2 mM, and 4 mM) on the proliferation, migration, and phenotypic switch of human VSMCs in vitro. Glutamine dose-dependently enhanced VSMC proliferation, and markedly increased VSMC migration. KEY FINDINGS: Notably, glutamine promoted the phenotypic switch of VSMCs towards a synthetic phenotype, as evidenced by significantly decreased expression of contractile markers myosin heavy chain 11 (MYH11) and calponin while increased expression of synthetic markers collagen I and vimentin. Importantly, these changes upon glutamine treatments were attenuated after additional treatments with glutamine metabolism inhibitor BPTES. Additionally, glutamine downregulated miR-143 expression, and miR-143 inactivation alone resulted in enhanced proliferation, migration, and promoted the synthetic phenotype of VSMCs. Moreover, Thy-1 cell surface antigen (THY1) was validated as a downstream target of miR-143, and THY1 expression was upregulated by glutamine in VSMCs. Furthermore, either miR-143 overexpression or THY1 silencing abolished the effect of glutamine on proliferation, migration, and phenotypic switch of VSMCs, supporting a novel glutamine-miR-143-THY1 pathway in modulating VSMC functions. SIGNIFICANCE: This study demonstrated a novel mechanism of glutamine in modulation of VSMC phenotypic switch by targeting miR-143 and THY1, and provides significant insight on targeted therapy of patients with cardiovascular diseases.

Tanshinone IIA attenuates high glucose induced human VSMC proliferation and migration through miR-21-5p-mediated tropomyosin 1 downregulation.

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the development and progression of diabetes-related vascular complications. Recently, microRNAs (miRNAs) have been suggested to be involved in the pathogenesis of vascular diseases. This study was designed to investigate the influences of tanshinone IIA, an active compound extracted from Chinese herb Salvia miltiorrhiza, on the proliferation and migration of human aortic VSMCs (HASMCs). cultured in a high glucose medium and the underlying mechanisms related miRNAs. Using a miRNA microarray method, we profiled the miRNA expression signature in human aortic VSMCs (HASMCs) exposed to normal glucose, high glucose with and without Tanshinone IIA. Cell proliferation was measured with 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell migration was evaluated using transwell migration assay and wound scratch assay. Western blot was used to examine the expression of tropomyosin 1 (TPM1) and miRNA level was quantified by real-time PCR. The results showed that several miRNAs that were highly expressed in the high glucose group were significantly decreased in the high glucose with Tanshinone IIA group compared with the normal glucose group (P < 0.05). Among these miRNAs, miR-21-5p was significantly upregulated in the high glucose group and downregulated after Tanshinone IIA treatment (P < 0.05). The depletion of miR-21-5p in HASMCs resulted in decreased cell proliferation and migration (P < 0.05). Moreover, we found that Tanshinone IIA inhibited proliferation and migration partly through miR-21-5p-mediated TPM1 downregulation (P < 0.05). In conclusion, the present study demonstrates that Tanshinone IIA is able to protect HASMCs from high glucose-induced proliferation and migration through regulating expression of miRNAs.

LncRNA-MIAT regulates fibrosis in hypertrophic cardiomyopathy (HCM) by mediating the expression of miR-29a-3p.

AIM: This study aimed to investigate the molecular mechanism underlying the fibrosis in hypertrophic cardiomyopathy (HCM). METHOD: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of potentially relevant microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in patients with HCM suffering from fibrosis and patients with HCM free of fibrosis. In addition, the regulatory relationship between lncRNAs and miR-29a was studied using a luciferase assay. Subsequently, area under the receiver-operating characteristics (ROC) curve (AUC) analysis was conducted to predict the diagnostic value of myocardial infarction-associated transcript (MIAT), miR-29a, H19, and MEG3 in patients with HCM. Finally, the predicted regulatory relationship betwe en miR-29a and MIAT was validated by transfecting cells with different plasmids. RESULT: miR-29a and MIAT were differently expressed between the fibrosis (+) HCM group and the fibrosis (-) HCM group, thus establishing a negative relationship between the expression of these two genes. In addition, both MIAT and miR-29a showed the ability to accurately predict the prognosis in patients with HCM. Furthermore, the luciferase activity of wild-type MIAT was evidently suppressed in cells transfected with miR-29a mimics, suggesting that the expression of miR-29a was apparently downregulated in the presence of MIAT. CONCLUSION: The results obtained in this study collectively indicated that the MIAT might be associated with the development of fibrosis (+) HCM via negatively regulating the expression of miR-29a.

Circ-SATB2 upregulates STIM1 expression and regulates vascular smooth muscle cell proliferation and differentiation through miR-939.

The prevention and treatment of coronary heart disease (CHD) is a difficult problem to be solved. More and more studies have found that circular RNAs (circRNAs) may play important roles in the development of CHD. Here detection of vascular smooth muscle cells (VSMCs) showed that circ-SATB2 and STIM1 were up-regulated in proliferative VSMCs, while miR-939 were down-regulated. Circ-SATB2 and miR-939 did not affect the expression of each other, but circ-SATB2 could promote while miR-939 inhibited the expression of STIM1 (a target gene of miR-939). Circ-SATB2 overexpression could inhibit the expression of SM22-alpha (SM22α, a marker of contractile VSMCs), while the expression of SM22α was promoted by miR-939. STIM1 could promote cell proliferation and migration, and circ-SATB2 had similar effects, but its linear sequence had no such functions. MiR-939 had the opposite effects, could promote cell apoptosis and inhibit cell proliferation and migration, and siRNAs targeting circ-SATB2 had similar effects. When co-transfected with circ-SATB2 over-expression vector and miR-939 mimics or STIM1 siRNAs, the changes of cell proliferation, apoptosis and migration were not significant. Therefore, circ-SATB2 can regulate VSMC phenotypic differentiation, proliferation, apoptosis and migration by promoting the expression of STIM1. This discovery will provide a theoretical reference for exploring the role of circRNA in VSMCs and the pathogenesis of CHD.

[Geographical distribution of the Serum creatinine reference values of healthy adults].

OBJECTIVE: To explore the relationship between serum creatinine (Scr) reference values in healthy adults and geographic factors and provide evidence for establishing Scr reference values in different regions. METHODS: We collected 29 697 Scr reference values from healthy adults measured by 347 medical facilities from 23 provinces, 4 municipalities and 5 autonomous regions. We chose 23 geographical factors and analyzed their correlation with Scr reference values to identify the factors correlated significantly with Scr reference values. According to the Principal component analysis and Ridge regression analysis, two predictive models were constructed and the optimal model was chosen after comparison of the two model's fitting degree of predicted results and measured results. The distribution map of Scr reference values was drawn using the Kriging interpolation method. RESULTS: Seven geographic factors, including latitude, annual sunshine duration, annual average temperature, annual average relative humidity, annual precipitation, annual temperature range and topsoil (silt) cation exchange capacity were found to correlate significantly with Scr reference values. The overall distribution of Scr reference values featured a pattern that the values were high in the south and low in the north, varying consistently with the latitude change. CONCLUSION: The data of the geographic factors in a given region allows the prediction of the Scr values in healthy adults. Analysis of these geographical factors can facilitate the determination of the reference values specific to a region to improve the accuracy for clinical diagnoses.

[Relationship between reference values of fibrinogen and geographical factors based on neural network analysis].

OBJECTIVE: To analyze the relationship between the reference values of fibrinogen (FIB) in healthy Chinese adults and geographical factors to provide scientific evidences for establishing the uniform standard. METHODS: The reference values of FIB of 10701 Chinese healthy adults from 103 cities were collected to investigate their relationship with 18 geographical factors including spatial index, terrain index, climate index, and soil index. Geographical factors that significantly correlated with the reference values were selected for constructing the BP neural network model. The spatial distribution map of the reference value of FIB of healthy Chinese adults was fitted by disjunctive kriging interpolation. We used the 5-layer neural network and selected 2000 times of training covering 11 hidden layers to build the simulation rule for simulating the relationship between FIB and geographical environmental factors using the MATLAB software. RESULTS: s The reference value of FIB in healthy Chinese adults was significantly correlated with the latitude, sunshine duration, annual average temperature, annual average relative humidity, annual precipitation, annual range of air temperature, average annual soil gravel content, and soil cation exchange capacity (silt). The artificial neural networks were created to analyze the simulation of the selected indicators of geographical factors. The spatial distribution map of the reference values of FIB in healthy Chinese adults showed a distribution pattern that FIB levels were higher in the South and lower in the North, and higher in the East and lower in the West. CONCLUSION: When the geographical factors of a certain area are known, the reference values of FIB in healthy Chinese adults can be obtained by establishing the neural network mode or plotting the spatial distribution map.

Effects of monocyte-endothelium interactions on the expression of type IV collagenases in monocytes.

The adhesion of monocytes to endothelial cells is one of the early stages in the development of atherosclerosis. The expression of type IV collagenases, which include matrix metalloproteinase (MMP)-2 and MMP-9, in monocytes is hypothesized to play an important role in monocyte infiltration and transformation into foam cells. The aim of the present study was to examine the effects of monocyte-endothelium interactions on the expression levels of type IV collagenases and their specific inhibitors in monocytes, and to investigate the roles of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in this process. Monocytes were single-cultured or co-cultured with endothelial cells. The expression of the type IV collagenases, MMP-2 and MMP-9, and their specific inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, in monocytes was determined by immunohistochemistry followed by image analysis. The expression levels of MMP-2 and MMP-9 were found to be low in the single-culture monocytes, but increased significantly when the monocytes and endothelial cells were co-cultured. However, treatment with monoclonal TNF-α or IL-1ß antibodies partially inhibited the upregulated expression of MMP-2 and MMP-9 in the co-cultured monocytes. Expression of TIMP-1 and TIMP-2 was observed in the single monocyte culture, and a small increase in the expression levels was observed when the monocytes were co-cultured with endothelial cells. Therefore, monocyte-endothlium interactions were shown to increase the expression of type IV collagenases in monocytes, resulting in the loss of balance between MMP-2 and -9 with TIMP-1 and -2. In addition, TNF-α and IL-1ß were demonstrated to play important roles in this process.

PI3K/AKT signaling pathway plays a role in enhancement of eNOS activity by recombinant human angiotensin converting enzyme 2 in human umbilical vein endothelial cells.

The aim of this study was to investigate the effect of PI3K/AKT signaling pathway in the activity of recombinant human angiotensin converting enzyme 2 (rhACE2) promoted the activity of endothelial nitric oxide synthase (eNOS). The human umbilical vein endothelial cells (HUVEC) were cultured in vitro. Then treated with Ang II (1×10(-6) mol/L) for 24 h. The rhACE2 (100 µmol/L) was added and incubated for 5, 10, 15, 30, 60 min respectively which was based on Ang II intervention. The effect of rhACE2 on phosphorylation eNOS level was also observed in the presence of LY294002 (10 µmol/L) (PI3K/AKT inhibitors). Griess reagent method was applied to measure NO contents in cell culture supernatant, RT-PCR to detect the expression of eNOSmRNA in HUVEC, and Western blot to detect the expression of eNOS and phosphorylated eNOS. In Ang II intervention group, NO contents were significantly lower than control group (P < 0.05). Through rhACE2 treatment, the NO contents in cell culture medium and the expression level of phosphorylated eNOS were significantly higher than in Ang II intervention group (P < 0.05), but eNOSmRNA and non-phosphorylated eNOS protein expression level showed no significant difference (P > 0.05). After HUVEC was intervened by PI3K/AKT pathway inhibitor LY294002, the expression level of phosphorylated eNOS was significantly lower than that in the rhACE2 30 min treatment group (P < 0.05). rhACE2 may reduce the activity of Ang II inhibited endothelial cell eNOS, which can be blocked by PI3K/AKT pathway inhibitor LY294002, suggesting PI3K/AKT signaling pathway plays an important role in rhACE2's promotion of the activity of endothelial cell eNOS.

Effect of peroxisome proliferator activated receptor γ agonist on angiotensin converting enzyme 2 mRNA expression in monocyte-derived macrophages of essential hypertensive patients.

OBJECTIVE: To study the effect of peroxisome proliferator activated receptor γ (PPAR-γ) agonist on the angiotensin converting enzyme 2 (ACE2) mRNA expression in monocyte-derived macrophages of essential hypertensive patients. METHODS: Totally 57 essential hypertensive patients were randomly divided into three groups: conventional treatment group (n=18), telmisartan group (n=19), and benazepril group (n=20); 20 patients with normal blood pressure were also selected as the control group. Monocyte-derived macrophages were isolated from blood samples of patients in all four groups. The expression of ACE2 mRNA in monocyte-derived macrophages was detected by RT-PCR before treatment and 4 and 12 weeks after treatment. RESULTS: Four and 12 weeks after treatment, the systolic pressure and diastolic pressure of telmisartan group and benazepril group were significantly lower than that of the conventional treatment group (all P<0.01), and the systolic pressure and diastolic pressure of telmisartan group were significantly lower than that of the benazepril group(both P<0.01) .The expression of ACE2 mRNA in monocyte-derived macrophages were significantly lower in essential hypertensive patients than that in control group (P<0.01). After having been treated for 4 weeks and 12 weeks, the expression of ACE2 mRNA in monocyte-derived macrophages of hypertensive patients in telmisartan and benazepril groups were significantly higher than that in conventional treatment group (all P<0.01), and the expression of ACE2 mRNA in telmisartan group was significantly higher than that in benazepril group (both P<0.01). CONCLUSION: PPAR-γ agonist could increase the ACE2 mRNA expression in monocyte-derived macrophages of essential hypertensive patients.

[Peroxisome proliferator-activated receptor activator troglitazone inhibits angiotensin II-stimulated secretion of vasoactive factors by endothelial cells].

OBJECTIVE: To investigate the effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand on angiotensin II (AngII)-induced endothelin-1 (ET-1) and NO secretion by endothelial cells in comparison with AngII type I receptor (AT1R) antagonist losartan, so as to reveal the relationship between PPAR gamma and essential hypertension. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were treated with AngII, PPAR gamma ligand troglitazone, AngII plus troglitazone, and AngII plus AT1R antagonist losartan, respectively, and the concentrations of NO and ET-1 in the cell culture supernatant were measured to evaluate the effects of troglitazone and losartan on AngII-induced NO and ET-1 production by human endothelial cells. RESULTS: Treatment of the HUVECs with troglitazone at 10 micromol/L and 50 micromol/L did not produce significant changes in ET-1 concentration in the cell culture supernatants, but significantly increased NO concentration as compared with the control group (P<0.05). Triglitazone at the concentration of 50 micromol/L significantly inhibited AngII (1x10(-6) mol/L)-induced ET-1 production (P<0.05), and at both 10 and 50 micromol/L, troglitazone inhibited the NO release-lowering effect of AngII in the endothelial cells (P<0.05). Both troglitazone and losartan inhibited AngII-induced ET-1 production by the endothelial cells, but losartan showed more potent effect (P<0.05). Similarly, both troglitazone and losartan inhibited decreased NO production in response to AngII treatment, and again losartan showed stronger effect (P<0.05). CONCLUSION: PPAR gamma ligand troglitazone can inhibit AngII-induced ET-1 production enhancement and decreased NO release by the endothelial cells, but its effect is not so strong as losartan, suggesting that troglitazone modulates blood pressure not solely through AT1R pathway.

[Comparison between anti-ouabain egg yolk(IgY) and rabbit antibody(IgG) in enzyme-linked immunosorbent assay].

AIM: To improve specificity and accuracy of endogenous ouabain measurement assay. METHODS: Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared. RESULTS: The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin. CONCLUSION: The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.