Single-cell RNA landscape of cell heterogeneity and immune microenvironment in ligation-induced vascular remodeling in rat.

BACKGROUND AND AIMS: Vascular remodeling is a common pathological basis for cardiovascular diseases. Although both immune and non-immune cells have been suggested to contribute to this process, the complex cellular heterogeneity and intercellular interactions remain largely uncharacterized. METHODS AND RESULTS: In this study, we simulated early and late vascular remodeling by ligating the rat carotid artery for 1 week and 4 weeks, respectively. Using single-cell RNA-sequencing, we characterized gene expression signatures and driver signals of major cell types involved in vascular remodeling. Focused analysis revealed a novel sub-population of Selenbp1hi smooth muscle cells (SMCs) associated with vascular remodeling. Results of intercellular communication analyses predicted several ligand-receptor pairs between immune cells with SMCs and endothelial cells (ECs), implicating SMCs apoptosis and repair, ECs aging and inflammatory responses. CONCLUSIONS: We present a comprehensive single-cell atlas of vascular cells in early and late stages of ligated rat carotid artery, providing valuable insights into the understanding of the initiation and progression of vascular remodeling.

Prevalence and Vascular Distribution of Multiterritorial Atherosclerosis Among Community-Dwelling Adults in Southeast China.

Importance: Data are limited on the prevalence and vascular distribution of multiterritorial atherosclerotic plaque and stenosis in community populations. Objective: To investigate the prevalence and vascular distribution of multiterritorial atherosclerotic plaque and stenosis in older, community-dwelling populations in China. Design, Setting, and Participants: This cross-sectional study was based on the baseline survey from the Polyvascular Evaluation for Cognitive Impairment and Vascular Events (PRECISE) study, a population-based prospective cohort study that enrolled community-dwelling adults aged 50 to 75 years based on cluster sampling from 6 villages and 4 living communities of Lishui city in southeast China. Data were collected from May 2017 to September 2019 and analyzed from September to November 2021. Main Outcomes and Measures: Atherosclerotic plaque and stenosis at baseline were assessed in multiple vascular territories. Brain vessel wall magnetic resonance imaging (MRI) for intracranial and extracranial arteries; computed tomography angiography (CTA) for coronary, subclavian, aorta, renal, and iliofemoral arteries; and ankle-brachial index for peripheral arteries were performed at baseline survey. The extent of atherosclerosis was assessed according to the number of these 8 vascular sites affected, and polyvascular lesions were defined as at least 2 affected sites. Results: A total of 3433 of 4202 invited individuals consented to participate in the study. After excluding 366 participants with contraindications for MRI or CTA scanning, with life expectancies of 4 years of fewer, or with mental disease, a total of 3067 community-dwelling adults were enrolled. The mean (SD) age was 61.2 (6.7) years; 1640 (53.5%) were women, and 74 (2.4%) had prevalent ASCVD. Most participants (2870 [93.6%]) had atherosclerotic plaques in at least 1 vascular territory. Atherosclerotic plaques were mostly detected in the aorta (2419 [79.6%]) and iliofemoral arteries (2312 [75.8%]), followed by subclavian (1500 [49.8%]), coronary (1366 [44.9%]), extracranial (1110 [36.4%]), renal (873 [28.7%]), and intracranial (542 [17.7%]) arteries. A substantial proportion of participants (1180 [38.5%]) had arterial stenosis of 50% or greater, predominantly affecting the coronary (542 [17.8%]) and iliofemoral (527 [17.3%]) arteries. Polyvascular atherosclerotic plaque was observed in 2541 participants (82.8%), with 1436 (46.8%) with plaque affecting 4 or more vascular territories, and polyvascular stenosis was observed in 412 patients (13.4%). Conclusions and Relevance: In this study, atherosclerotic plaque was highly prevalent in the older community population in China, and a substantial proportion of individuals reach stenosis of 50% or greater.

circFAT1(e2) Inhibits Cell Apoptosis and Facilitates Progression in Vascular Smooth Muscle Cells through miR-298/MYB Axis.

Presently, as one of the three types of muscles in the human body, smooth muscle carries out many biological activities. Meanwhile, its abnormal development also leads to many diseases. Circular RNA, belonging to the noncoding RNA family, is demonstrated to function importantly in various diseases including smooth muscle. Here, we assumed circFAT1(e2) probably exhibited a primary role in vascular smooth muscle. Therefore, we conducted cell viability and cell apoptosis assay to validate the effects of circFAT1(e2) on vascular smooth muscle progression. Then, we supposed miR-298 was one target of circFAT1(e2) and executed corresponding experiments to test this hypothesis. Dual-luciferase reporter assay indicated miR-298 could bind to circFAT1(e2) and then modulated MYB level, thus regulating smooth muscle progression. Subsequently, based on the GSE41177 dataset, we identified 1982 differentially expressed genes (DEGs) in atrial fibrillation, and all DEGs were upregulated, including MYB. Finally, enrichment analysis of upregulated genes indicated that they were related to endodermal cell differentiation. The protein-protein interaction network revealed that EGFR, GNG2, and FPR2 were related to atrial fibrillation. In conclusion, our data find that circFAT1(e2) sponges miR-298 and then regulates MYB expression, thus affecting atrial fibrillation progression. Our findings provide a newly produced indicator and target for vascular smooth muscle diagnosis and treatment.

Estimated plasma volume status (ePVS) is a predictor for acute myocardial infarction in-hospital mortality: analysis based on MIMIC-III database.

BACKGROUND: Estimated plasma volume status (ePVS) has been reported that associated with poor prognosis in heart failure patients. However, no researchinvestigated the association of ePVS and prognosis in patients with acute myocardial infarction (AMI). Therefore, we aimed to determine the association between ePVS and in-hospital mortality in AMI patients. METHODS AND RESULTS: We extracted AMI patients data from MIMIC-III database. A generalized additive model and logistic regression model were used to demonstrate the association between ePVS levels and in-hospital mortality in AMI patients. Kaplan-Meier survival analysis was used to pooled the in-hospital mortality between the various group. ROC curve analysis were used to assessed the discrimination of ePVS for predicting in-hospital mortality. 1534 eligible subjects (1004 males and 530 females) with an average age of 67.36 ± 0.36 years old were included in our study finally. 136 patients (73 males and 63 females) died in hospital, with the prevalence of in-hospital mortality was 8.9%. The result of the Kaplan-Meier analysis showed that the high-ePVS group (ePVS ≥ 5.28 mL/g) had significant lower survival possibility in-hospital admission compared with the low-ePVS group (ePVS < 5.28 mL/g). In the unadjusted model, high-level of ePVS was associated with higher OR (1.09; 95% CI 1.06-1.12; P < 0.001) compared with low-level of ePVS. After adjusted the vital signs data, laboratory data, and treatment, high-level of ePVS were also associated with increased OR of in-hospital mortality, 1.06 (95% CI 1.03-1.09; P < 0.001), 1.05 (95% CI 1.01-1.08; P = 0.009), 1.04 (95% CI 1.01-1.07; P = 0.023), respectively. The ROC curve indicated that ePVS has acceptable discrimination for predicting in-hospital mortality. The AUC value was found to be 0.667 (95% CI 0.653-0.681). CONCLUSION: Higher ePVS values, calculated simply from Duarte's formula (based on hemoglobin/hematocrit) was associated with poor prognosis in AMI patients. EPVS is a predictor for predicting in-hospital mortality of AMI, and could help refine risk stratification.

Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization.

SHP2 mediates the activities of multiple receptor tyrosine kinase signaling and its function in endothelial processes has been explored extensively. However, genetic studies on the role of SHP2 in tumor angiogenesis have not been conducted. Here, we show that SHP2 is activated in tumor endothelia. Shp2 deletion and pharmacological inhibition reduce tumor growth and microvascular density in multiple mouse tumor models. Shp2 deletion also leads to tumor vascular normalization, indicated by increased pericyte coverage and vessel perfusion. SHP2 inefficiency impairs endothelial cell proliferation, migration, and tubulogenesis through downregulating the expression of proangiogenic SRY-Box transcription factor 7 (SOX7), whose re-expression restores endothelial function in SHP2-knockdown cells and tumor growth, angiogenesis, and vascular abnormalization in Shp2-deleted mice. SHP2 stabilizes apoptosis signal-regulating kinase 1 (ASK1), which regulates SOX7 expression mediated by c-Jun. Our studies suggest SHP2 in tumor associated endothelial cells is a promising anti-angiogenic target for cancer therapy.

Naringin Ameliorates Monocrotaline-Induced Pulmonary Arterial Hypertension Through Endothelial-To-Mesenchymal Transition Inhibition.

Pulmonary arterial hypertension (PAH) caused by enhanced arterial pressure increases vessel resistance in the lung. Endothelial-to-mesenchymal transition (EndMT) plays key roles in the vascular remodeling in PAH. Naringin, a protective gaseous mediator is commonly extracted from tomatoes and citrus fruits (such as grapefruits), and demonstrates anti-inflammation, anti-oxidant, anti-proliferation, and anti-tumor effects. Meanwhile, the association of Naringin and the process of EndMT is still unclear. In this study, monocrotaline (MCT) administration (60 mg/kg) was delivered for the induction of PAH in rats. Following this, Naringin (concentrations: 25, 50, and 100 mg/kg/day) was used for treatments. Human Umbilical Vein Endothelial Cells (HUVECs) were stimulated with Naringin and transforming growth factor ß1 (TGFß1, 10 ng/ml). As the result, Naringin was demonstrated to inhibit EndMT and alleviate PAH progression. In particular, in HUVECs, Naringin significantly suppressed the mesenchymal marker expression induced by TGFß1 treatment, enhanced the endothelial marker expression, and inhibited the activation of ERK and NF-κB signaling pathways. To conclude, this study provided novel evidence suggesting the beneficial effects of Naringin in PAH through the inhibition of the ERK and NF-κB signaling pathways and the EndMT progression in pulmonary arteries.

Sodium Selenite Attenuates Balloon Injury-Induced and Monocrotaline-Induced Vascular Remodeling in Rats.

Vascular remodeling (VR), induced by the massive proliferation and reduced apoptosis of vascular smooth muscle cells (VSMCs), is primarily responsible for many cardiovascular conditions, such as restenosis and pulmonary arterial hypertension. Sodium selenite (SSE) is an inorganic selenium, which can block proliferation and stimulate apoptosis of tumor cells; still, its protective effects on VR remains unknown. In this study, we established rat models with carotid artery balloon injury and monocrotaline induced pulmonary arterial hypertension and administered them SSE (0.25, 0.5, or 1 mg/kg/day) orally by feeding tube for 14 consecutive days. We found that SSE treatment greatly ameliorated the development of VR as evidenced by an improvement of its characteristic features, including elevation of the ratio of carotid artery intimal area to medial area, right ventricular hypertrophy, pulmonary arterial wall hypertrophy and right ventricular systolic pressure. Furthermore, PCNA and TUNEL staining of the arteries showed that SSE suppressed proliferation and enhanced apoptosis of VSMCs in both models. Compared with the untreated VR rats, lower expression of PCNA and CyclinD1, but higher levels of Cleaved Caspase-3 and Bax/Bcl-2 were observed in the SSE-treated rats. Moreover, the increased protein expression of MMP2, MMP9, p-AKT, p-ERK, p-GSK3ß and ß-catenin that occurred in the VR rats were significantly inhibited by SSE. Collectively, treatment with SSE remarkably attenuates the pathogenesis of VR, and this protection may be associated with the inhibition of AKT and ERK signaling and prevention of VSMC's dysfunction. Our study suggest that SSE is a potential agent for treatment of VR-related diseases.

The association of fracture risk in atrial fibrillation patients and long-term anticoagulant therapy category: a systematic review and meta-analysis.

OBJECTIVE: Our study aimed to assess the risk of all fractures and hip fractures in patients with atrial fibrillation (AF) who took non-vitamin K antagonist oral anticoagulants (NOACs) compared to warfarin. METHODS: We searched PubMed, Embase, and Cochrane Library and Clinical Trials.gov Website. Reviewed related researches up to January 31, 2020, to identify studies with more than 12 months of follow-up data. The protocol for this systematic review and meta-analysis has been registered in the International Prospective Register of Systematic Reviews (PROSPERO Number: CRD42020156893). RESULTS: We included five RCT studies, and five observational studies that contained a total of 326,846 patients in our meta-analysis. Our meta-analysis showed that patients taken NOACs had no significant all fracture risk (RR = 0.91, 95% CI [0.81-1.01]) and hip fracture risk (RR = 0.92, 95% CI [0.82-1.03]) compared with those taken warfarin. Subanalysis showed that the risk of all fractures and hip fractures treated by NOACs were significant lower compared with warfarin in observational studies compared with RCT studies. Also, a subanalysis across the duration of anticoagulation showed the NOACs users have lower all fracture risk than warfarin users when the duration of anticoagulation ≤2 years (RR = 0.89, 95% CI [0.80-0.99]). Further analysis, significant lower all fracture risk in the rivaroxaban therapy (RR = 0.81; 95% CI [0.76-0.86]) compared with warfarin but no statistical significance in hip fracture. There were no significant difference of all fracture risk and hip fracture risk in dabigatran, apixaban, and edoxaban therapy compared with warfarin. CONCLUSION: The meta-analysis demonstrated that NOACs associated with a significantly lower all fracture risk compared with warfarin when the duration of anticoagulation more than 2 years. Rivaroxaban users had lower risk of all fracture than warfarin users in AF patients. But there was no evidence to verify apixaban, edoxaban, and dabigatranin could decrease all fracture and hip fracture risk compared with warfarin.

Blood Urea Nitrogen and In-Hospital Mortality in Critically Ill Patients with Cardiogenic Shock: Analysis of the MIMIC-III Database.

The association between blood urea nitrogen (BUN) and prognosis has been the focus of recent research. Therefore, the objective of this study was to investigate the association between BUN and hospital mortality in critically ill patients with cardiogenic shock (CS). This was a retrospective cohort study, in which data were obtained from the Medical Information Mart for Intensive Care III V1.4 database. Data from 697 patients with CS were analyzed. Logistic regression and subgroup analyses were used to assess the association between BUN and hospital mortality in patients with CS. The average age of the 697 participants was 71.14 years, and approximately 42.18% were men. In the multivariate logistic regression model, after adjusting for age, sex, diabetes, cardiac arrhythmias, urine output, simplified acute physiology score II, sequential organ failure assessment, creatinine, anion gap, and heart rate, high BUN demonstrated strong associations with increased in-hospital mortality (per standard deviation increase: odds ratio [OR] 1.47, 95% confidence interval [CI] 1.13-1.92). A similar result was observed in BUN tertile groups (BUN 23-37 mg/dL versus 6-22 mg/dL: OR [95% CI], 1.42 [0.86-2.34]; BUN 38-165 mg/dL versus 6-22 mg/dL: OR [95% CI], 1.99 [1.10-3.62]; P trend 0.0272). Subgroup analysis did not reveal any significant interactions among various subgroups, and higher BUN was associated with adverse clinical outcomes in patients with CS.

PolyvasculaR Evaluation for Cognitive Impairment and vaScular Events (PRECISE)-a population-based prospective cohort study: rationale, design and baseline participant characteristics.

BACKGROUND AND PURPOSE: Cardiovascular diseases and dementia are two major diseases in the elderly. Atherosclerosis is associated with future vascular events and cognitive impairment. The PolyvasculaR Evaluation for Cognitive Impairment and vaScular Events (PRECISE) study is a population-based prospective cohort study with comprehensive evaluation of multiterritorial artery stenosis and plaque using advanced vascular imaging techniques and prospective collection of vascular events and cognitive assessments. METHODS: Between May 2017 and September 2019, the PRECISE study enrolled 3067 community-dwelling adults with ages between 50 and 75 years cluster sampled from six villages and four communities of Lishui city in China. Data are collected in face-to-face interviews at baseline, 2-year and 4-year follow-up visits. Brain MRI including high-resolution sequences for intracranial and carotidal arteries and CT angiography for thoracoabdominal arteries were performed at baseline and will be rescanned after 4 years. Cardiovascular/cerebrovascular events and cognitive assessment will be prospectively collected after the enrollment. Blood and urine samples were collected and biomarkers were tested at baseline. RESULTS: A total of 3067 subjects were enrolled, among which 53.5% were female with an average age of 61.2±6.7 years. Among them, 2.8%, 8.1%, 43.1% and 21.6% had a history of stroke, coronary heart diseases, hypertension and diabetes mellitus, respectively. CONCLUSIONS: The PRECISE study is a population-based prospective cohort study with comprehensive evaluation of atherosclerotic stenosis and plaque using advanced vascular imaging techniques. Data from this cohort provide us an opportunity to precisely evaluate polyvascular atherosclerosis and its association with future vascular events and cognitive impairment. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT03178448).

Correlation between Renal Artery Anatomy and Hypertension: A Retrospective Analysis of 3000 Patients.

OBJECTIVE: To assess the correlation between renal artery anatomy and blood pressure in Undiagnosed Hypertension and Diagnosed Hypertension. METHODS: The renal artery CT scanning imaging data and laboratory data of 3000 inpatients and outpatients were collected retrospectively in 4 centers of China. Morphometric parameters were assessed using the quantitative vascular analysis (unit: mM). RESULTS: 687 cases (23.2%) had accessory renal arteries unilaterally, and 216 cases (7.3%) had bilateral accessory renal arteries, including left kidney 825 (27.9%) and right kidney 798 (27.0%). The presence of accessory renal arteries and renal artery branches was higher in the diagnosed hypertension group as compared with the undiagnosed hypertension group (MARB, pp < 0.001; ARA, p < 0.001; others, p < 0.001). Consequently, multivariate regression analysis showed that age (OR = 2.519 (95% CI: 0.990-6.411, p < 0.001)), dyslipidemia (OR = 1.187 (95% CI: 0.960-1.454, p = 0.007)), renal hilum Outside the main renal artery branch (MRAB) (OR = 2.069 (95% CI: 1.614-2.524, p = 0.002)), and accessory renal artery (ARA) (OR = 2.071 (95% CI: 1.614-2.634, p = 0.001)) were risk factors of hypertension. In addition, higher renin activity was associated with ARA patients (2.19 ± 2.91 vs. 1.75 ± 2.85, p < 0.001). CONCLUSIONS: When comparing renal arteries side by side, the anatomical length of the renal arteries is significantly different. In addition, the prevalence of accessory renal arteries and renal artery branches is higher in the hypertension group. The auxiliary renal artery and the main renal artery branch outside the renal portal are independent factors of hypertension. Renal sympathetic nerve activity is affected by renin activity and is related to the accessory renal artery.

MiR-124-3p reduces angiotensin II-dependent hypertension by down-regulating EGR1.

Through previous literature studies, we found that miR-124-3p may be associated with hypertension. Therefore, we investigated the relationship between miR-124-3p and hypertension in Human Umbilical Vein Endothelial Cells (HUVECs) induced by angiotensin II (AngII). AngII-induced HUVECs model was constructed and the expression of miR-124-3p was detected by qRT-PCR. After transfected cells, apoptosis and ROS production were detected by flow cytometry, caspase-3 kit, and DCFH-DA staining. The target genes of miR-124-3p were predicted and verified by TargrtScan, Luciferase assay, qRT-PCR, and western blot. After silencing Early growth response factor 1 (siEGR1), its effects on apoptosis and ROS production were explored. Finally, the rescue experiments were conducted to explore the mechanism of miR-124-3p to reduce hypertension. MiR-124-3p was underexpressed in the cell model. In Ang II-induced HUVECs, the number of apoptosis increased, the content of caspase-3 was higher, and ROS production increased. However, these effects could be partially inhibited by miR-124-3p mimic. EGR1 was down-regulated by miR-124-3p, and siEGR1 was able to inhibit apoptosis and ROS production of cell model. In the final rescue experiments, miR-124-3p partially reversed the effect of Ang-II on the viability, migration, invasion and apoptosis and ROS production in HUVECs by down-regulating EGR1. MiR-124-3p inhibits Ang II-induced apoptosis and ROS production in HUVECs by down-regulating EGR1.

Icariin Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension via the Inhibition of TGF-ß1/Smads Pathway in Rats.

BACKGROUND: Pulmonary artery remodeling is important in the development of pulmonary artery hypertension. The TGF-ß1/Smads signaling pathway is activated in pulmonary arterial hypertension (PAH) in rats. Icariin (ICA) suppresses the TGF-ß1/Smad2 pathway in myocardial fibrosis in rats. Therefore, we investigated the role of icariin in PAH by inhibiting the TGF-ß1/Smads pathway. METHODS: Rats were randomly divided into control, monocrotaline (MCT), MCT + ICA-low, and MCT + ICA-high groups. MCT (60 mg/kg) was subcutaneously injected to induce PAH, and icariin (50 or 100 mg/kg.d) was orally administered for 2 weeks. At the end of the fourth week, right ventricular systolic pressure (RVSP) was obtained and the right ventricular hypertrophy index (RI) was determined as the ratio of the right ventricular weight to the left ventricular plus septal weight (RV/LV + S). Western blots were used to determine the expression of TGF-ß1, Smad2/3, P-Smad2/3, and matrix metalloproteinase-2 (MMP2) in lung tissues. RESULTS: Compared to the control group, RVSP and RI were increased in the MCT group (ρ < 0.05). Additionally, TGF-ß1, Smad2/3, P-Smad2/3, and MMP2 expressions were obviously increased (ρ < 0.01). Compared to the MCT group, RVSP and RI were decreased in the MCT + ICA group (ρ < 0.05). TGF-ß1, Smad2/3, P-Smad2/3, and MMP2 expressions were also inhibited in the icariin treatment groups (ρ < 0.05). Conclusions. Icariin may suppress MCT-induced PAH via the inhibition of the TGFß1-Smad2/3 pathway.

PDLIM5 inhibits STUB1-mediated degradation of SMAD3 and promotes the migration and invasion of lung cancer cells.

Transforming growth factor ß (TGFß) signaling plays an important role in regulating tumor malignancy, including in non-small cell lung cancer (NSCLC). The major biological responses of TGFß signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFß-SMAD signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDLIM5 (PDZ and LIM domain protein 5, ENH) critically promotes TGFß signaling by maintaining SMAD3 stability in NSCLC. First, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro and lung metastasis in vivo In addition, TGFß signaling and TGFß-induced epithelial-mesenchymal transition was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFß-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2 and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration, and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFß signaling and NSCLC progression.

Endothelial Scaffolding Protein ENH (Enigma Homolog Protein) Promotes PHLPP2 (Pleckstrin Homology Domain and Leucine-Rich Repeat Protein Phosphatase 2)-Mediated Dephosphorylation of AKT1 and eNOS (Endothelial NO Synthase) Promoting Vascular Remodeling.

OBJECTIVE: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. CONCLUSIONS: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.

Inhibitory effects of formononetin on the monocrotaline­induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a fatal syndrome resulting from enhanced pulmonary arterial pressure and pulmonary vessel resistance. Perivascular inflammation and extracellular matrix deposition are considered to be the crucial pathophysiologic bases of PAH. Formononetin (FMN), a natural phytoestrogen isolated from red clover (Trifolium pratense), has a variety of proapoptotic, anti­inflammatory and anti­tumor activities. However, the therapeutic effectiveness of FMN for PAH remains unclear. In the present study, 60 mg/kg monocrotaline (MCT) was first used to induce PAH in rats, and then all rats were treated with different concentrations of FMN (10, 30 and 60 mg/kg/day). At the end of this study, the hemodynamics and pulmonary vascular morphology of rats were evaluated. Specifically, matrix metalloproteinase (MMP)2, transforming growth factor ß1 (TGFß1) and MMP9 were measured using western blot and immunohistochemical staining. Collagen type I, collagen type III, fibronectin, monocyte chemotactic protein­1, tumor necrosis factor­α, interleukin­1ß, ERK and NF­κB were quantified using western blotting. The results demonstrated that FMN significantly alleviated the changes of hemodynamics and pulmonary vascular morphology, and decreased the MCT­induced upregulations of TGFß1, MMP2 and MMP9 expression levels. Meanwhile, the expression levels of collagen type I, collagen type III and fibronectin in rat lungs decreased after FMN treatment. Furthermore, the phosphorylated ERK and NF­κB also decreased after FMN treatment. Taken together, the present study indicated that FMN serves a therapeutic role in the MCT­induced PAH in rats via suppressing pulmonary vascular remodeling, which may be partially related to ERK and NF­κB signals.

Salvianolic acid a inhibits platelet activation and aggregation in patients with type 2 diabetes mellitus.

BACKGROUND: Platelets in patients with type 2 diabetes mellitus (DM2) are characterized by increased activation and aggregation, which tends to be associated with a high morbidity and mortality due to cardiovascular disease (CVD). Moreover, a large proportion of DM2 patients show an inadequate response to standard antiplatelet treatments, contributing to recurrent cardiovascular events. In our previous study, we indicated that Salvianolic acid A (SAA) presents an antiplatelet effect in healthy volunteers. However, whether it can inhibit "activated platelets" with a pathologic status has not been explored. Therefore, this study was designed to investigate the antiplatelet effect of SAA and its diabetic complication-related difference in DM2. METHODS: Forty patients diagnosed with DM2 from January 2018 to April 2018 were recruited. Fibrinogen-binding (PAC-1) and P-selectin (CD62p) flow cytometry reagents were measured under resting and stimulated conditions by flow cytometry, while agonist-induced platelet aggregation was conducted by light transmission aggregometry. Before all these measurements were conducted, all platelet samples were preincubated with a vehicle or SAA for 10 min. Additionally, the diabetic complication-related difference in the antiplatelet effect of SAA was further studied in enrolled patients. RESULTS: The expressions of PAC-1 and CD62p were elevated in DM2, as well as the maximal platelet aggregation. In addition, SAA decreased the expressions of PAC-1 and CD62p, which were enhanced by ADP and thrombin (all P < 0.01). It also reduced the platelet aggregation induced by ADP (P < 0.001) and thrombin (P < 0.05). Comparing the antiplatelet effect of SAA on DM2, with and without diabetic complications, no statistically significant difference was found (all P > 0.05). CONCLUSIONS: The present study demonstrated that SAA can inhibit platelet activation and aggregation in patients with DM2, and the inhibition did not abate for the existence of diabetic complications.

Association between Triglyceride-Glucose Index and Type 2 Diabetes Mellitus in the Japanese Population: A Secondary Analysis of a Retrospective Cohort Study.

Triglyceride-glucose index (TyG index) is associated with type 2 diabetes mellitus (T2DM), but research on this relationship is limited in Japan. The purpose of this study was to evaluate the correlation between TyG index and the risk of T2DM in the Japanese population. Here, 12732 participants were selected from the NAGALA study (NAfld in the Gifu Area, Longitudinal Analysis) conducted between 2004 and 2015 for a retrospective cohort analysis. The association between TyG index and T2DM was assessed using the Cox proportional-hazard model. Subgroup analyses were conducted according to age, sex, smoking status, alcohol consumption, waist circumference, BMI, and follow-up duration. The formula for TyG index was expressed as ln [fasting triglyceride level (mg/dL) × fasting plasma glucose level (mg/dL)/2]. After follow-up, 150 (1.18%) patients developed T2DM. After adjusting for potential confounders, a linear relationship was observed between TyG and the risk of T2DM. After adjusting for age, sex, BMI, waist circumference, HDL-cholesterol, total cholesterol, systolic blood pressure, regular exercise, smoking status, and alcohol consumption, TyG index, as a continuous variable, was associated with an increased risk of T2DM (adjusted hazard ratio (aHR), 1.79; 95% confidence interval (95% CI), 1.25-2.57). Compared with the first quartile of TyG index, subjects in the fourth quartile were 2.33-fold more likely to develop T2DM (aHR 2.33, 95% CI 1.09-4.96; P for trend 0.0224). Subgroup analyses showed that the association between TyG index and incident T2DM stably existed in different subgroups according to the variables tested. Therefore, TyG index was linearly related to the risk of incident T2DM in the Japanese population and may be used as a monitoring tool.

Cerebral microbleeds are associated with blood pressure levels in individuals with hypertension.

Objective: Cerebral microbleeds (CMBs), which appear as small dot-like hypointense lesions, are strongly associated with cerebrovascular disease. Recently, numerous investigations have suggested that hypertension and age are risk factors for CMBs; however, whether blood pressure grade and age rank are related to the severity of CMBs remains unclear. The purpose of this research was to assess the association between cerebral microbleeds and blood pressure levels.Methods: In total, 460 consecutive hypertension patients (214 males and 246 females; aged 44-96 years, mean age 60.95 ± 6.82 years) from Lishui Central Hospital were enrolled and classified as CMB or non-CMB patients according to magnetic resonance imaging (MRI). Gradient echo T2*-weighted MRI was used to detect CMBs. Differences in blood pressure, CMB severity, and other patient characteristics were compared between the two groups. Multifactorial logistic regression was used to analyze the correlation between blood pressure and microbleeds.Results: In our study, CMB lesions were identified in 123 patients (26.7%), including 39 patients with CMB lesions located deep in the brain. In the hypertensive population, smoking is an independent risk factor for CMBs. Additionally, systolic blood pressure (SBP), diastolic blood pressure (DBP) and age are also independent risk factors for CMBs. Furthermore, a modest correlation was noted between the number of microbleeds and grade of hypertension.Conclusions: This study provides novel evidence that microbleed severity is associated with hypertension grade. This conclusion emphasizes the importance of antihypertensive therapy in hypertension patients to avoid an increase in CMBs.

Formononetin attenuates monocrotaline­induced pulmonary arterial hypertension via inhibiting pulmonary vascular remodeling in rats.

Pulmonary arterial hypertension (PAH) is a life­threatening disease induced by the excessive proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Formononetin (FMN) is a natural isoflavone with numerous cardioprotective properties, which can inhibit the proliferation and induce the apoptosis of tumor cells; however, whether FMN has a therapeutic effect on PAH remains unclear. In the present study, PAH was induced in rats with monocrotaline (MCT, 60 mg/kg); rats were then administered FMN (10, 30 or 60 mg/kg/day). At the end of the experiment, hemodynamic changes, right ventricular hypertrophy and lung morphological characteristics were evaluated. α­smooth muscle actin (α­SMA), proliferating cell nuclear antigen (PCNA), and TUNEL were detected by immunohistochemical staining. The expression of PCNA, Bcl­2­associated X protein (Bax), Bcl­2 and, cleaved caspase­3, and activation of AKT and ERK were examined by western blot analysis. The results demonstrated that FMN significantly ameliorated the right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling induced by MCT. FMN also attenuated MCT­induced increased expression of α­SMA and PCNA. The ratio of Bax/Bcl­2 and cleaved caspase­3 expression increased in rat lung tissue in response to FMN treatment. Furthermore, reduced phosphorylation of AKT and ERK was also observed in FMN­treated rats. Therefore, FMN may provide protection against MCT­induced PAH by preventing pulmonary vascular remodeling, potentially by suppressing the PI3K/AKT and ERK pathways in rats.