Construction of a novel lower-extremity peripheral artery disease subtype prediction model using unsupervised machine learning and neutrophil-related biomarkers.

Lower-extremity peripheral artery disease (LE-PAD) is a prevalent circulatory disorder with risks of critical limb ischemia and amputation. This study aimed to develop a prediction model for a novel LE-PAD subtype to predict the severity of the disease and guide personalized interventions. Additionally, LE-PAD pathogenesis involves altered immune microenvironment, we examined the immune differences to elucidate LE-PAD pathogenesis. A total of 460 patients with LE-PAD were enrolled and clustered using unsupervised machine learning algorithms (UMLAs). Logistic regression analyses were performed to screen and identify predictive factors for the novel subtype of LE-PAD and a prediction model was built. We performed a comparative analysis regarding neutrophil levels in different subgroups of patients and an immune cell infiltration analysis to explore the associations between neutrophil levels and LE-PAD. Through hematoxylin and eosin (H&E) staining of lower-extremity arteries, neutrophil infiltration in patients with and without LE-PAD was compared. We found that UMLAs can helped in constructing a prediction model for patients with novel LE-PAD subtypes which enabled risk stratification for patients with LE-PAD using routinely available clinical data to assist clinical decision-making and improve personalized management for patients with LE-PAD. Additionally, the results indicated the critical role of neutrophil infiltration in LE-PAD pathogenesis.

Identification of Clinical Heterogeneity and Construction of Prediction Models for Novel Subtypes in Patients with Abdominal Aortic Aneurysm: An Unsupervised Machine Learning Study.

BACKGROUND: Abdominal aortic aneurysm (AAA) is one of the most common diseases in vascular surgery. Endovascular aneurysm repair (EVAR) can effectively treat AAA. It is essential to accurately classify patients with AAA who need EVAR. METHODS: We enrolled 266 patients with AAA who underwent EVAR. Unsupervised machine learning algorithms (UMLAs) were used to cluster subjects according to similar clinical characteristics. To verify UMLA's accuracy, the operative and postoperative results of the 2 clusters were analyzed. Finally, a prediction model was developed using binary logistic regression analysis. RESULTS: UMLAs could correctly classify patients based on their clinical characteristics. Patients in Cluster 1 were older, had a higher BMI, and were more likely than patients in Cluster 2 to develop pneumonia, chronic obstructive pulmonary disease, and cerebrovascular disease. The aneurysm diameter, neck angulation, diameter and angulation of bilateral common iliac arteries, and incidence of iliac artery aneurysm were significantly higher in cluster 1 patients than in cluster 2. Cluster 1 had a longer operative time, a longer length of stay in the intensive care unit and hospital, a higher medical expense, and a higher incidence of reintervention. A nomogram was established based on the BMI, neck angulation, left common iliac artery (LCIA) diameter and angulation, and right common iliac artery (RCIA) diameter and angulation. The nomogram was evaluated using receiver operating characteristic curve analysis, with an area under the curve of 0.933 (95% confidence interval, 0.902-0.963) and a C-index of 0.927. CONCLUSIONS: Our findings demonstrate that UMLAs can be used to rationally classify a heterogeneous cohort of patients with AAA effectively, and the analysis of postoperative variables also verified the accuracy of UMLAs. We established a prediction model for new subtypes of AAA, which can improve the quality of management of patients with AAA.

Immune-dysregulated neutrophils characterized by upregulation of CXCL1 may be a potential factor in the pathogenesis of abdominal aortic aneurysm and systemic lupus erythematosus.

Background: The abdominal aortic aneurysm (AAA) incidence is closely related to systemic lupus erythematosus (SLE). However, the common mechanisms between AAA and SLE are still unknown. The purpose of this research was to examine the main molecules and pathways involved in the immunization process that lead to the co-occurrence of AAA and SLE through the utilization of quantitative bioinformatics analysis of publicly available RNA sequencing databases. Moreover, routine blood test information was gathered from 460 patients to validate the findings. Materials and methods: Datasets of both AAA (GSE57691 and GSE205071) and SLE (GSE50772 and GSE154851) were downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were analyzed using bioinformatic tools. To determine the functions of the common differentially expressed genes (DEGs), Gene Ontology (GO) and Kyoto Encyclopedia analyses were conducted. Subsequently, the hub gene was identified through cytoHubba, and its validation was carried out in GSE47472 for AAA and GSE81622 for SLE. Immune cell infiltration analysis was performed to identify the key immune cells correlated with AAA and SLE, and to evaluate the correlation between key immune cells and the hub gene. Subsequently, the routine blood test data of 460 patients were collected, and the result of the immune cell infiltration analysis was further validated by univariate and multivariate logistic regression analysis. Results: A total of 25 common DEGs were obtained, and three genes were screened by cytoHubba algorithms. Upon validation of the datasets, CXCL1 emerged as the hub gene with strong predictive capabilities, as evidenced by an area under the curve (AUC) > 0.7 for both AAA and SLE. The infiltration of immune cells was also validated, revealing a significant upregulation of neutrophils in the AAA and SLE datasets, along with a correlation between neutrophil infiltration and CXCL1 upregulation. Clinical data analysis revealed a significant increase in neutrophils in both AAA and SLE patients (p < 0.05). Neutrophils were found to be an independent factor in the diagnosis of AAA and SLE, exhibiting good diagnostic accuracy with AUC >0.7. Conclusion: This study elucidates CXCL1 as a hub gene for the co-occurrence of AAA and SLE. Neutrophil infiltration plays a central role in the development of AAA and SLE and may serve to be a potential diagnostic and therapeutic target.

Curcumin attenuates vascular calcification via the exosomal miR-92b-3p/KLF4 axis.

Vascular calcification (VC) is the most widespread pathological change in diseases of the vascular system. However, we do not have a good understanding of the molecular mechanisms and effective therapeutic approaches for VC. Curcumin (CUR) is a natural polyphenolic compound that has hypolipidemic, anti-inflammatory, and antioxidant effects on the cardiovascular system. Exosomes are known to have extensive miRNAs for intercellular regulation. This study investigated whether CUR attenuates VC by affecting the secretion of exosomal miRNAs. Calcification models were established in vivo and in vitro using vitamin D3 and ß-glycerophosphate, respectively. Appropriate therapeutic concentrations of CUR were detected on vascular smooth muscle cells (VSMCs) using a cell counting kit 8. Exosomes were extracted by super speed centrifugation from the supernatant of cultured VSMCs and identified by transmission electron microscopy and particle size analysis. Functional and phenotypic experiments were performed in vitro to verify the effects of CUR and exosomes secreted by VSMCs treated with CUR on calcified VSMCs. Compared with the calcified control group, both CUR and exosomes secreted by VSMCs after CUR intervention attenuated calcification in VSMCs. Real-Time quantitative PCR (RT-qPCR) experiments showed that miR-92b-3p, which is important for alleviating VC, was expressed highly in both VSMCs and exosomes after CUR intervention. The mimic miR-92b-3p significantly decreased the expression of transcription factor KLF4 and osteogenic factor RUNX2 in VSMCs, while the inhibitor miR-92b-3p had the opposite effect. Based on bioinformatics databases and dual luciferase experiments, the prospective target of miR-92b-3p was determined to be KLF4. Both mRNA and protein of RUNX2 were decreased and increased in VSMCs by inhibiting and overexpressing of KLF4, respectively. In addition, in the rat calcification models, CUR attenuated vitamin D3-induced VC by increasing miR-92b-3p expression and decreasing KLF4 expression in the aorta. In conclusion, our study suggests that CUR attenuates vascular calcification via the exosomal miR-92b-3p/KLF4 axis.

The Cell Origin and Role of Osteoclastogenesis and Osteoblastogenesis in Vascular Calcification.

Arterial calcification refers to the abnormal deposition of calcium salts in the arterial wall, which results in vessel lumen stenosis and vascular remodeling. Studies increasingly show that arterial calcification is a cell mediated, reversible and active regulated process similar to physiological bone mineralization. The osteoblasts and chondrocytes-like cells are present in large numbers in the calcified lesions, and express osteogenic transcription factor and bone matrix proteins that are known to initiate and promote arterial calcification. In addition, osteoclast-like cells have also been detected in calcified arterial walls wherein they possibly inhibit vascular calcification, similar to the catabolic process of bone mineral resorption. Therefore, tilting the balance between osteoblast-like and osteoclast-like cells to the latter maybe a promising therapeutic strategy against vascular calcification. In this review, we have summarized the current findings on the origin and functions of osteoblast-like and osteoclast-like cells in the development and progression of vascular progression, and explored novel therapeutic possibilities.

The miR-29b/Matrix Metalloproteinase 2 Axis Regulates Transdifferentiation and Calcification of Vascular Smooth Muscle Cells in a Calcified Environment.

BACKGROUND: Vascular calcification (VC) is a common pathological lesion that promotes progress and mortality in cardiovascular disease. Vascular smooth muscle cells (VSMCs) acquiring an osteogenic phenotype facilitate VC occurrence and development. We recently reported that miR-29b-3p directly regulates the expression of matrix metalloproteinase 2 (MMP2). Herein, we test whether miR-29b-3p functions in the phenotypic transition and calcification in a calcified environment. METHODS AND RESULTS: VSMC calcification in vitro was induced with calcification medium containing ß-glycerophosphoric acid or high calcium. MiR-29b-3p expression in VSMCs tended to decrease during culturing in calcification medium. MiR-29b-3p overexpression ameliorated VSMC calcification, whereas miR-29b-3p knockdown exacerbated VSMC calcification. Furthermore, ectopic expression of miR-29b-3p inhibited the expression of osteogenic markers and MMP2 (a known target gene of miR-29b-3p). By contrast, miR-29b-3p deficiency facilitated VSMC osteogenesis differentiation and upregulated MMP2 expression. CONCLUSION: Our research suggests that miR-29b-3p regulates VSMC calcification and osteogenesis differentiation, at least in part, by targeting MMP2. Regulation of miR-29b-3p expression is therefore a potential therapeutic target for VSMC calcification.

Schisandrin B ameliorates high-glucose-induced vascular endothelial cells injury by regulating the Noxa/Hsp27/NF-κB signaling pathway.

BACKGROUND: To address the molecular mechanism of the anti-inflammation effects of schisandrin B (Sch B) in atherosclerosis, we examined injured HMEC-1, HBMEC, and HUVEC-12 cells induced by high glucose (HG). METHODS: Western blot was performed to detect the levels of the proteins Hsp27, Noxa, TLR5, p-IκBα, and p-p65 in HG-induced cells, while ELISA was used to analyze the inflammatory cytokines TNF-α, IL-6, MCP-1, and IL-1ß in cells with Hsp27 or Noxa stable expression. RESULTS: Overexpression of Hsp27 upregulated the inflammatory cytokines and the release of IκBα, promoted transportation of p65 into the nucleus, and lastly, affected the inflammation process, while Sch B counteracted the upregulation. In addition, the effect of Noxa overexpression, which is different from Hsp27 overexpression, was consistent with that of Sch B treatment. CONCLUSIONS: Sch B may inhibit the inflammatory cascade and alleviate the injury to HMEC-1, HBMEC, and HUEVC-12 cells caused by HG by regulating the Noxa/Hsp27/NF-κB signaling pathway.

The miR-182/SORT1 axis regulates vascular smooth muscle cell calcification in vitro and in vivo.

Arterial calcification is a common feature of cardiovascular disease. Sortilin is involved in the development of atherosclerosis, but the specific mechanism is unclear. In this study, we established calcification models in vivo and in vitro by using vitamin D3 and ß-glycerophosphate, respectively. In vivo, the expression of SORT1 was up-regulated and the expression of miR-182 was down-regulated in calcified arterial tissues. Meanwhile there was a negative correlation between SORT1 expression and miR-182 levels. In vitro, downregulating SORT1 expression using shRNA inhibited ß-glycerophosphoric induced vascular smooth muscle cells (VSMCs) calcification. Moreover, reduced sortilin levels followed transfection of miR-182 mimics, whereas there was a significant increase in sortilin levels after transfection of miR-182 inhibitors. A luciferase reporter assay confirmed that SORT1 is the direct target of miR-182. Our study suggests that SORT1 plays a vital role in the development of arterial calcification and is regulated by miR-182.

The Involvement of miR-29b-3p in Arterial Calcification by Targeting Matrix Metalloproteinase-2.

Vascular calcification is a risk predictor and common pathological change in cardiovascular diseases that are associated with elastin degradation and phenotypic transformation of vascular smooth muscle cells via gelatinase matrix metalloproteinase-2 (MMP2). However, the mechanisms involved in this process remain unclear. In this study, we investigated the relationships between miR-29b-3p and MMP2, to confirm miR-29b-3p-mediated MMP2 expression at the posttranscriptional level in arterial calcification. In male Sprague Dawley rats, arterial calcification was induced by subcutaneous injection of a toxic dose of cholecalciferol. In vivo, the quantitative real-time polymerase chain reaction (qRT-PCR) showed that MMP2 expression was upregulated in calcified arterial tissues, and miR-29b-3p expression was downregulated. There was a negative correlation between MMP2 mRNA expression and miR-29b-3p levels (P = 0.0014, R2 = 0.481). Western blotting showed that MMP2 expression was significantly increased in rats treated with cholecalciferol. In vitro, overexpression of miR-29b-3p led to decreased MMP2 expression in rat vascular smooth muscle cells, while downregulation of miR-29b-3p expression led to increased MMP2 expression. Moreover, the luciferase reporter assay confirmed that MMP2 is the direct target of miR-29b-3p. Together, our results demonstrated that a role of miR-29b-3p in vascular calcification involves targeting MMP2.