Platelet TGF-ß1 inhibits the migration and proliferation of smooth muscle cells in aneurysms.

BACKGROUND: The study explored the role of platelet TGF-ß1 from the perspective of inhibiting the excessive proliferation, migration and invasion of murine aortic vascular smooth muscle cells (MASMCs). METHOD: The platelets were first extracted from C57BL/6 mice, and the TGF-ß1 protein was obtained after the purification of protein. In vitro, the concentrations of angiotensin Ⅱ (Ang Ⅱ) and TGF-ß1 for intervention were screened by testing the viability of MASMCs, followed by the analysis concerning the effects of platelets, Ang Ⅱ and TGF-ß1 on the proliferation, migration, invasion, and the expressions of pathway-related proteins in MASMCs. In vivo, an Ang Ⅱ-induced mouse model was established. TGF-ß1 was injected into the tail of mice as a therapeutic agent, and its action mechanism was further verified by the treatment of inhibitor SB505124. The results of the cell experiment were validated by evaluating the maximum diameter of abdominal aorta, the proportion of total weight, the changes of both pathology and the expressions of pathway-related proteins in the mice. RESULT: 0.5 ng/mL Ang Ⅱ and 15 ng/mL TGF-ß1 were chosen for treatment. The following results of cell functional experiments and Western blot assay demonstrated that Ang Ⅱ promoted the proliferation, migration and invasion of MASMCs via regulating related pathways, the effects of which were evidently reversed by TGF-ß1 and platelets. Consistent results were also observed in the animal experiments, where TGF-ß1 effectively alleviated Ang Ⅱ-induced abdominal aortic injury in mice. CONCLUSION: TGF-ß1 in platelets inhibits Ang Ⅱ-induced proliferation, migration and invasion of MASMCs.

Sulfated Galactofucan from Sargassum Thunbergii Attenuates Atherosclerosis by Suppressing Inflammation Via the TLR4/MyD88/NF-κB Signaling Pathway.

PURPOSE: Sulfated galactofucan (SWZ-4), which was extracted from Sargassum thunbergii, has recently been reported to show anti-inflammatory and anticancer properties. The present study aimed to evaluate whether SWZ-4 attenuates atherosclerosis in apolipoprotein E-knockout (ApoE-KO) mice by suppressing the inflammatory response through the TLR4/MyD88/NF-κB signaling pathway. METHODS: Male ApoE-KO mice were fed with a high-fat diet for 16 weeks and intraperitoneally injected with SWZ-4. RAW246.7 cells were treated with lipopolysaccharide (LPS) and SWZ-4. Atherosclerotic lesions were measured by Sudan IV and oil red O staining. Serum lipid profiles, inflammatory cytokines, and mRNA and protein expression levels were evaluated. RESULTS: SWZ-4 decreased serum TNF-α, IL-6 and IL-1 levels, but did not reduce blood lipid profiles. SWZ-4 downregulated the mRNA and protein expression of TLR4 and MyD88, reduced the phosphorylation of p65, and attenuated atherosclerosis in the ApoE-KO mice (p < 0.01). In LPS-stimulated RAW 264.7 cells, SWZ-4 inhibited proinflammatory cytokine production and the mRNA expression of TLR4, MyD88, and p65 and reduced the protein expression of TLR4 and MyD88 and the phosphorylation of p65 (p < 0.01). CONCLUSION: These results suggest that SWZ-4 may exert an anti-inflammatory effect on ApoE-KO atherosclerotic mice by inhibiting the TLR4/MyD88/NF-κB signaling pathway in macrophages and therefore may be a treatment for atherosclerosis.

Single-Cell RNA Sequencing Technology Revealed the Pivotal Role of Fibroblast Heterogeneity in Angiotensin II-Induced Abdominal Aortic Aneurysms.

The mechanism of abdominal aortic aneurysm (AAA) has not been fully elucidated. In this study, we aimed to map the cellular heterogeneity, molecular alteration, and functional transformation of angiotensin (Ang) II-induced AAA in mice based on single-cell RNA sequencing (sc-RNA seq) technology. sc-RNA seq was performed on suprarenal abdominal aorta tissue from male Apoe-/- C57BL/6 mice of Ang II-induced AAA and shame models to determine the heterogeneity and phenotypic transformation of all cells. Immunohistochemistry was used to determine the pathophysiological characteristics of AAA. The single-cell trajectory was performed to predict the differentiation of fibroblasts. Finally ligand-receptor analysis was used to evaluate intercellular communication between fibroblasts and smooth muscle cells (SMCs). More than 27,000 cells were isolated and 25 clusters representing 8 types of cells were identified, including fibroblasts, macrophages, endothelial cells, SMCs, T lymphocytes, B lymphocytes, granulocytes, and natural killer cells. During AAA progression, the function and phenotype of different type cells altered separately, including activation of inflammatory cells, alternations of macrophage polarization, phenotypic transformation of vascular smooth muscle cells, and endothelial to mesenchymal transformation. The alterations of fibroblasts were the most conspicuous. Single-cell trajectory revealed the critical reprogramming genes of fibroblasts mainly enriched in regulation of immune system. Finally, the ligand-receptor analysis confirmed that disorder of collagen metabolism led by fibroblasts was one of the most prominent characteristics of Ang II-induced AAA. Our study revealed the cellular heterogeneity of Ang II-induced AAA. Fibroblasts may play a critical role in Ang II-induced AAA progression according to multiple biological functions, including immune regulation and extracellular matrix metabolic balance. Our study may provide us with a different perspective on the etiology and pathogenesis of AAA.

Platelet protects angiotensin II-driven abdominal aortic aneurysm formation through inhibition of inflammation.

BACKGROUND: Inflammation is the primary cause of abdominal aortic aneurysm (AAA) formation and development. It has been reported that platelets protect against septic shock by inhibiting inflammation. However, it is unclear whether platelets protect AAA progress via suppressing inflammation. METHODS: A mouse model of AAA was established by a daily administration of angiotensin II (Ang II, 1000 ng/kg/min) for 28-day. The AAA mice received 1 × 109 platelets transfusion in normal saline every 3rd day for 1 month. Hematoxylin and eosin, Masson's trichrome, and elastic van Gieson staining techniques were used to analyze the morphology of the abdominal aorta. Immunohistochemistry was used to detect any infiltration of inflammatory cells, inflammatory factors, and matrix metalloproteins (MMPs) in the aortic tissue. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to examine the inflammatory factor proteins levels in the aortic wall and peripheral blood, respectively. RESULTS: Platelets infusion significantly suppressed the Ang II-driven elevation of aortic diameter, AAA formation (52.38% decrease, P < 0.05), aortic expansion, elastic lamina destruction, and inflammatory response. In addition, MMP-2 and MMP-9 production were also reduced by platelets transfusion. CONCLUSIONS: For the first time, our study reported the beneficial effect of platelet transfusion in suppressing the Ang II-driven AAA progress in mice through the inhibition of inflammation.

The Association Between Extracellular Matrix Metalloproteinase Inducer Polymorphisms and Coronary Heart Disease: A Potential Way to Predict Disease.

Extracellular matrix metalloproteinase inducer (EMMPRIN) had been reported to be involved in the occurrence and development of coronary heart disease (CHD) in previous studies. This study aimed to investigate whether single nucleotide polymorphisms of EMMPRIN and matrix metalloproteinase-9 (MMP-9) contributed to the onset and severity of CHD. One thousand seventy patients suspected to have CHD were enrolled into the study. Each patient had undergone coronary angiogram, and the severity of coronary artery stenosis was assessed by Gensini score. Eight hundred twelve patients were confirmed to have CHD, while 258 patients were selected as non-CHD control. All patients were genotyped for five EMMPRIN polymorphisms (rs8259, rs28915400, rs4919859, rs6758, and rs8637) and one MMP-9 polymorphism (rs3918242) by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing. EMMPRIN polymorphism rs8259 and MMP-9 polymorphism rs3918242 were found to be associated with CHD (rs8259: AT vs. AA, adjusted odds ratio [OR] = 2.038, adjusted 95% confidence interval [CI] = 1.080-3.847, p = 0.028; rs3918242: CT vs. CC, adjusted OR = 0.607, adjusted 95% CI = 0.403-0.916, p = 0.017, TT vs. CC, adjusted OR = 2.559, adjusted 95% CI = 1.326-4.975, p = 0.006). No crossover effects were observed although a single environmental or genetic factor had an impact on the occurrence of CHD. The value of the Gensini score revealed that severity of CHD decreased in the rs3918242 CT carriers in both the male and female population. Our study suggested that EMMPRIN rs8259 and MMP-9 rs3918242 polymorphisms may contribute to pathological process of CHD. It could play a critical role in the prediction of CHD.

Effects of high glucose on proliferation and function of circulating fibrocytes: Involvement of CXCR4/SDF­1 axis.

The present study aimed to further investigate the effects of high glucose on the function of circulating fibrocytes and its underlying mechanisms. The total peripheral blood mononuclear cells were obtained from normal glucose tolerance patients and type 2 diabetic mellitus patients. Circulating fibrocytes were stimulated with different glucose concentrations for different time periods (24, 48 and 72 h). Cell proliferation was determined by Cell Counting Kit­8 assay. The expression of connective tissue growth factor (CTGF) was detected by western blotting. The expression of COL­I was detected by flow cytometry. The apoptotic bodies of cells were detected by fluorescence microscopy after Hoechst33258 staining. The invasive and migration abilities of fibrocytes were detected by Transwell chamber assay. Secretion of stromal cell­derived factor 1 (SDF­1) was measured by ELISA. The circulating fibrocytes showed a typical spindle­shape and were double­positive for cluster of differentiation 45 (green) and COL­I (red). Compared with the 5.5 mmol/l glucose group, a high glucose concentration significantly promoted the proliferation of circulating fibrocytes and showed the most significant effects at 30 mmol/l after treatment for 48 h. AMD3100 showed no effects on the proliferation of circulating fibrocytes. Flow cytometry revealed that 30 mmol/l glucose significantly promoted the expression of COL­I vs. 5.5 mmol/l glucose group (P<0.01), while AMD3100 reversed this (P<0.05). Hoechst33258 staining showed no differences in the apoptotic bodies between experimental groups (P>0.05). Western blotting revealed that the expression of CTGF was decreased significantly by AMD3100 pretreatment (P<0.01). Transwell chamber assay showed that 30 mmol/l glucose significantly promoted the invasive and transfer abilities (P<0.01) of fibrocytes when compared with the 5.5 mmol/l glucose group. While AMD3100 reversed the cell migratory effects induced by high glucose (P<0.01). In addition, the secretion of SDF­1 stimulated by 30 mmol/l glucose DMEM showed no differences compared with 5.5 mmol/l glucose DMEM (P>0.05). High glucose stimulated the expressions of CTGF and COL­I, and promoted migration of circulating fibrocytes via the CXC chemokine receptor 4/SDF­1 axis.

Isobaric tags for relative and absolute quantitation­based proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3 h.

Acute myocardial infarction (AMI) is one of the most common and life­threatening cardiovascular diseases. However, the ability to diagnose AMI within 3 h is currently lacking. The present study aimed to identify the differentially expressed proteins of AMI within 3 h and to investigate novel biomarkers using isobaric tags for relative and absolute quantitation (ITRAQ) technology. A total of 30 beagle dogs were used for establishing the MI models successfully by injecting thrombin powder and a polyethylene microsphere suspension. Serum samples were collected prior to (0 h) and following MI (1, 2 and 3 h). ITRAQ­coupled liquid chromatography­mass spectrometry (LC­MS) technology was used to identify the differentially expressed proteins. The bioinformatics analysis selected several key proteins in the initiation of MI. Further analysis was performed using STRING software. Finally, western blot analysis was used to evaluate the results obtained from ITRAQ. In total, 28 proteins were upregulated and 23 were downregulated in the 1 h/0 h group, 28 proteins were upregulated and 26 were downregulated in the 2 h/0 h group, and 24 proteins were upregulated and 19 were downregulated in the 3 h/0 h group. The Gene Ontology (GO) annotation and functional enrichment analysis identified 19 key proteins. Protein­protein interactions (PPIs) were investigated using the STRING database. GO enrichment analysis revealed that a number of key proteins, including ATP synthase F1 subunit ß (ATP5B), cytochrome c oxidase subunit 2 and cytochrome c, were components of the electron transport chain and were involved in energy metabolism. The western blot analysis demonstrated that the expression of ATP5B decreased significantly at all three time points (P<0.01), which was consistent with the ITRAQ results, whereas the expression of fibrinogen γ chain increased at 2 and 3 h (P<0.01) and the expression of integrator complex subunit 4 increased at all three time points (P<0.01), which differed from the ITRAQ results. According to the proteomics of the beagle dog MI model, ATP5B may serve as the potential biomarkers of AMI. Mitochondrial dysfunction and disruption of the electron transport chain may be critical indicators of early MI within 3 h. These finding may provide a novel direction for the diagnosis of AMI.