Shiming Liu

Myocyte enhancer factor 2A (MEF2A), a transcription factor implicated in coronary artery disease, remains unexplored in vascular redox regulation. To address this gap and overcome the limitations of current antioxidant therapies, we investigate the role of MEF2A in oxidative defense via human umbilical vein endothelial cells (HUVECs) and murine models. Adenoviral vectors encoding MEF2A-specific shRNAs or mRNAs are used to silence or overexpress MEF2A in HUVECs. For in vivo validation, endothelial-targeted MEF2A knockdown is achieved via AAV1-shRNA delivery in mice fed a high-fat diet. Systemic redox status is assessed by measuring reactive oxygen species (ROS), glutathione homeostasis (GSH/GSSG ratio), the NADH/NAD⁺ balance, the mitochondrial membrane potential (ΔΨm), and 8-hydroxy-2′-deoxyguanosine (8-OHdG). Mechanistic insights are derived from immunofluorescence, qPCR, western blotting, and dual-luciferase reporter assays. MEF2A silencing induced redox imbalance, characterized by elevated ROS, a reduced GSH/GSSG ratio, and ΔΨm collapse. Conversely, MEF2A overexpression synergizes with SIRT1 to restore the glutathione pool, maintain NAD⁺ homeostasis, and suppress ROS under oxidative stress. Chromatin immunoprecipitation confirm that MEF2A directly binds to two cis-elements in the SIRT1 promoter, driving transcriptional activation. In vivo, MEF2A-deficient mice present increased vascular oxidative damage, as indicated by elevated DNA damage marker (8-OHdG) and ROS levels. The downregulation of SIRT1/PGC-1α in MEF2A-silenced cells is verified in vivo. Our findings establish MEF2A as a master regulator of endothelial redox defense via the SIRT1-PGC-1α axis, providing a mechanistic foundation for the treatment of oxidative cardiovascular disorders. This work suggests that pharmacological MEF2A activation is a novel strategy for precision antioxidant therapy in vascular medicine.

Endothelial dysfunction is a pivotal contributor to cardiovascular complications in individuals with diabetes. However, the precise role of macrophages and their exosomes in the diabetic milieu remains elusive. Exosomes (Exos) were isolated from the supernatants of macrophages treated with advanced glycation end products (AGE) or bovine serum albumin (BSA) using ultracentrifugation. Following coculture with AGE-Exos or BSA-Exos, human umbilical vein endothelial cells (HUVECs) were subjected to CCK-8, EdU, cell migration, monocyte adhesion, and tube formation assays. ELISA and Western blotting were employed to assess inflammatory cytokine release and protein expression levels in HUVECs. The miRNA expression profiles of AGE-Exos and BSA-Exos were analysed using miRNA arrays. Potential targets of miR-22-5p were predicted via miRNA databases and validated through RT‒qPCR, dual-luciferase reporter assays, and rescue experiments. Furthermore, a Rab27a knockout mouse model of type 2 diabetes mellitus (T2DM) was established by intraperitoneal injection of Streptozotocin. Aortic tissues were analysed via immunofluorescence for CD63 and CD31 expression, immunohistochemistry for VCAM-1 and ICAM-1 expression, and Western blotting for FOXP1 expression. AGE stimulation increased the secretion of exosomes from macrophages. Compared with BSA-Exos, AGE-Exos significantly impaired endothelial cell proliferation, migration, and tube formation capabilities while increasing monocyte adhesion and proinflammatory cytokine release without affecting cell viability. miR-22-5p was enriched in AGE-Exos, which were subsequently transferred to HUVECs, specifically targeting FOXP1, resulting in endothelial dysfunction. Overexpression of miR-22-5p in HUVECs using lentiviral vectors recapitulated the inflammatory effects observed with AGE-Exos, whereas anti-miR-22-5p conferred protective effects. Rab27a knockout significantly reduced exosome accumulation in T2DM model mouse aortic tissues, alleviating endothelial discontinuity, downregulating VCAM-1 and ICAM-1 expression, and upregulating FOXP1 expression. AGE-induced release of macrophage-derived exosomes may partially depend on Rab27a transport, which delivers miR-22-5p to ECs. This miR-22-5p targets FOXP1 in ECs, leading to inflammation and resulting in endothelial dysfunction that accelerates the development of diabetic vascular lesions.

Background Endothelial dysfunction is a pivotal contributor to cardiovascular complications in individuals with diabetes. However, the precise role of macrophages and their exosomes in the diabetic milieu remains elusive. Methods Exosomes (Exos) were isolated from the supernatants of macrophages treated with advanced glycation end products (AGE) or bovine serum albumin (BSA) using ultracentrifugation. Following coculture with AGE-Exos or BSA-Exos, human umbilical vein endothelial cells (HUVECs) were subjected to CCK-8, EdU, cell migration, monocyte adhesion, and tube formation assays. ELISA and Western blotting were employed to assess inflammatory cytokine release and protein expression levels in HUVECs. The miRNA expression profiles of AGE-Exos and BSA-Exos were analysed using miRNA arrays. Potential targets of miR-22-5p were predicted via miRNA databases and validated through RT‒qPCR, dual-luciferase reporter assays, and rescue experiments. Furthermore, a Rab27a knockout mouse model of type 2 diabetes mellitus (T2DM) was established. Aortic tissues were analysed via immunofluorescence for CD63 and CD31 expression, immunohistochemistry for VCAM-1 and ICAM-1 expression, and Western blotting for FOXP1 expression. Results AGE stimulation increased the secretion of exosomes from macrophages. Compared with BSA-Exos, AGE-Exos significantly impaired endothelial cell proliferation, migration, and tube formation capabilities while increasing monocyte adhesion and proinflammatory cytokine release without affecting cell viability. miR-22-5p was enriched in AGE-Exos, which were subsequently transferred to HUVECs, specifically targeting FOXP1, resulting in endothelial dysfunction. Overexpression of miR-22-5p in HUVECs using lentiviral vectors recapitulated the inflammatory effects observed with AGE-Exos, whereas anti-miR-22-5p conferred protective effects. Rab27a knockout significantly reduced exosome accumulation in T2DM model mouse aortic tissues, alleviating endothelial discontinuity, downregulating VCAM-1 and ICAM-1 expression, and upregulating FOXP1 expression. Conclusions AGE-induced release of macrophage-derived exosomes may partially depend on Rab27a transport, which delivers miR-22-5p to ECs. This miR-22-5p targets FOXP1 in ECs, leading to inflammation and resulting in endothelial dysfunction that accelerates the development of diabetic vascular lesions.

Heart regeneration was mainly achieved by intrinsic capacity. Exosomes are crucial in cardiovascular disease, yet their involvement in myocardial regeneration remains underexplored. To understand the role of cardiomyocyte‐derived exosomes (CM‐Exos) in heart regeneration. We established mouse models of myocardial infarction and apical resection in neonates to investigate the potential benefits of exosomes in response to injury. Rab27a knockout (KO) mice were constructed as an exosome decrease model. Distinct fibrosis appears in the infarcted and resection area in the KO mice 21 days after heart injury. The proliferation marker pH 3, Ki67, and Aurora B were detected 3 days after surgery, which decreased in KO mice compared to WT mice. Intravenous injection of CM‐Exos increased cardiomyocyte proliferation and partially restored heart function in KO mice. Rab27a knockdown in vitro reduced the expression of pH 3, Ki67, and Aurora B positive cardiomyocytes. However, the supplementation of CM‐Exos increased the proliferation of cardiomyocytes. Exosomal miRNA sequencing was subsequently applied, and miR‐21‐5p was a promising candidate that promoted cardiomyocyte proliferation through its target genes Spry‐1 and PDCD4. Intravenous injection of miR‐21‐5p exhibited similar proliferative effects as CM‐Exos. Our results indicate that CM‐Exos promotes cardiomyocyte cycle reentry by delivering miR‐21‐5p, highlighting the endogenous factors of myocardial regeneration.

The commercially available drug-eluting stent with limus (rapamycin, everolimus, etc.) or paclitaxel inhibits smooth muscle cell (SMC), reducing the in-stent restenosis, whereas damages endothelial cell (EC) and delays stent reendothelialization, increasing the risk of stent thrombosis (ST) and sudden cardiac death. Here we present a new strategy for promoting stent reendothelialization and preventing ST by exploring the application of precise molecular targets with EC specificity. Proteomics was used to investigate the molecular mechanism of EC injury caused by rapamycin. Endothelial protein C receptor (EPCR) was screened out as a crucial EC-specific effector. Limus and paclitaxel repressed the EPCR expression, while overexpression of EPCR protected EC from coating (eluting) drug-induced injury. Furthermore, the ligand activated protein C (APC), polypeptide TR47, and compound parmodulin 2, which activated the target EPCR, promoted EC functions and inhibited platelet or neutrophil adhesion, and enhanced rapamycin stent reendothelialization in the simulated stent environment and in vitro. In vivo, the APC/rapamycin-coating promoted reendothelialization rapidly and prevented ST more effectively than rapamycin-coating alone, in both traditional metal stents and biodegradable stents. Additionally, overexpression or activation of the target EPCR did not affect the cellular behavior of SMC or the inhibitory effect of rapamycin on SMC. In conclusion, EPCR is a promising therapeutical agonistic target for pro-reendothelialization and anti-thrombosis of eluting stent. Activation of EPCR protects against coating drugs-induced EC injury, inflammatory cell, or platelet adhesion onto the stent. The novel application formula for APC/rapamycin-combined eluting promotes stent reendothelialization and prevents ST.

Sex characteristics exhibit significant disparities in various human diseases, including prevalent cardiovascular diseases, cancers, metabolic disorders, autoimmune diseases, and neurodegenerative diseases. Risk profiles and pathological manifestations of these diseases exhibit notable variations between sexes. The underlying reasons for these sex disparities encompass multifactorial elements, such as physiology, genetics, and environment. Recent studies have shown that human body systems demonstrate sex-specific gene expression during critical developmental stages and gene editing processes. These genes, differentially expressed based on different sex, may be regulated by androgen or estrogen-responsive elements, thereby influencing the incidence and presentation of cardiovascular, oncological, metabolic, immune, and neurological diseases across sexes. However, despite the existence of sex differences in patients with human diseases, treatment guidelines predominantly rely on male data due to the underrepresentation of women in clinical trials. At present, there exists a substantial knowledge gap concerning sex-specific mechanisms and clinical treatments for diverse diseases. Therefore, this review aims to elucidate the advances of sex differences on human diseases by examining epidemiological factors, pathogenesis, and innovative progress of clinical treatments in accordance with the distinctive risk characteristics of each disease and provide a new theoretical and practical basis for further optimizing individualized treatment and improving patient prognosis.

Background: M1 macrophages are closely associated with cardiac injury after myocardial infarction (MI). Increasing evidence shows that exosomes play a key role in pathophysiological regulation after MI, but the role of M1 macrophage-derived exosomes (M1-Exos) in myocardial regeneration remains unclear. In this study, we explored the impact of M1 macrophage-derived exosomes on cardiomyocytes regeneration in vitro and in vivo. Methods: M0 macrophages were induced to differentiate into M1 macrophages with GM-CSF (50 ng/mL) and IFN-γ (20 ng/mL). Then M1-Exos were isolated and co-incubated with cardiomyocytes. Cardiomyocyte proliferation was detected by pH3 or ki67 staining. Quantitative real-time PCR (qPCR) was used to test the level of miR-155 in macrophages, macrophage-derived exosomes and exosome-treated cardiomyocytes. MI model was constructed and LV-miR-155 was injected around the infarct area, the proliferation of cardiomyocytes was counted by pH3 or ki67 staining. The downstream gene and pathway of miR-155 were predicted and verified by dual-luciferase reporter gene assay, qPCR and immunoblotting analysis. IL-6 (50 ng/mL) was added to cardiomyocytes transfected with miR-155 mimics, and the proliferation of cardiomyocytes was calculated by immunofluorescence. The protein expressions of IL-6R, p-JAK2 and p-STAT3 were detected by Western blot. Results: The results showed that M1-Exos suppressed cardiomyocytes proliferation. Meanwhile, miR-155 was highly expressed in M1-Exos and transferred to cardiomyocytes. miR-155 inhibited the proliferation of cardiomyocytes and antagonized the pro-proliferation effect of interleukin 6 (IL-6). Furthermore, miR-155 targeted gene IL-6 receptor (IL-6R) and inhibited the Janus kinase 2(JAK)/Signal transducer and activator of transcription (STAT3) signaling pathway. Conclusion: M1-Exos inhibited cardiomyocyte proliferation by delivering miR-155 and inhibiting the IL-6R/JAK/STAT3 signaling pathway. This study provided new insight and potential treatment strategy for the regulation of myocardial regeneration and cardiac repair by macrophages.

Ubiquitin-proteasome system (UPS) is involved in vascular smooth muscle cell (VSMC) proliferation. Deubiquitinating enzymes (DUBs) have an essential role in the UPS-regulated stability of the substrate; however, the function of DUBs in intimal hyperplasia remains unclear. We screened DUBs to identify a protein responsible for regulating VSMC proliferation and identified USP14 protein that mediates cancer development, inflammation, and foam cell formation. USP14 promotes human aortic smooth muscle cell and A7r5 cell growth in vitro, and its inhibition or deficiency decreases the intimal area in the mice carotid artery ligation model. In addition, USP14 stabilizes Skp2 expression by decreasing its degradation, while Skp2 overexpression rescues USP14 loss-induced issues. The current findings suggested an essential role of USP14 in the pathology of vascular remodeling, deeming it a promising target for arterial restenosis therapy.

Purpose: Inflammation associated endothelial cell (EC) dysfunction is key to atherosclerotic disease. Recent studies have demonstrated a protective role of amitriptyline in cardiomyocytes induced by hypoxia/reoxygenation. However, the mechanism by which amitriptyline regulates the inflammatory reaction in ECs remains unknown. Thus, the aim of this study was to investigate whether amitriptyline protects against inflammation in TNF-α-treated ECs. Methods: HUVECs were incubated with amitriptyline (2.5 μM) or TNF-α (20 ng/ml) for 24 h. EdU, tube formation, transwell, DHE fluorescence staining, and monocyte adhesion assays were performed to investigate endothelial function. Thoracic aortas were isolated from mice, and vascular tone was measured with a wire myograph system. The levels of ICAM-1, VCAM-1, MCP-1, phosphorylated MAPK and NF-κB were detected using western blotting. Results: Amitriptyline increased the phosphorylation of nitric oxide synthase (eNOS) and the release of NO. Amitriptyline significantly inhibited TNF-α-induced increases in ASMase activity and the release of ceramide and downregulated TNF-α-induced expression of proinflammatory proteins, including ICAM-1, VCAM-1, and MCP-1 in ECs, as well as the secretion of sICAM-1 and sVCAM-1. TNF-α treatment obviously increased monocyte adhesion and ROS production and impaired HUVEC proliferation, migration and tube formation, while amitriptyline rescued proliferation, migration, and tube formation and decreased monocyte adhesion and ROS production. Additionally, we demonstrated that amitriptyline suppressed TNF-α-induced MAPK phosphorylation as well as the activity of NF-κB in HUVECs. The results showed that the relaxation response of aortic rings to acetylcholine in the WT-TNF-α group was much lower than that in the WT group, and the sensitivity of aortic rings to acetylcholine in the WT-TNF-α group and WT-AMI-TNF-α group was significantly higher than that in the WT-TNF-α group. Conclusion: These results suggest that amitriptyline reduces endothelial inflammation, consequently improving vascular endothelial function. Thus, the identification of amitriptyline as a potential strategy to improve endothelial function is important for preventing vascular diseases.