Exogeneous metal ions as therapeutic agents in cardiovascular disease and their delivery strategies.

Metal ions participate in many metabolic processes in the human body, and their homeostasis is crucial for life. In cardiovascular diseases (CVDs), the equilibriums of metal ions are frequently interrupted, which are related to a variety of disturbances of physiological processes leading to abnormal cardiac functions. Exogenous supplement of metal ions has the potential to work as therapeutic strategies for the treatment of CVDs. Compared with other therapeutic drugs, metal ions possess broad availability, good stability and safety and diverse drug delivery strategies. The delivery strategies of metal ions are important to exert their therapeutic effects and reduce the potential toxic side effects for cardiovascular applications, which are also receiving increasing attention. Controllable local delivery strategies for metal ions based on various biomaterials are constantly being designed. In this review, we comprehensively summarized the positive roles of metal ions in the treatment of CVDs from three aspects: protecting cells from oxidative stress, inducing angiogenesis, and adjusting the functions of ion channels. In addition, we introduced the transferability of metal ions in vascular reconstruction and cardiac tissue repair, as well as the currently available engineered strategies for the precise delivery of metal ions, such as integrated with nanoparticles, hydrogels and scaffolds.

Voltage-dependent anion channel 1 (VDAC1) overexpression alleviates cardiac fibroblast activation in cardiac fibrosis via regulating fatty acid metabolism.

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix in the myocardium with cardiac fibroblast activation, leading to chronic cardiac remodeling and dysfunction. However, little is known about metabolic alterations in fibroblasts during cardiac fibrosis, and there is a lack of pharmaceutical treatments that target metabolic dysregulation. Here, we provided evidence that fatty acid ß-oxidation (FAO) dysregulation contributes to fibroblast activation and cardiac fibrosis. With transcriptome, metabolome, and functional assays, we demonstrated that FAO was downregulated during fibroblast activation and cardiac fibrosis, and that perturbation of FAO reversely affected the fibroblast-to-myofibroblast transition. The decrease in FAO may be attributed to reduced long-chain fatty acid (LCFA) uptake. Voltage-dependent anion channel 1 (VDAC1), the main gatekeeper of the outer mitochondrial membrane (OMM), serves as the transporter of LCFA into the mitochondria for further utilization and has been shown to be decreased in myofibroblasts. In vitro, the addition of exogenous VDAC1 was shown to ameliorate cardiac fibroblast activation initiated by transforming growth factor beta 1 (TGF-ß1) stimuli, and silencing of VDAC1 displayed the opposite effect. A mechanistic study revealed that VDAC1 exerts a protective effect by regulating LCFA uptake into the mitochondria, which is impaired by an inhibitor of carnitine palmitoyltransferase 1A. In vivo, AAV9-mediated overexpression of VDAC1 in myofibroblasts significantly alleviated transverse aortic constriction (TAC)-induced cardiac fibrosis and rescued cardiac function in mice. Finally, we treated mice with the VDAC1-derived R-Tf-D-LP4 peptide, and the results showed that R-Tf-D-LP4 prevented TAC-induced cardiac fibrosis and dysfunction in mice. In conclusion, this study provides evidence that VDAC1 maintains FAO metabolism in cardiac fibroblasts to repress fibroblast activation and cardiac fibrosis and suggests that the VDAC1 peptide is a promising drug for rescuing fibroblast metabolism and repressing cardiac fibrosis.

The long noncoding RNA THBS1-AS1 promotes cardiac fibroblast activation in cardiac fibrosis by regulating TGFBR1.

Cardiac fibrosis is associated with an adverse prognosis in cardiovascular disease that results in a decreased cardiac compliance and, ultimately, heart failure. Recent studies have identified the role of long noncoding RNA (lncRNA) in cardiac fibrosis. However, the functions of many lncRNAs in cardiac fibrosis remain to be characterized. Through a whole-transcriptome sequencing and bioinformatics analysis on a mouse model of pressure overload-induced cardiac fibrosis, we screened a key lncRNA termed thrombospondin 1 antisense 1 (THBS1-AS1), which was positively associated with cardiac fibrosis. In vitro functional studies demonstrated that the silencing of THBS1-AS1 ameliorated TGF-ß1 effects on cardiac fibroblast (CF) activation, and the overexpression of THBS1-AS1 displayed the opposite effect. A mechanistic study revealed that THBS1-AS1 could sponge miR-221/222 to regulate the expression of TGFBR1. Moreover, under TGF-ß1 stimulation, the forced expression of miR-221/222 or the knockdown TGFBR1 significantly reversed the THBS1-AS1 overexpression induced by further CF activation. In vivo, specific knockdown of THBS1-AS1 in activated CFs significantly alleviated transverse aorta constriction-induced (TAC-induced) cardiac fibrosis in mice. Finally, we demonstrated that the human THBS1-AS1 can also affect the activation of CFs by regulating TGFBR1. In conclusion, this study reveals that lncRNA THBS1-AS1 is a potentially novel regulator of cardiac fibrosis and may serve as a target for the treatment of cardiac fibrosis.

Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases.

Fibrotic cardiac diseases are characterized by myocardial fibrosis that results in maladaptive cardiac remodeling. Cardiac fibroblasts (CFs) are the main cell type responsible for fibrosis. In response to stress or injury, intrinsic CFs develop into myofibroblasts and produce excess extracellular matrix (ECM) proteins. Myofibroblasts are mechanosensitive cells that can detect changes in tissue stiffness and respond accordingly. Previous studies have revealed that some mechanical stimuli control fibroblast behaviors, including ECM formation, cell migration, and other phenotypic traits. Further, metabolic alteration is reported to regulate fibrotic signaling cascades, such as the transforming growth factor-ß pathway and ECM deposition. However, the relationship between metabolic changes and mechanical stress during fibroblast-to-myofibroblast transition remains unclear. This review aims to elaborate on the crosstalk between mechanical stress and metabolic changes during the pathological transition of cardiac fibroblasts.

Identification of circular RNAs in cardiac hypertrophy and cardiac fibrosis.

Cardiac hypertrophy initially serves as an adaptive response to physiological and pathological stimuli. Sustained hypertrophy progress to pathological cardiac hypertrophy, cardiac fibrosis and ultimately lead to heart failure, one of the leading medical causes of mortality worldwide. Intervention of pathological cardiac hypertrophy can effectively reduce the occurrence of heart failure. Abundant factors, such as adrenergic, angiotensin, and endothelin (ET-1) receptors, have been shown to participate in the regulation of pathological cardiac hypertrophy. Recently, an increasing number of studies have indicated that circRNA and circRNA-miRNA-mRNA network regulation is indispensable for the posttranscriptional regulation of mRNA in cardiac hypertrophy. In our study, the morphological, cardiac function and pathological changes during cardiac hypertrophy were investigated. RNA sequencing identified 93 circRNAs that were differentially expressed in the TAC_2w group, and 55 circRNAs in the TAC_4w group compared with the sham group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified several significant pathways, including hypertrophic cardiomyopathy, extracellular matrix (ECM)-receptor interaction and focal adhesion. Coexpression analyses were performed for differentially expressed circRNAs and differentially expressed mRNAs. Based on gene set enrichment analysis (GSEA), 8 circRNAs (mmu-Nfkb1_0001, mmu-Smad4_0007, mmu-Hecw2_0009, mmu-Itgbl1_0002, mmu-Lrrc2_0005, mmu-Cpeb3_0007, mmu-Ryr2_0040, and mmu-Rtn4_0001) involved in cardiac hypertrophy and cardiac fibrosis were identified. We validated some key circRNAs by qPCR. The crucial coexpression of circRNA-mRNA and its interaction with miRNA showed the possible mechanism of circRNAs in the process of cardiac dysfunction. Our results may provide promising targets for the treatment of pathological cardiac hypertrophy and fibrosis.

Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma.

Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

[The potential role of long non-coding RNA Dnm3os in the activation of cardiac fibroblasts].

Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the in vitro cardiac fibroblast activation model induced by transforming growth factor-ß1 (TGF-ß1). Quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, and collagen gel contraction test were used to identify the changes of activation phenotype and the expression of Dnm3os in cardiac fibroblasts. Small interfering RNA was used to silence Dnm3os to explore its role in the activation of cardiac fibroblasts. The results showed that the expression of Dnm3os was increased significantly in myocardial fibrotic tissues and in the activated cardiac fibroblasts. And the activation of cardiac fibroblasts could be alleviated by Dnm3os silencing. Furthermore, the TGF-ß1/Smad2/3 pathway was activated during the process of cardiac fibroblasts activation, while was inhibited after silencing Dnm3os. The results suggest that Dnm3os silencing may affect the process of cardiac fibroblast activation by inhibiting TGF-ß1/Smad2/3 signal pathway. Therefore, interfering with the expression of lncRNA Dnm3os may be a potential target for the treatment of cardiac fibrosis.

Purinergic P2 Receptors: Novel Mediators of Mechanotransduction.

Mechanosensing and mechanotransduction are vital processes in mechanobiology and play critical roles in regulating cellular behavior and fate. There is increasing evidence that purinergic P2 receptors, members of the purinergic family, play a crucial role in cellular mechanotransduction. Thus, information on the specific mechanism of P2 receptor-mediated mechanotransduction would be valuable. In this review, we focus on purinergic P2 receptor signaling pathways and describe in detail the interaction of P2 receptors with other mechanosensitive molecules, including transient receptor potential channels, integrins, caveolae-associated proteins and hemichannels. In addition, we review the activation of purinergic P2 receptors and the role of various P2 receptors in the regulation of various pathophysiological processes induced by mechanical stimuli.

P2Y1 Receptor Agonist Attenuates Cardiac Fibroblasts Activation Triggered by TGF-ß1.

Cardiac fibroblasts (CFs) activation is a hallmark feature of cardiac fibrosis caused by cardiac remodeling. The purinergic signaling molecules have been proven to participate in the activation of CFs. In this study, we explored the expression pattern of P2Y receptor family in the cardiac fibrosis mice model induced by the transverse aortic constriction (TAC) operation and in the activation of CFs triggered by transforming growth factor ß1 (TGF-ß1) stimulation. We then investigated the role of P2Y1receptor (P2Y1R) in activated CFs. The results showed that among P2Y family members, only P2Y1R was downregulated in the heart tissues of TAC mice. Consistent with our in vivo results, the level of P2Y1R was decreased in the activated CFs, when CFs were treated with TGF-ß1. Silencing P2Y1R expression with siP2Y1R accelerated the effects of TGF-ß1 on CFs activation. Moreover, the P2Y1R selective antagonist BPTU increased the levels of mRNA and protein of profibrogenic markers, such as connective tissue growth factor (CTGF), periostin (POSTN). periostin (POSTN), and α-smooth muscle actin(α-SMA). Further, MRS2365, the agonist of P2Y1R, ameliorated the activation of CFs and activated the p38 MAPK and ERK signaling pathways. In conclusion , our findings revealed that upregulating of P2Y1R may attenuate the abnormal activation of CFs via the p38 MAPK and ERK signaling pathway.

Inhibition of P2X7 Purinergic Receptor Ameliorates Cardiac Fibrosis by Suppressing NLRP3/IL-1ß Pathway.

P2X7 purinergic receptor (P2X7R) has been implicated in several cardiovascular diseases. However, whether it regulates cardiac fibrosis remains elusive. Herein, its involvement in the development of cardiac fibrosis was examined using a transverse aortic constriction (TAC) mice model and cardiac fibroblasts (CFs) hyperstimulated by TGF-ß1 for 48 hours. Results showed that TAC and TGF-ß1 treatment increased the expression of P2X7R. Silencing of P2X7R expression with siP2X7R ameliorated TGF-ß1 effects on fibroblasts activation. Similarly, P2X7R inhibition by Brilliant Blue G (BBG) reduced mRNA and protein levels of profibrosis markers, while the P2X7R agonist BzATP accelerated the TGF-ß1-induced CFs activation. Moreover, it was found that TGF-ß1-induced CFs activation was mediated by the NLRP3/IL-1ß inflammasome pathway. BBG or siP2X7R treatment suppressed NLRP3/IL-1ß pathway signaling. In vivo, BBG significantly alleviated TAC-induced cardiac fibrosis, cardiac dysfunction, and NLRP3/IL-1ß activation. Collectively, our findings imply that suppressing P2X7R may limit cardiac fibrosis and abnormal activation of CFs.

[The Observation of Liver and Kidney Injury and the Activation of Macrophages in the Overload Pressure Induced Cardiac Hypertrophy/Heart Failure Mouse Model].

OBJECTIVE: The purpose of this study is to investigate the injury of liver and kidney tissues in overload pressure induced cardiac hypertrophy/heart failure mice model and the changes of macrophage activation level. METHODS: 6-8 week-old C57BL/6 mice were subjected to transverse aortic constriction (TAC) surgery to establish the cardiac hypertrophy/heart failure mouse model induced by pressure overload, while the aortic was not ligated in the Sham group. At 4 weeks and 8 weeks after TAC, the mice of each group were subjected to echocardiography and blood collection. And mice were sacrificed to collect samples of the heart, liver, and kidney tissues. The contents of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and serum creatinine (Scr) in Sham group and two operation groups were determined. The histological changes of liver, heart and kidney tissues were observed by HE staining, and the expression of the marker of macrophage activation, F4/80 protein, was detected in the heart, liver and kidney tissue by immunohistochemical staining. RESULTS: Cardiac hypertrophy occurred at 4 weeks after TAC operation in C57BL/6 mice and developed into heart failure at 8 weeks after TAC. The echocardiography showed that, compared with the Sham group, the left ventricular end-diastolic posterior wall thickness (LVPWd) and the left ventricular internal diameter in diastole (LVIDd) were significantly increased, while the left ventricular ejection fraction (EF) and the left ventricular fractional shortening (FS) were significantly decreased ( P<0.05) in the 4-week-TAC group and 8-week-TAC group. The plasma content of ALT, AST, TBil and Scr in the 4-week-TAC group and 8-week-TAC group were significantly higher than those in the Sham group ( P<0.05). HE staining showed obvious liver pathological changes in TAC mice, such as vacuolation, mild hepatic sinusoid congestion and inflammatory infiltration in mice post 4 weeks after surgery, and such liver injury was worse in mice post 8 weeks after surgery. Besides, there was a slight damage in renal tissue shown by HE staining, such as slight glomerular injury and slight bleeding. F4/80 protein immunohistochemical staining results demonstrated that the activation of macrophages in the heart and liver in the 4-week-TAC group and 8-week-TAC group was significantly increased than that in the sham group ( P<0.05), but there was no significant difference in kidney tissues in groups. CONCLUSION: Macrophages are involved in the process of liver and kidney injury in cardiac hypertrophy/heart failure.

[The potential role of calnexin in the activation of cardiac fibroblasts].

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca 2+ homeostasis, and Ca 2+ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor ß1 (TGFß1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFß1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Mitochondrial ROS-Modulated mtDNA: A Potential Target for Cardiac Aging.

Mitochondrial DNA (mtDNA) damage is associated with the development of cardiovascular diseases. Cardiac aging plays a central role in cardiovascular diseases. There is accumulating evidence linking cardiac aging to mtDNA damage, including mtDNA mutation and decreased mtDNA copy number. Current wisdom indicates that mtDNA is susceptible to damage by mitochondrial reactive oxygen species (mtROS). This review presents the cellular and molecular mechanisms of cardiac aging, including autophagy, chronic inflammation, mtROS, and mtDNA damage, and the effects of mitochondrial biogenesis and oxidative stress on mtDNA. The importance of nucleoid-associated proteins (Pol γ), nuclear respiratory factors (NRF1 and NRF2), the cGAS-STING pathway, and the mitochondrial biogenesis pathway concerning the development of mtDNA damage during cardiac aging is discussed. Thus, the repair of damaged mtDNA provides a potential clinical target for preventing cardiac aging.

Mechanoresponse of stem cells for vascular repair.

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.

Enhanced anticancer effect of oncostatin M combined with salinomycin in CD133+ HepG2 liver cancer cells.

Oncostatin M (OSM) induces the differentiation of liver cancer stem cells (LCSCs) and increases sensitivity to the chemotherapeutic agent 5-fluorouracil, whereas salinomycin (Sal) induces apoptosis in cancer stem cells and inhibits the proliferation of liver cancer cells. However, there have been no studies investigating the anticancer effects of combination treatment with OSM and Sal. In the present study, we investigated the synergistic effects of OSM and Sal on LCSCs, the CD133+ subpopulations from HepG2 human liver cancer cells. CD133+ LCSCs were isolated using an immunomagnetic bead technique and identified through colony formation. After incubating with OSM and Sal, the ability of LCSC proliferation and invasion, as well as apoptosis rates were evaluated, and the expression of stemness-related genes was examined by quantitative real-time polymerase chain reaction. Additionally, the secretion of α-fetoprotein (AFP) and albumin (ALB) were analyzed by enzyme-linked immunosorbent assay. Our results indicated that OSM combined with Sal significantly suppressed LCSC proliferation and invasion and induced apoptosis, as determined by flow cytometry and increases in cleaved caspase-3 levels detected by western blotting. The results of the JC-1 staining assay indicated that this effect involved the mitochondrial pathway. Moreover, combination treatment reduced the expression of CD133 in LCSCs and suppressed stemness-related gene expression. Furthermore, the LCSCs produced lower levels of AFP and higher levels of ALB following combination treatment. In all experiments, combination treatment elicited more efficient anticancer effects on LCSCs as compared with single-drug treatment; therefore, our results demonstrated that combined treatment with OSM and Sal inhibited proliferation and induced differentiation and apoptosis in LCSCs, suggesting combined use of OSM and Sal as a therapeutic strategy for liver cancer.