Donor circadian clock influences the long-term survival of heart transplantation by immunoregulation.

AIMS: Circadian clocks play important role in immunoregulation. We aimed to investigate cardiac circadian clock specific pathways and compare cardiac grafts procured at different timing on survival after transplantation to explore novel criteria for donor selection. METHODS AND RESULTS: In primate heart, phase set enrichment analysis (PSEA) showed rhythmic transcripts were enriched in antigen processing and presentation during activation of circadian rhythm. Digital sorting of immune cell composition and single-sample gene set enrichment analysis (ssGSEA) in unused donor transcriptomes showed the pathway, positive regulation of circadian rhythm significantly correlates with allograft rejection and antigen presentation pathways as well as with increased compositions of matured dendritic cell, CD4+ T cell, and naive B cell. Single-centre retrospective cohort of 390 adult heart transplants between 1 January 2015 and 31 December 2020 was used to generate a propensity score matching (PSM) cohort. Survival curve differed significantly showing inferior long-term survival when donor hearts were procured at activation group (12 pm to 12 am) compared to repression group (12 am to 12 pm) (6-year survival: 64.2% vs. 75.8%, P = 0.0065). Activation group was also associated with significantly higher rates of in-hospital death, cardiopulmonary resuscitation, and usage of mechanical circulatory support after heart transplantation compared to repression group. Furthermore, tendency for post-transplant free of rejection rates was higher in repression group compared to activation group (acute rejection, Gehan-Breslow P = 0.11 and 0.04; chronic rejection, Log rank P = 0.077 and 0.15, in full and PSM cohorts, respectively). Adjusted Cox regression analysis showed that activation group was associated with 2.20 times increased hazard of death (hazard ratio: 2.20; 95% confidence interval: 1.23-3.95; P = 0.008) compared to repression group. CONCLUSIONS: Circadian immunity may represent donor-related risk factors for cardiac allograft rejection through activating genes related to antigen presentation pathway and immune cells oscillation at specific time of day. Molecular circadian clock should be considered during retrieval of cardiac allografts in order to maximize graft durability.

Platelet membrane-coated alterbrassicene A nanoparticle inhibits calcification of the aortic valve by suppressing phosphorylation P65 NF-κB.

Rationale: Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and morbidity with increasing prevalence and incidence. The pathobiology of CAVD involves valvular fibrocalcification, and osteogenic and fibrogenic activities are elevated in aortic valve interstitial cells (VICs) from diseased valves. It has been demonstrated that activated NF-κB pathway was present in the early stage of CAVD process. There is currently no effective clinical drugs targeting NF-κB pathway for CAVD treatment. Therefore, it is of great clinical significance to seek effective treatments for valve calcification. Methods: In this study, we established immortal human valve interstitial cells (im-hVICs) with pGMLV-SV40T-puro lentivirus. Alizarin red staining and western blotting were performed to evaluate the calcification of immortal VICs supplemented with different compounds. The natural fusicoccane diterpenoid alterbrassicene A (ABA) was found to have potential therapeutic functions. Ribonucleic acid sequencing was used to identify the potential target of ABA. Platelet membrane-coated nanoparticle of ABA (PNP-ABA) was fabricated and the IBIDI pump was used to evaluate the adhesion ability of PNP-ABA. Murine wire-induced aortic valve stenosis model was conducted for in vivo study of PNP-ABA. Results: The natural fusicoccane diterpenoid ABA was found to significantly reduce the calcification of human VICs during osteogenic induction via inhibiting the phosphorylation P65. Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein-2 (BMP2) were down regulated with the treatment of ABA in human VICs. Additionally, molecular docking results revealed that ABA bound to RelA (P65) protein. Phosphorylation of P65 (Ser536) was alleviated by ABA treatment, as well as the nuclear translocation of P65 during osteogenic induction in human VICs. Alizarin red staining showed that ABA inhibited osteogenic differentiation of VICs in a dose-dependent manner. PNP-ABA attenuated aortic valve calcification in murine wire-induced aortic valve stenosis model in vivo. Conclusions: The establishment of im-hVICs provides a convenient cell line for the study of CAVD. Moreover, our current research highlights a novel natural compound, ABA, as a promising candidate to prevent the progression of CAVD.

Natural Oncolysis of Enterovirus 71 in Antitumor Therapy of Colorectal Cancer.

Colorectal cancer (CRC) is an intestinal malignant tumor with high morbidity and mortality worldwide. Inoperability or resistanance to radiation and chemotherapy occur in the conventional treatments against CRC. Oncolytic viruses (OVs) are one kind of virus that selectively infects and lyses cancer cells, which is considered to be a new anticancer therapy with biological and immune-based approaches. Enterovirus 71 (EV71), belonging to the enterovirus genus in the family Picornaviridae, is a single positive-stranded RNA virus. EV71 is transmitted in a fetal-oral route and infects gastrointestinal tract in infants. Here, EV71 is exploited to be a novel oncolytic virus in colorectal cancer. It is revealed that EV71 infection can selectively cause colorectal cancer cells cytotoxicity but not primary intestinal epithelial cells. Consistently, EV71 injection significantly inhibits tumor growth in nude mice xenografted colorectal cancer cells. In detail, EV71 infects colorectal cancer cells to repress the expression of Ki67 and B-cell leukemia 2 (Bcl-2) leading to the inhibition of cell proliferation, while activating the cleavage of poly-adenosine diphosphatase-ribose polymerase and Caspase-3 protein resulting in the promotion of cell apoptosis. The findings demonstrate the oncolytic feature of EV71 in CRC treatment and may provide a potential clue for clinical anticancer therapy.

Indoloquinazoline alkaloids with cardiomyocyte protective activity against cold ischemic injury from Aspergillus clavatonanicus.

The arthropod-associated fungi have been demonstrated to be a remarkable producer of structurally captivating and bioactive secondary metabolites for drug discovery. In this study, eleven new indoloquinazoline alkaloids, namely aspergilloids A-K (1-11), along with five known congeners (12-16), were obtained from fungus Aspergillus clavatonanicus, which was isolated from the gut of a centipede collected in our Tongji campus. All these compounds were rarely defined by a 6/5/5 indolone ring system in conjugation with a five-membered spiral ring (1-5 and 10-16) or an opening five-membered spiral ring (6-9). Their structures were elucidated by widespread spectroscopic analyses, mainly including HRESIMS and 1D and 2D NMR data, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) data analyses. The cardiomyocyte protective activity assay revealed that compounds 1, 2, 5, 12-14, and 16 ameliorated cold ischemic injury at 48 h post cold ischemia (CI), and compounds 1, 5, and 14 prevented cold ischemia induced Ser9 dephosphorylation of GSK3ß at 12 h post CI. Our current study highlights indoloquinazoline alkaloids as the first class of natural cardiomyocyte protective agents against cold ischemic injury, which furnishes promising lead molecules for the development of new cardioprotectants in heart transplantation medicine.

Epicardial Adipose Tissue in Patients with Coronary Artery Disease: A Meta-Analysis.

Objective: The aim of this study is to assess the association between epicardial adipose tissue (EAT) and coronary artery disease (CAD) via meta−analysis. Methods: Specific searches of online databases from January 2000 to May 2022 were conducted. All observational studies evaluating the association between EAT and CAD in PubMed, Web of Science, and the Cochrane Library databases were screened. A meta-analysis was conducted following the Preferred Reporting Items for Systematic Reviews and Meta−Analyses guidelines (PRISMA). In total, 21 studies encompassing 4975 subjects met the inclusion criteria, including 2377 diagnosed and assigned as the CAD group, while the other 2598 were assigned as the non−CAD group. Subjects in the CAD group were further divided into the severe stenosis group (stenosis ≥ 50%, n = 846) and the mild/moderate stenosis group (stenosis < 50%, n = 577). Results: Both the volume and thickness of EAT in the CAD group were larger compared to the non−CAD group (p < 0.00001). In a subgroup analysis within the CAD group, the severe stenosis group had a larger volume and thickness with respect to EAT when compared to the mild/moderate group (p < 0.001). Conclusions: The enlargement of EAT presented in CAD patients with an association with CAD severity. Although limited by different CAD types and measuring methods for EAT, as well as a smaller sample size, our results suggest that EAT is a novel predictor and a potential therapeutic target for CAD.

Roles and potential clinical implications of tissue transglutaminase in cardiovascular diseases.

Cardiovascular disease (CVD)-related mortality and morbidity are among the most critical disease burdens worldwide. CVDs encompass many diseases and involve complex pathogenesis and pathological changes. While research on these diseases has advanced significantly, treatments and their efficacy remain rather limited. New therapeutic strategies and targets must, therefore, be explored. Tissue transglutaminase (TG2) is pivotal to the pathological development of CVDs, including participating in the cross-linking of extracellular proteins, activation of fibroblasts, hypertrophy and apoptosis of cardiomyocytes, proliferation and migration of smooth muscle cells (SMCs), and inflammatory reactions. Regulating TG2 activity and expression could ensure remarkable improvements in disorders like heart failure (HF), pulmonary hypertension (PH), hypertension, and coronary atherosclerosis. In this review, we summarize recent advances in TG2: we discuss its role and mechanisms in the progression of various CVDs and its potential as a diagnostic and therapeutic target.

Intracellular AGR2 transduces PGE2 stimuli to promote epithelial-mesenchymal transition and metastasis of colorectal cancer.

Human anterior gradient homolog 2 (AGR2) reportedly acts as an oncogene in multiple types of cancers. As a secreted protein, the oncogenic roles of extracellular AGR2 have been the focus of the increasing number of studies. In contrast, the oncological functions of intracellular AGR2 (iAGR2) remain elusive. Here, we report that intracellular AGR2 (iAGR2) is sufficient to promote CRC metastasis. iAGR2 binds to KDEL receptors (KDELRs) via its KTEL motif to activate downstream Gs-PKA signaling. Activated PKA upregulates the expression of NF-κB subunit c-Rel (REL) and acetylates histone H3 at lysine 9 (H3K9ac) to promote the transcription of SNAIL and SLUG. AGR2 can be upregulated by prostaglandin E2 (PGE2) via EP4-PI3K-AKT pathway and is indispensable for PGE2-induced CRC metastasis. AGR2 knockdown enhances therapeutic effects of a COX-2 inhibitor, celecoxib, in CRC metastasis. Collectively, our study reveals a promoting role and molecular mechanisms of iAGR2 in CRC metastasis and uncovers a new tumor microenvironment signal regulating AGR2 expression, which may provide new targets for treating metastatic CRC.

AGR3 promotes the stemness of colorectal cancer via modulating Wnt/ß-catenin signalling.

Cancer cells with stem cell properties have been acknowledged to be responsible for cancer initiation and progression. Wnt/ß-catenin signalling is a major signal pathway promoting the stemness of cancer cells. Anterior gradient 3 (AGR3), a member of the protein disulfide isomerase (PDI) family, was found to be overexpressed in several cancers. However, the roles and mechanisms of AGR3 in colorectal cancer (CRC) have not been previously described. In our study, we find that AGR3 is highly expressed in CRC and associated with poor prognosis. Functional studies show that AGR3 promotes the stemness of CRC cells. Mechanically, AGR3 activates Wnt/ß-catenin signalling and promotes the nuclear translocation of ß-catenin to upregulate stemness related genes. Wnt/ß-catenin signalling inhibition counteracts the promoting effect of AGR3 on cancer stemness. Moreover, the effect of AGR3 on Wnt/ß-catenin signalling and cancer stemness depends on the presence of frizzled 4 (FZD4). Thus, our study first uncovers the stemness-promoting role and the oncogenic mechanism of AGR3 in CRC, which might provide a novel target for designing anti-CRC strategies.

AGR2 is controlled by DNMT3a-centered signaling module and mediates tumor resistance to 5-Aza in colorectal cancer.

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is upregulated in various human cancers and reportedly has oncogenic activities. However, the functional roles of AGR2 and its regulation in colorectal cancer (CRC) remain unclear. Here, we showed that AGR2 promoted CRC tumorigenesis and progression in vitro and in vivo and acted as an independent prognostic factor of poor outcome. AGR2 was negatively regulated by DNA methyltransferase 3a (DNMT3a) through directly methylating AGR2 promoter and by a DNMT3a-SPRY2-miR-194 axis. Moreover, AGR2 mediated the resistance to 5-Aza-2'-deoxycytidine (5-Aza) treatment. Knockdown of AGR2 improved the therapeutic effect of 5-Aza in human CRC xenograft tumor model. Thus, our work supports AGR2's oncogenic role in CRC, reveals DNMT3a-mediated epigenetic modulation on AGR2 promoter, and uncovers a new DNMT3a signaling module controlling expression of AGR2. Upregulated AGR2 offset 5-Aza mediated epigenetic therapy. This work might provide potential targets for clinical anti-cancer therapy.

A novel mechanism of action for salidroside to alleviate diabetic albuminuria: effects on albumin transcytosis across glomerular endothelial cells.

Salidroside (SAL) is a phenylethanoid glycoside isolated from the medicinal plant Rhodiola rosea. R. rosea has been reported to have beneficial effects on diabetic nephropathy (DN) and high-glucose (HG)-induced mesangial cell proliferation. Given the importance of caveolin-1 (Cav-1) in transcytosis of albumin across the endothelial barrier, the present study was designed to elucidate whether SAL could inhibit Cav-1 phosphorylation and reduce the albumin transcytosis across glomerular endothelial cells (GECs) to alleviate diabetic albuminuria as well as to explore its upstream signaling pathway. To assess the therapeutic potential of SAL and the mechanisms involved in DN albuminuria, we orally administered SAL to db/db mice, and the effect of SAL on the albuminuria was measured. The albumin transcytosis across GECs was explored in a newly established in vitro cellular model. The ratio of albumin to creatinine was significantly reduced upon SAL treatment in db/db mice. SAL decreased the albumin transcytosis across GECs in both normoglycemic and hyperglycemic conditions. SAL reversed the HG-induced downregulation of AMP-activated protein kinase and upregulation of Src kinase and blocked the upregulation Cav-1 phosphorylation. Meanwhile, SAL decreased mitochondrial superoxide anion production and moderately depolarized mitochondrial membrane potential. We conclude that SAL exerts its proteinuria-alleviating effects by downregulation of Cav-1 phosphorylation and inhibition of albumin transcytosis across GECs. These studies provide the first evidence of interference with albumin transcytosis across GECs as a novel approach to the treatment of diabetic albuminuria.

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway.

Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE(-/-) male mice were fed a high-fat diet for 8weeks and then were administered with SAL for another 8weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on mitochondria, which played critical roles in improving endothelial function in atherosclerosis.

Salidroside ameliorates insulin resistance through activation of a mitochondria-associated AMPK/PI3K/Akt/GSK3ß pathway.

BACKGROUND AND PURPOSE: Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms. EXPERIMENTAL APPROACH: The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg(-1) ·day(-1)) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro. KEY RESULTS: Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3ß (GSK3ß) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio. CONCLUSIONS AND IMPLICATIONS: Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3ß pathway.

Salidroside stimulates mitochondrial biogenesis and protects against H2O2-induced endothelial dysfunction.

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2(∙-)) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF- κ B). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1 α ), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δ ψ m) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways.

TNF-α promotes early atherosclerosis by increasing transcytosis of LDL across endothelial cells: crosstalk between NF-κB and PPAR-γ.

Tumor necrosis factor-α (TNF-α) is an established pro-atherosclerotic factor, but the mechanism is not completely understood. We explored whether TNF-α could promote atherosclerosis by increasing the transcytosis of lipoproteins (e.g., LDL) across endothelial cells and how NF-κB and PPAR-γ were involved in this process. TNF-α significantly increased the transcytosis of LDL across human umbilical vein endothelial cells (HUVECs) and stimulated an increase of subendothelial retention of LDL in vascular walls. These effects of TNF-α were substantially blocked not only by transcytosis inhibitors, but also by NF-κB inhibitors and PPAR-γ inhibitors. In ApoE(-/-) mice, both NF-κB and PPAR-γ inhibitors alleviated the early atherosclerotic changes promoted by TNF-α. NF-κB and PPAR-γ inhibitors down-regulated the transcriptional activities of NF-κB and PPAR-γ induced by TNF-α. Furthermore, cross-binding activity assay revealed that NF-κB and PPAR-γ could form an active transcription factor complex containing both the NF-κB P65 subunit and PPAR-γ. The increased expressions of LDL transcytosis-related proteins (LDL receptor and caveolin-1, -2) stimulated by TNF-α were also blocked by both NF-κB inhibitors and PPAR-γ inhibitors. TNF-α promotes atherosclerosis by increasing the LDL transcytosis across endothelial cells and thereby facilitating LDL retention in vascular walls. In this process, NF-κB and PPAR-γ are activated coordinately to up-regulate the expression of transcytosis-related proteins. These observations suggest that inhibitors of either NF-κB or PPAR-γ can be used to target atherosclerosis.

C-reactive protein promotes atherosclerosis by increasing LDL transcytosis across endothelial cells.

BACKGROUND AND PURPOSE: The retention of plasma low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Whether or not C-reactive protein (CRP) can directly affect the transcytosis of LDL is not clear. Here we have examined the effect of CRP on transcytosis of LDL across endothelial cells and have explored the underlying mechanisms. EXPERIMENTAL APPROACH: Effects of CRP on transcytosis of FITC-labelled LDL were examined with human umbilical vein endothelial cells and venous rings in vitro and, in vivo, ApoE(-/-) mice. Laser scanning confocal microscopy, immunohistochemistry and Oil Red O staining were used to assay LDL. KEY RESULTS: CRP increased transcytosis of LDL. An NADPH oxidase inhibitor, diphenylene iodonium, and the reducing agent, dithiothreitol partly or completely blocked CRP-stimulated increase of LDL transcytosis. The PKC inhibitor, bisindolylmaleimide I and the Src kinase inhibitor, PP2, blocked the trafficking of the molecules responsible for transcytosis. Confocal imaging analysis revealed that CRP stimulated LDL uptake by endothelial cells and vessel walls. In ApoE(-/-) mice, CRP significantly promoted early changes of atherosclerosis, which were blocked by inhibitors of transcytosis. CONCLUSIONS AND IMPLICATIONS: CRP promoted atherosclerosis by directly increasing the transcytosis of LDL across endothelial cells and increasing LDL retention in vascular walls. These actions of CRP were associated with generation of reactive oxygen species, activation of PKC and Src, and translocation of caveolar or soluble forms of the N-ethylmaleimide-sensitive factor attachment protein.