Exploring the mechanism of icariin in regulat⁃ing cardiac microvascular endothelial cells based on network pharmacology,molecular docking and in vitro experiments

OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro⁃vascular endothelial cells(CMEC)after oxygenglucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemiareperfusion model,and were divided into normal,model,low(10μmol·L^(-1)),medium(20μmol·L^(-1))and high(40μmol·L^(-1))ICA group,and high ICA+inhibitor group(40μmol·L^(-1)+20 nmol·L^(-1)).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col⁃lect ICA-related targets,the GeneCards data⁃base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar⁃get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft⁃ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec⁃tive effect at 10^(-1)60μmol·L^(-1);the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P<0.01,P<0.01);and the results of tube-for⁃mation assay showed that each group of ICA could significantly promote the generation of branches(P<0.01)and the capillary length exten⁃sion(P<0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar⁃gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich⁃ment analysis found 53 results,involving AGERAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu⁃lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra⁃tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha⁃nism of action of ICA against myocardial isch⁃emia-reperfusion injury may be related to the reg⁃ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.

Cardiac stem cells: Current knowledge and future prospects

Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs.Since the inception of the field several decades ago,regenerative medicine therapies,namely stem cells,have received significant attention in preclinical studies and clinical trials.Apart from their known potential for differentiation into the various body cells,stem cells enhance the organ's intrinsic regenerative capacity by altering its environment,whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration.Recently,research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells(CSCs/CPCs).The global burden of cardiovascular diseases’morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy.This review will discuss the nature of each of the CSCs/CPCs,their environment,their interplay with other cells,and their metabolism.In addition,important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells.Moreover,the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration.Finally,the novel role of nanotechnology in cardiac regeneration will be explored.

Effect of Hypoxia on Ca^(2+) Concentration in Broiler's Cardiac Muscle Cells

The purpose of this research was to study the effect of hypoxia on the Ca^2＋ concentration in broiler＇s cardiac muscle cells （CMCs）. The concentration of Ca^2＋ in the CMC was observed using a laser scanning confocal microscope （LSCM）. The results showed that hypoxia could significantly increase intracellular Ca^2＋（normal oxygen, 99.3 ＋_ 13.1; hypoxia, 129.4 ＋_ 24.3, P 〈 0.01） in CMCs. The Ca^2＋ antagonist （nifedipine, verapamil） could significantly restrain the Ca^2＋ influx across the cell membrane of CMC treated by hypoxia （CMC： hypoxia ＋ verapamil, 100.9± 28.2; hypoxia ＋ nifedipine, 107.6± 27.7; P 〈 0.01）. The results showed hypoxia could increase intracellular Ca^2＋ concentration of CMC, and the Ca^2＋ antagonist could restrain the Ca^2＋ influx across the cell membrane of CMC treated by hypoxia.

The Effect of Fructose-1,6-diphosphate and HTK Solution on Protecting Primary Cardiac Muscle Cells of Rat with Cold Preservation

In this study we tried to investigate the effect of fructose-1,6-diphosphate and HTK solution on protecting primary cardiac muscle cells of rat with cold preservation. The primary cardiac muscle cells of rat were cultured in vitro with four preservation solutions respectively: 0.9 % sodium chloride solution (group A), FDP (group B), HTK solution (group C) and a mixture of FDP and HTK solution (group D). The cells were preserved for 6, 8 and 10 h at 0-4 ℃. The values of AST and LDH-L and the Na+-K+ ATPase activity in cardiac muscle cells were detected, and the survival rate of cardiac muscle cells was detected with trypan blue staining. The values of AST and LDH-L in group C and group D were remarkable lower those in group A and group B (P<0.001), while the Na+-K+ ATPase activity and the survival rate of cells in group C and group D were much higher than those in group A and group B (P<0.001). The values of AST and LDH-L after 6 hours in group D decreased much more than those in group C (P<0.01), while the Na+-K+ ATPase activity and the survival rate of cells in group D improved more than those in group C (P<0.01). Both of the HTK solution and the mixture of HTK and FDP solution have an evident effect on protecting the primary cardiac muscle cells of rat in vitro with cold preservation, Compared with the HTK solution, the mixture solution has a better short-term protective effect.

Stem cell therapies in cardiac diseases: Current status and future possibilities

Cardiovascular diseases represent the world’s leading cause of death. In thisheterogeneous group of diseases, ischemic cardiomyopathies are the mostdevastating and prevalent, estimated to cause 17.9 million deaths per year.Despite all biomedical efforts, there are no effective treatments that can replacethe myocytes lost during an ischemic event or progression of the disease to heartfailure. In this context, cell therapy is an emerging therapeutic alternative to treatcardiovascular diseases by cell administration, aimed at cardiac regeneration andrepair. In this review, we will cover more than 30 years of cell therapy in cardiology,presenting the main milestones and drawbacks in the field and signalingfuture challenges and perspectives. The outcomes of cardiac cell therapies arediscussed in three distinct aspects: The search for remuscularization byreplacement of lost cells by exogenous adult cells, the endogenous stem cell era,which pursued the isolation of a progenitor with the ability to induce heart repair,and the utilization of pluripotent stem cells as a rich and reliable source ofcardiomyocytes. Acellular therapies using cell derivatives, such as microvesiclesand exosomes, are presented as a promising cell-free therapeutic alternative.

Rbfox1 controls alternative splicing of focal adhesion genes in cardiac muscle cells

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants.However,it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing.Hence,we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail.By using a combination of in silico target prediction and in-cell validation,we identified several focal adhesion proteins as alternative splicing targets of Rbfox1.We focused on the alternative splicing patterns of vinculin(metavinculin isoform)and paxillin(extended paxillin isoform)and identified both as potential Rbfox1 targets.Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns.Focal adhesions play an important role in the cardiac muscle context,since they mainly influence cell shape,cytoskeletal organization,and cell–matrix association.Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology,cytoskeletal organization,and multinuclearity after differentiation,which might be due to changes in alternative splicing of focal adhesion proteins.Hence,our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells,which might contribute to heart disease progression,where downregulation of Rbfox1 is frequently observed.

Diabetic cardiomyopathy:Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis

Recently,the roles of pyroptosis,a form of cell death induced by activated NODlike receptor protein 3(NLRP3)inflammasome,in the pathogenesis of diabetic cardiomyopathy(DCM)have been extensively investigated.However,most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models,and whether various medications and natural products prevent the development of DCM,associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis.The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies,with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors.We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link,given that several studies have provided both direct and indirect evidence under specific conditions.This editorial emphasizes that the current investigation should be circumspect in its conclusion,i.e.,not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models.Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM,targeting these biomarkers.

Transdifferentiation of Fetal Liver-delivered Mesenchymal Stem Cells into Cardiomyocyte-like Cells

Objectives To explore the possibility to induce mesenchymal stem cells from human fetal livers （FMSCs） to differentiate along cardiac lineage and the way to obtain high rate of differentiation. Methods Cells from passage 6-9 were plated at the density of 1.5 × 10^4/cm^2 and were treated with the combination of 5-azacytine（5-aza）, retinoitic acid（RA） and Dimethylsulfoxide （DMSO） in different doses when near confluence. 24 hours later, the treatment was removed by changing into normal medium without inducers. Different culture conditions were tried, including temperature, oxygen content and medium. Results When FMSCs were treated with highdose combination （ 5-aza 50 μM ＋RA 10-1 μM ＋ DMSO 1%） and modified combination（5-aza 50 μM ＋RA 10-3 μM ＋ DMSO 0.8 %） in cardiac differentiation medium （CDM）, at 37℃ and 20% 02, the cardiac differentiation was induced. When near confluence, cells became round and tended to gather together to form ball-like structures. 3 weeks after treatment, the cells were harvested and stained with anti-desmin and cardiac troponin I antibodies, and about 40% of the cells were positively stained. No beating cells observed during observation. Conclusions FMSCs cardiac have lineage the potential to differentiate along , and the stimulus for the cardiac differentiation is different from those for MSCs from different species.

EFFECTS OF EPCs OR b-FGF INTRAMYOCARDIAL INFUSION ON CARDIAC FUNCTION AND NEOVASCULARIZATION FOR DILATED CARDIOMYOPATHY RATS

Objective To compare the different effects of endothelia progenitor cells （ EPCs ） or basic fibroblast growth factor （b-FGF） intromyocardial infusion on cardiac function and neovascularization for dilated cardiomyopathy（ DCM） rats. Methods Fifty adult female rats received inguinal subcutaneous injections of isoproterenol （ISO, 250 mg/kg） for induction of DCM. Four weeks later, the model rats were randomly divided into EPCs group, b-FGF group and control group. The 2×106 EPCs （ resolved in 100 μL PBS） , 100 μL b-FGF （ lO0 μg/mL ） and 100 μL PBS were evenly transplanted into the myocardium of EPCs group, b-FGF group and control group, respectively. Three months later, echocardiographic examination and regional myocardial blood flow （RMBF） measurement were performed. EPCs were traced by fluorescence in situ hybridization （FISH）. The protein and mRNA expression of b-FGF in each group was measured by ELISA assay and reverse transcription-polymerase chain reaction （ RT-PCR ） . Results Three months after transplantation, sry positive cells were detected only in EPCs group. The cardiac function as well as RMBF was significantly improved in EPCs group compared with b-FGF group or control group. There was higher capillary density in EPCs group. The protein and mRNA expression of b-FGF was stronger than b-FGF group and control group. Conclusion Transplantation of EPCs can improve cardiac function, induce neovascularization and increase RMBF for DCM rats. The treatment with EPCs has better effect than administration of b-FGF alone.

Sanguinarine Attenuates Lipopolysaccharide-induced Inflammation and Apoptosis by Inhibiting the TLR4/NF-KB Pathway in H9c2 Cardiomyocytes

The inflammatory response is involved in the pathogenesis of the most common types of heart disease. Sanguinarine （SAN） has various pharmacological properties such as anti-inflammatory, antioxidant, antibacterial, antitumor, and immune-enhancing properties. However, few studies have investigated the effects of SAN on lipopolysaceharide （LPS）-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes. Therefore, in this study, H9c2 cells were co-treated with SAN and LPS, and the mRNA levels of pro-inflammation markers and the apoptosis rate were measured to clarify the effect of SAN on cardiac inflammation. The underlying mechanism was further investigated by detecting the activation of Toll-like receptor （TLR）4/nuclear faetor-κB （NF-κB） signaling pathways. As a result, increased mRNA expression of interleukin （IL）-1β, IL-6, and TNFα induced by LPS was attenuated after SAN treatment; LPS-induced apoptosis ofHge2 cardiomyocytes and cleaved-caspase 8, 9, 3 were all significantly reduced by SAN. Further experiments showed that the beneficial effect of SAN on blocking the inflammation and apoptosis of H9c2 cardiomyocytes induced by LPS was associated with suppression of the TLR4/NF-κB signaling pathway. It was suggested that SAN suppressed the LPS-induced inflammation and apoptosis of H9c2 cardiomyocytes, which may be mediated by inhibition of the TLR4/NF-κB signaling pathway. Thus, SAN may be a feasible therapy to treat sepsis patients with cardiac dysfunction.

Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells

Background： Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease.Although tongxinluo （TXL） has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-1 1 in hypoxia-stimulated human cardiac microvascular endothelial cells （HCMECs） have not been reported.Methods： In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL.First, the messenger RNA （mRNA） expression of claudin-5, claudin-9, and claudin-l 1 was confirmed.Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment.Furthermore, the distribution and content histone H3K9 acetylation （H3K9ac） in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.Results： We found that hypoxia-suppressed claudin-9 gene expression in HCMECs （F=7.244;P =0.011） and the hypoxia-suppressed claudin-9 could be reversed by TXL （F=61.911;P =0.000）, which was verified by its protein content changes （F=29.142;P =0.000）.Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury.Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter （F=37.766;P =0.000）, activating claudin-9 transcription.Conclusions： The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.

Gene Expression Profiling of the Proliferative Effect of Periplocin on Mouse Cardiac Microvascular Endothelial Cells

Objective： Periplocin is an active digitalis-like component from Cortex Periplocae, which has been widely used in the treatment of heart diseases in China for many years. According to the recommendations on the cardiovascular effect of periplocin from in vivo experiments, subsequent in vitro experiments are greatly needed for the global assessment of periplocin. The objective of this study is to investigate the cell proliferation effect and the mechanism of periplocin on endothelial cells. Methods： The proliferative activity of periplocin （0.4, 2, 10, 50, 250 pmol/L; 6, 12, 24, 48, 72 h） was investigated by a comparison with the well-reported cardiac glycoside, ouabain, on mouse cardiac microvascular endothelial cells （CMEC）. 3-（4,5-dimethylthiazolyl）- 2,5-diphenyltetrazolium bromide （MTT）, lactate dehydrogenase （LDH） and 5-bromo-2-deoxyuridine （BrdU） assays were used to evaluate cell proliferation and viability. Subsequently, cDNA microarray experiments were performed on periplocin- （50 pmol/L） and ouabain- （50 pmol/L） treated cells, and data was analyzed by ArrayTrack software. Results： Periplocin could increase cell viability to a level lower than ouabain in the MIF analysis, but decrease LDH release simultaneously. The BrdU incorporation assay showed an increase in cell proliferation with 2-50 μmol/L periplocin. Genes related to protein serine/threonine kinase were the most significantly enriched in the 160 genes identified in periplocin versus the control. In the 165 genes regulated by periplocin versus ouabain, GTP-binding was the most altered term. Conclusions： The results demonstrated the proliferation action of periplocin on CMEC. Meanwhile, its lower cytotoxicity compared to ouabain provides a new insight into the treatment of heart failure.

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart

Cannabinoid receptor type 2(CB2)activation is recently reported to promote proliferation of some types of resident stem cells(e.g.,hematopoietic stem/progenitor cell or neural progenitor cell).Resident cardiac progenitor cell(CPC)activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction(MI).This study aims to explore the role and possible mechanisms of CB2receptor activation in enhancing myocardial repair.Our results revealed that CB2receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation.AM1241 also decreased serum levels of MDA,TNF-αand IL-6 after MI.In addition,AM1241 can ameliorate left ventricular ejection fraction and fractional shortening,and reduce fibrosis.Moreover,AM1241 treatment markedly increased p-Akt and HO-1 expression,and promoted Nrf-2 nuclear translocation.However,PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241.In conclusion,AM1241 could induce myocardial regeneration and improve cardiac function,which might be associated with PI3K/Akt/Nrf2 signaling pathway activation.Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.

Dual human iPSC-derived cardiac lineage cell-seeding extracellular matrix patches promote regeneration and long-term repair of infarcted hearts

Human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) and cardiomyocytes (hCMs) possess therapeutic potential for infarcted hearts;however, their efficacy needs to be enhanced. Here we tested the hypotheses that the combination of decellularized porcine small intestinal submucosal extracellular matrix (SIS-ECM) with hCVPCs, hCMs, or dual of them (Mix, 1:1) could provide better therapeutic effects than the SIS alone, and dual hCVPCs with hCMs would exert synergic effects in cardiac repair. The data showed that the SIS patch well supported the growth of hCVPCs and hCMs. Epicardially implanted SIS-hCVPC, SIS-hCM, or SIS-Mix patches at 7-day post-myocardial infarction significantly ameliorated functional worsening, ventricular dilation and scar formation at 28- and 90-day post-implantation in C57/B6 mice, whereas the SIS only mildly improved function at 90-day post-implantation. Moreover, the SIS and SIS-cell patches improved vascularization and suppressed MI-induced cardiomyocyte hypertrophy and expression of Col1 and Col3, but only the SIS-hCM and the SIS-Mix patches increased the ratio of collagen III/I fibers in the infarcted hearts. Further, the SIS-cell patches stimulated cardiomyocyte proliferation via paracrine action. Notably, the SIS-Mix had better improvements in cardiac function and structure, engraftments, and cardiomyocyte proliferation. Proteomic analysis showed distinct biological functions of exclusive proteins secreted from hCVPCs and hCMs, and more exclusive proteins secreted from co-cultivated hCVPCs and hCMs than mono-cells involving in various functional processes essential for infarct repair. These findings are the first to demonstrate the efficacy and mechanisms of mono- and dual-hCVPC- and hCM-seeding SIS-ECM for repair of infarcted hearts based on the side-by-side comparison.

The therapeutic potential of stem cell therapy for myocardial infraction

It has been researched that myocardial infarction(MI)has drastically affected patients all over the world.The current guidelines of the medical treatments including PTCA or CABG just improve the condition and reduce damage to an extent.In the new studies and recent updates on myocardial stem cells,it has been researched that myocardial stem cells have regenerative capacity.Stem cell therapy used in cardiac disease management shows a promising and novel approach for cardiac tissues,cardiac muscle repair,and regeneration.Furthermore,it’s been observed that the stem cell-derived paracrine factors help in regulating and remodeling the coronary artery inflammation and cardiac tissue generation in the MI region.Here,we highlight recent findings and discuss how they use stem cell therapy during MI and heart disease.

Polymorphisms in tumor necrosis factor genes and susceptibility to esophageal squamous cell carcinoma and gastric cardiac adenocarcinoma in a population of high incidence region of North China

Background We investigated the possible association of the functional polymorphisms in the tumor necrosis factor （TNF） genes with susceptibility to esophageal squamous cell carcinoma （ESCC） and gastric cardiac adenocarcinoma （GCA）.Methods The TNF-α-308G/A and TNF-β ＋ 252G/A single nucleotide polymorphisms （SNPs） were genotyped using polymerase-chain reaction-restriction fragment length polymorphism （PCR-RFLP） analysis, in 555 cancer patients （291 ESCC and 264 GCA） and 437 healthy controls in a high incidence region of North China. Results Among healthy controls, frequencies of the TNF-α 1/1, 1/2 and 2/2 genotypes were 89.4% ,9.2% and 1.4% respectively, while frequencies of the TNF-β B1/B1, B1/B2 and B2/B2 genotypes were 12.6% , 32.3% and 55.1%, respectively. No significant difference was found in the overall genotype and allelotype distribution of the TNF-α-308G/A and TNF-β ＋ 252G/A SNPs among cancer patients and controls. However, both the B1/B1 genotype and B1/B2 genotype significantly increased the risk of developing ESCC [ the age and gender adjusted odds ratio （OR） =2.04 and 1.91, 95% confidence interval （CI） = 1.04 -4.43 and 1.14 - 2.60, respectively] and GCA （the age and gender adjusted OR =2. 68 and 2. 64, 95% CI = 1.14 -6.29 and 1.47 -4.72, respectively） in individuals with negative family history of UGIC, in comparison with the B2/B2 genotype. When the two TNF polymorphisms were combined and analyzed, individuals with the TNF-β B1/B2 and TNF-α1/2 or 2/2 genotypes significantly reduced the risk of developing ESCC and GCA, in comparison with those harboring the TNF-β B2/B2 and TNF-α 1/1 genotypes （ the age and gender adjusted OR = 0.37 and 0. 34, 95% CI =0. 15 -0.92 and 0. 13 -0.90, respectively）. Conclusions Therefore, the TNF-α -308G/A and TNF-β ＋ 252G/A genotyping may be used as a stratification markers to predicate the risk of ESCC and GCA development in North China.

Effects of hypoxia on promoter of telomerase reverse transcriptase and cell cycle distribution in neonatal rat cardiac myocytes

On the hypothesis that telomerase reverse transcriptase (TERT) of cardiac myocytes (CMs) is consistent with cell cycle distribution as well as tumour cells, we plan to investigate the expression of TERT in CMs and how TERT is in keeping with CMs cycle distribution after birth and under hypoxia, and roughly understand how hypoxia affects activity of TERT promoter.