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Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

A complete and prompt cardiac arrest using a cold cardioplegic solution is routinely used in heart transplantation to protect the graft function. However, warm ischemic time is still inevitable during the procedure to isolate donor hearts in the clinical setting. Our knowledge of the mechanism changes prevented by cold storage, and how warm ischemia damages donor hearts, is extremely poor. The potential consequences of this inevitable warm ischemic time to grafts, and the underlying potential protective mechanism of prompt graft cooling, have been studied in order to explore an advanced graft protection strategy. To this end, a surgical procedure, including 10–15 min warm ischemic time during procurement, was performed in mouse models to mimic the clinical situation (Group I), and compared to a group of mice that had the procurement performed with prompt cooling procedures (Group II). The myocardial morphologic changes (including ultrastructure) were then assessed by electron and optical microscopy after 6 h of cold preservation. Furthermore, syngeneic heart transplantation was performed after 6 h of cold preservation to measure the graft heart function. An electron microscopy showed extensive damage, including hypercontracted myofibers with contraction bands, and damaged mitochondria that released mitochondrial contents in Group I mice, while similar patterns of damage were not observed in the mice from Group II. The results from both the electron microscopy and immunoblotting verified that cardiac mitophagy (protective mitochondrial autophagy) was present in the mice from Group II, but was absent in the mice from Group I. Moreover, the mice from Group II demonstrated faster rebeating times and higher beating scores, as compared to the mice from Group I. The pressure catheter system results indicated that the graft heart function was significantly more improved in the mice from Group II than in those from Group I, as demonstrated by the left ventricle systolic pressure (31.96 ± 6.54 vs. 26.12 ± 8.87 mmHg), the +dp/dt (815.6 ± 215.4 vs. 693.9 ± 153.8 mmHg/s), and the -dp/dt: (492.4 ± 92.98 vs. 418.5 ± 118.9 mmHg/s). In conclusion, the warm ischemic time during the procedure impaired the graft function and destroyed the activation of mitophagy. Thus, appropriate mitophagy activation has emerged as a promising therapeutic target that may be essential for graft protection and functional improvement during heart transplantation.

CeO2-AgI, synthesized via depositing AgI nanoparticles onto CeO2 nanorods, was utilized for bacterial disinfection and organic contaminant degradation. Escherichia coli (E. coli) and Bisphenol A (BPA) were used as the model bacteria and emerging organic contaminant to test the photocatalytic activity of CeO2-AgI, respectively. Results showed that CeO2-AgI with the optimal AgI content exhibited superior photocatalytic activity over pure CeO2 or AgI for both inactivation of E. coli cells and BPA removal. However, the photocatalytic mechanisms for E. coli inactivation and BPA degradation were different. Specifically, the photo-generated holes (h+), photo-generated electrons (e−) and superoxide radicals ( O2−) were the dominated active species for E. coli inactivation, whereas, BPA degradation relied on the generation of O2− and e−. Cell membrane disruption was found to be the main disinfection mechanism. The decomposition of BPA was clarified by detecting the degradation intermediates by LC–MS and DFT calculation. The facile synthesized CeO2-AgI exhibited good photocatalytic stability in four reused cycles and thus could be potentially applied to purify water.

Heart transplantation continues to be the gold standard clinical intervention to treat patients with end-stage heart failure. However, there are major complications associated with this surgical procedure that reduce the survival prognosis of heart transplant patients, including allograft rejection, malignancies, infections, and other complications that arise from the use of broad-spectrum immunosuppression drugs. Recent studies have demonstrated the use of mesenchymal stem cells (MSCs) against allotransplantation rejection in both in vitro and in vivo settings due to their immunomodulatory properties. Therefore, utilization of MSCs provides new and exciting strategies to improve heart transplantation and potentially reduce the use of broad-spectrum immunosuppression drugs while alleviating allograft rejection. In this review, we will discuss the current research on the mechanisms of cardiac allograft rejection, the physiological and immunological characteristics of MSCs, the effects of MSCs on the immune system, and immunomodulation of heart transplantation by MSCs.

Carbamazepine (CBZ), a widely used antiepileptic drug, is refractory to biological wastewater treatment. Rapid removal of CBZ is possible using synthetic manganese oxide (δ-MnO2) but the removal mechanisms require further investigation. In this study, CBZ degradation by δ-MnO2 was carried out at different pH to further explore the degradation mechanisms. Results show that CBZ degradation by δ-MnO2 was highly pH dependent, and rapid degradation occurred when pH

Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system. They are derived from differentiation of oligodendrocyte progenitors through a process requiring cell cycle exit and histone modifications. Here we identify the histone arginine methyl-transferase PRMT5, a molecule catalyzing symmetric methylation of histone H4R3, as critical for developmental myelination. PRMT5 pharmacological inhibition, CRISPR/cas9 targeting, or genetic ablation decrease p53-dependent survival and impair differentiation without affecting proliferation. Conditional ablation of Prmt5 in progenitors results in hypomyelination, reduced survival and differentiation. Decreased histone H4R3 symmetric methylation is followed by increased nuclear acetylation of H4K5, and is rescued by pharmacological inhibition of histone acetyltransferases. Data obtained using purified histones further validate the results obtained in mice and in cultured oligodendrocyte progenitors. Together, these results identify PRMT5 as critical for oligodendrocyte differentiation and developmental myelination by modulating the cross-talk between histone arginine methylation and lysine acetylation., Myelin-forming cells derive from oligodendrocyte progenitors. Here the authors identify histone arginine methyl-transferase PRMT5 as critical for developmental myelination by modulating the cross-talk between histone arginine methylation and lysine acetylation, to favor differentiation.

BiOBr-AgBr hybrid photocatalysts were synthesized via a facile precipitation and in-situ ion exchange method at room temperature. The as-synthesized photocatalysts were well characterized with different techniques. The disinfection activities of BiOBr-AgBr towards model bacterial strain Gram-negative Escherichia coli (E. coli) were tested under both dark and visible light irradiation conditions. 1 × 107 CFU/mL of E. coli cells could be completely inactivated by BiOBr-AgBr with the optimal silver amount (BiOBr-0.5AgBr) in 24 min under visible light irradiation, while 1.5 log CFU/mL decrease of viable cell density was achieved at the same experiment duration under dark condition. Obviously, with the visible light irradiation, the bactericidal efficiency BiOBr-0.5AgBr was significantly enhanced. The subsequent disinfection mechanism analysis showed that Ag+ released from BiOBr-0.5AgBr did not contribute to E. coli inactivation, while reactive oxygen species including h+, surfaced bounded OH, O2−, and H2O2 were found to play important roles in the disinfection process. The presence of dissolved O2 and the direct contact of cells with photocatalysts was found to be essential for the disinfection process. The reactive species induced the destruction of cell membrane, thus led to the death of bacteria. In addition, BiOBr-0.5AgBr exhibited good bactericidal activity within a wide pH range (5–9) and contained effective antibacterial activity even in fifth consecutive reuse cycles.

In this study, we determined the mechanisms and kinetics of the degradations of ibuprofen (IBP) and sulfamethoxazole (SMX), and identified the active species contributions in ferrous ion (Fe(II))/free chlorine (FC) system. Reactive chlorine species (RCS) were the major contributor to the degradations of IBP (73.0%) and SMX (59.3%), respectively, at pH 3. Due to the low reaction rates between Fe(IV) and target pollutants (kFe(IV), IBP = (1.5 ± 0.03) × 103 M−1 s−1 and kFe(IV), SMX = (4.8 ± 0.2) × 103 M−1 s−1) and the low [Fe(IV)]ss ((5.0 ± 0.6) × 10−8 M), Fe(IV) was not the main contributor and only contributed 0.17% and 0.86% to the degradation of IBP and SMX, respectively, at pH 3. The degradations of pharmaceuticals were facilitated by acidic conditions. Chloride (Cl−) accelerated the degradation of SMX and had a weak effect on the degradation of IBP. Natural organic matter limited the degradation of IBP and SMX. Overall, we demonstrated that multiple active oxidants (Fe(IV) and RCS) are produced by Fe(II)/FC and elucidated the mechanism of active oxidants degradation of pollutants.

The interplay of transcription and epigenetic marks is essential for oligodendrocyte progenitor cell (OPC) proliferation and differentiation during development. Here, we review the recent advances in this field and highlight mechanisms of transcriptional repression and activation involved in OPC proliferation, differentiation and plasticity. We also describe how dysregulation of these epigenetic events may affect demyelinating disorders, and consider potential ways to manipulate NG2 cell behavior through modulation of the epigenome. This article is part of a Special Issue entitled SI:NG2-glia(Invited only).

Limited adult cardiac cell proliferation after cardiovascular disease, such as heart failure, hampers regeneration, resulting in a major loss of cardiomyocytes (CMs) at the site of injury. Recent studies in cellular reprogramming approaches have provided the opportunity to improve upon previous techniques used to regenerate damaged heart. Using these approaches, new CMs can be regenerated from differentiation of iPSCs (similar to embryonic stem cells), the direct reprogramming of fibroblasts [induced cardiomyocytes (iCMs)], or induced cardiac progenitors. Although these CMs have been shown to functionally repair infarcted heart, advancements in technology are still in the early stages of development in research laboratories. In this review, reprogramming-based approaches for generating CMs are briefly introduced and reviewed, and the challenges (including low efficiency, functional maturity, and safety issues) that hinder further translation of these approaches into a clinical setting are discussed. The creative and combined optimal methods to address these challenges are also summarized, with optimism that further investigation into tissue engineering, cardiac development signaling, and epigenetic mechanisms will help to establish methods that improve cell-reprogramming approaches for heart regeneration.

Depleted oxygen (O2) in the sediment and overlying water of malodorous black water poses a potential threat to aquatic ecosystems. This study presents a method for sustainable regulation of the dissolved oxygen (DO) levels towards the malodorous black water. Oxygen-loaded natural porous materials were prepared by vacuum degassing to remove air from the pores and fill them with pure O2. Capping anaerobic sediment with the prepared 6 oxygen-loaded porous materials was effective in prompting the DO concentration of the malodorous black water. Although granules activated carbon (GAC) displayed the highest oxygen-loading capability, oxygen-loaded volcanic stone additive was more efficient for long-lasting combating of the anaerobic condition because the DO level at sediment-water interface (SWI) and the DO penetration depth showed approximately 5.38- and 3.75-fold increase, respectively, compared with the untreated systems. The improvement in DO was substantially enhanced in the presence of submerged macrophyte (Vallisneria natans), during which the release of O2 from oxygen-loaded volcanic stone facilitated the plant growth. With the joint efforts of the O2 released from volcanic stone and photosynthesis by the macrophytes, the DO levels were maintained at approximately 6.80 mg/L after a 41-day incubation, which exceeded (P

Pluripotent stem cells (PSCs) are an attractive, reliable source for generating functional cardiomyocytes for regeneration of infarcted heart. However, inefficient cell engraftment into host tissue remains a notable challenge to therapeutic success due to mechanical damage or relatively inhospitable microenvironment. Evidence has shown that excessively formed scar tissues around cell delivery sites present as mechanical and biological barriers that inhibit migration and engraftment of implanted cells. In this review, we focus on the functional responses of stem cells and cardiomyocytes during the process of cardiac fibrosis and scar formation. Survival, migration, contraction, and coupling function of implanted cells may be affected by matrix remodeling, inflammatory factors, altered tissue stiffness, and presence of electroactive myofibroblasts in the fibrotic microenvironment. Although paracrine factors from implanted cells can improve cardiac fibrosis, the transient effect is insufficient for complete repair of an infarcted heart. Furthermore, investigation of interactions between implanted cells and fibroblasts including myofibroblasts helps the identification of new targets to optimize the host substrate environment for facilitating cell engraftment and functional integration. Several antifibrotic approaches, including the use of pharmacological agents, gene therapies, microRNAs, and modified biomaterials, can prevent progression of heart failure and have been developed as adjunct therapies for stem cell-based regeneration. Investigation and optimization of new biomaterials is also required to enhance cell engraftment of engineered cardiac tissue and move PSCs from a laboratory setting into translational medicine.

Citrullination, the deimination of arginine residues into citrulline, has been implicated in the aetiology of several diseases. In multiple sclerosis (MS), citrullination is thought to be a major driver of pathology, through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show citrullination by peptidylarginine deiminase 2 (PADI2) is required for normal oligodendrocyte differentiation, myelination and motor function. We identify several targets for PADI2, including myelin-related proteins and chromatin-associated proteins, implicating PADI2 in epigenetic regulation. Accordingly, we observe that PADI2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PADI2, display motor dysfunction and decreased number of myelinated axons in the corpus callosum. We conclude that citrullination is required for oligodendrocyte lineage progression and myelination and suggest its targeted activation in the oligodendrocyte lineage might be beneficial in the context of remyelination.

The AgI/AgBr/BiOBr0.75I0.25 nanocomposites were synthesized by a solvothermal process, followed by an in-situ ion exchange reaction. The disinfection activities of the as-synthesized photocatalyst to model cell type, Gram-negative Escherichia coli (E. coli), were investigated under visible light irradiation condition (λ≥400 nm). Results showed that 80 mg/L AgI/AgBr/BiOBr0.75I0.25 could completely inactivate 3×10(7) CFU mL(-1)E. coli cells within 30 min under visible light irradiation. Moreover, the bactericidal mechanisms involved in the photocatalytic disinfection process were systematically investigated. Ag(+) ions released from the nanocomposites negligibly contributed to the bactericidal activity, while active species including h(+), e(-) and ·O2(-) played important roles in the disinfection system. Direct contact of bacterial cells and nanoparticles was found to be the prerequisite for both the generation of ·O2(-) and the disinfection processes. The disruption of cell membrane and emission of cytoplasm directly inactivated E. coli cells. In addition, AgI/AgBr/BiOBr0.75I0.25 exhibited strong antibacterial activity toward E. coli even in four consecutive reused cycles.

The BiOI–AgI nanocomposites were synthesized by a solvothermal process, followed by an in-situ ion exchange reaction. The disinfection activities of BiOI–AgI on model cell type, Gram-negative Escherichia coli ( E. coli ), were examined under visible light irradiation conditions. BiOI–AgI could completely inactivate 5 × 10 7 CFU mL −1 E. coli cells in 18 min under visible light irradiation ( λ ⩾ 400 nm). The bactericidal mechanisms involved in this photocatalytic disinfection process were systematically investigated. Ag + ions released from the nanocomposites did not contribute to the bactericidal activity of BiOI–AgI. Active species including h + , O 2 − , e − , and H 2 O 2 generated by BiOI–AgI played important roles in the inactivation of bacteria. Direct contact of bacterial cells and nanoparticles was found to be essential for the disinfection processes. The destruction of cell membrane and emission of cytoplasm directly inactivated the cells. The influence of solution chemistry (pH and ionic strength) on disinfection process was also estimated. Low pH condition was found to be favorable for the inactivation process. High disinfection efficiencies were achieved at a wide range of solution ionic strength (0–1000 mM). The reusability of BiOI–AgI were also determined. BiOI–AgI exhibited strong antibacterial activity toward E. coli even in five consecutively reused cycles. This study indicated that the fabricated BiOI–AgI could be potentially utilized to disinfect bacteria in water (even with high salinity).

Au@TNBs nanocomposites were synthesized by depositing Au nanoparticles onto the surfaces of TiO2 nanobelts (TNBs). The disinfection activities of Au@TNBs on model cell type, Gram-negative Escherichia coli (E. coli), were examined under visible light irradiation conditions. Au@TNBs exhibited stronger bactericidal properties toward E. coli than those of TNBs and Au NPs under visible light irradiation. The bactericidal mechanisms of Au@TNBs under light conditions were explored, specifically, the specific active species controlling the inactivation of bacteria were determined. Active species (H2O2, diffusing ∙OH, ∙O2-, 1O2, and e-) generated by Au@TNBs were found to play important roles on the inactivation of bacteria. Moreover, the concentrations of H2O2, ·OH, ·O2-, and 1O2 generated in the antimicrobial system were estimated. Without the presence of active species, the direct contact of Au@TNBs with bacterial cells was found to have no bactericidal effect. The reusability of Au@TNBs were also determined. Au@TNBs exhibited strong antibacterial activity toward E. coli even in five consecutively reused cycles. This study indicated that the fabricated Au@TNBs could be potentially utilized to inactivate bacteria in water.

The chemokine (C-X-C motif) receptor 4 (CXCR4) is expressed on native cardiomyocytes and can modulate isolated cardiomyocyte contractility. This study examines the role of CXCR4 in cardiomyocyte response to ischaemia-reperfusion (I/R) injury. Isolated adult rat ventricular cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. In response to H/R injury, the decrease in CXCR4 expression was associated with dysfunctional energy metabolism indicated by an increased adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio. CXCR4-overexpressing cardiomyocytes were used to determine whether such overexpression (OE) can prevent bio-energetic disruption-associated cell death. CXCR4 OE was performed with adenoviral infection with CXCR4 encoding-gene or non-translated nucleotide sequence (Control). The increased CXCR4 expression was observed in cardiomyocytes post CXCR4-adenovirus transduction and this OE significantly reduced the cardiomyocyte contractility under basal conditions. Although the same extent of H/R-provoked cytosolic calcium overload was measured, the hydrogen peroxide-induced decay of mitochondrial membrane potential was suppressed in CXCR4 OE group compared with control group, and the mitochondrial swelling was significantly attenuated in CXCR4 group, implicating that CXCR4 OE prevents permeability transition pore opening exposure to overload calcium. Interestingly, this CXCR4-induced mitochondrial protective effect is associated with the enhanced signal transducer and activator of transcription 3 (expression in mitochondria. Consequently, in the presence of H/R, mitochondrial dysfunction was mitigated and cardiomyocyte death was decreased to 65% in the CXCR4 OE group as compared with the control group. I/R injury leads to the reduction in CXCR4 in cardiomyocytes associated with the dysfunctional energy metabolism, and CXCR4 OE can alleviate mitochondrial dysfunction to improve cardiomyocyte survival.

Aims: This study is designed to assess the protective cardiac effects after myocardial infarction (MI) of (i) cardiovascular progenitor cells (PC) differentiated directly into cardiomyocytes (CM) and endothelial cells (ECs) at the injury site, as separable from the effects of (ii) paracrine factors released from PC. Results: In vivo: bi-cell patch containing induced pluripotent stem cell (iPSC)-derived CM and EC (BIC) was transplanted onto the infarcted heart. BIC were transduced with herpes simplex virus thymidine kinase “suicide” gene driven by cardiac NCX1 or endothelial vascular endothelium-cadherin promoter. IGF-1α and VEGF levels released from ischemic tissues were significantly enhanced in the BIC patch treatment group. Heart function, infarction size, and vessel density were significantly improved after BIC patch treatment. These effects were completely abolished in the group given ganciclovir (GCV) at week 1 as a suicide gene activator, and partially abolished in the group given GCV at week 3 as compared with the untreated cell patch group. Innovation: This study was designed to distinguish between cell-based and noncell-based therapeutic effects of PC lineages after MI. PCs derived from iPSC were genetically modified to express “suicide” gene. iPSC-derived CM and EC were then ablated in situ at week 1 and 3 by intraperitoneal administration of GCV. This enabled direct assessment of the effects of iPSC transplantation on myocardial function and tissue regeneration potential. Conclusions: Data support a mechanism in which iPSC-derived cardiovascular lineages contribute directly to improved cardiac performance and attenuated remodeling. Paracrine factors provide additional support to the restoration of heart function. Antioxid. Redox Signal. 21, 2177–2191.

Electrolysis and permanganate (PM) oxidation are two commonly used technologies for water treatment. However, they are often handicapped by their slow reaction rates. To improve the removal efficiency of refractory contaminants, we combined electrolysis with PM using an activated carbon fiber (ACF) as cathode (E-ACF-PM) for the first time to treat diclofenac (DCF) in aqueous solution. Up to 90% DCF was removed in 5 min by E-ACF-PM process. In comparison, only 3.95 and 27.35% of DCF was removed by individual electrolysis and PM oxidation at the same time, respectively. Acidic condition was more conducive to DCF removal. Surprisingly, soluble Mn(III) (aq) formed on the surface of ACF was demonstrated as the principal oxidizing agent in E-ACF-PM process. Further studies showed that all three components (electrolysis + ACF + PM) were necessary to facilitate the heterogeneous generation of reactive Mn(III) (aq). Moreover, SEM images and XPS spectra of ACF before and after treatment revealed that the morphologies and elemental compositions of reacted ACF were nearly unchanged during the E-ACF-PM process. ACF can be remained active and utilized to the rapid degradation of DCF in E-ACF-PM process even after reused for 20 times. Therefore, the E-ACF-PM process may provide a novel and effective alternative on the generation of reactive Mn(III) (aq) in situ for water treatment by green electrochemical reactions.

Citrullination, the deimination of peptidylarginine residues into peptidylcitrulline, has been implicated in the etiology of several diseases. In multiple sclerosis, citrullination is thought to be a major driver of pathology through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show that citrullination by peptidylarginine deiminase 2 (PAD2) contributes to normal oligodendrocyte differentiation, myelination, and motor function. We identify several targets for PAD2, including myelin and chromatin-related proteins, implicating PAD2 in epigenomic regulation. Accordingly, we observe that PAD2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PAD2 display motor dysfunction and a decreased number of myelinated axons in the corpus callosum. We conclude that citrullination contributes to proper oligodendrocyte lineage progression and myelination.

Selenium (Se) could enter the environment through different anthropogenic sources, posing potential health risk. The removal of trace Se(IV) from water by hematite coated magnetic nanoparticle (MNP@hematite) under different solution conditions was investigated through batch experiments. The adsorption capacity of Se(IV) by MNP@hematite was 25.0 mg/g.100 μg/L of Se(IV) could be rapidly decreased to below 10 μg/L by 0.1 g/L of MNP@hematite in 10 min. MNP@hematite could effectively remove Se(IV) in a wide pH range from 4 to 9. Se(IV) could form inner-sphere complex with MNP@hematite. Thus, the presence of chloride, nitrate, and sulfate (concentration of each anions

It has been reported that CXCR4-overexpressing mesenchymal stem cells (MSCCX4 ) can repair heart tissue post myocardial infarction. This study aims to investigate the MSCCX4-derived paracrine cardio-protective signaling in the presence of myocardial infarction. Mesenchymal stem cells (MSCs) were divided into 3 groups: MSC only, MSCCX4 , and CXCR4 gene-specific siRNA-transduced MSC. Mesenchymal stem cells were exposed to hypoxia, and then MSCs-conditioned culture medium was incubated with neonatal and adult cardiomyocytes, respectively. Cell proliferation-regulating genes were assessed by real-time polymerase chain reaction (RT-PCR). In vitro: The number of cardiomyocytes undergoing DNA synthesis, cytokinesis, and mitosis was increased to a greater extent in MSCCX4 medium-treated group than control group, while this proproliferative effect was reduced in CXCR4 gene-specific siRNA-transduced MSC-treated cells. Accordingly, the maximal enhancement of vascular endothelial growth factor, cyclin 2, and transforming growth factor-β2 was observed in hypoxia-exposed MSCCX4 . In vivo: MSCs were labeled with enhanced green fluorescent protein (EGFP) and engrafted into injured myocardium in rats. The number of EGFP and CD31 positive cells in the MSCCX4 group was significantly increased than other 2 groups, associated with the reduced left ventricular (LV) fibrosis, the increased LV free wall thickness, the enhanced angiogenesis, and the improved contractile function. CXCR4 overexpression can mobilize MSCs into ischemic area, whereby these cells can promoted angiogenesis and alleviate LV remodeling via paracrine signaling mechanism.

Aims: Therapeutic angiogenesis has emerged as a promising strategy to regenerate the damaged blood vessels resulting from ischemic diseases such as myocardial infarction (MI). However, the functional integration of implanted endothelial cells (ECs) in infarcted heart remains challenging. We herein develop an EC generation approach by inhibiting microRNA-495 (miR-495) in human induced pluripotent stem cells (hiPSCs) and assess the angiogenic potential for MI treatment. Methods and Results: The anti-angiogenic miR-495 belonging to Dlk1-Dio3 miR cluster was identified through expression profiling and computational analysis. Loss-of-function experiments for miR-495 were performed using a lentiviral transfer of antisense sequence in hiPSCs. The pluripotency of hiPSCs was not impacted by the genetic modification. Induced with differentiation medium, miR-495 inhibition enhanced the expression of EC genes of hiPSCs, as well as the yield of ECs. Newly derived ECs displayed prominent angiogenic characteristics including tube formation, cell migration, and proliferation. Mechanistically, miR-495 mediated the expression of endothelial or angiogenic genes by directly targeting vascular endothelial zinc finger 1 (VEZF1). After transplantation in immunodeficient MI mice, the derived ECs significantly increased neovascularization in the infarcted heart, prevented functional worsening, and attenuated expansion of infarct size. The functional integration of the implanted ECs into coronary networks was also enhanced by inhibiting miR-495. Conclusion: miR-495 represents a new target not only for promoting EC generation from hiPSCs but also for enhancing angiogenesis and engraftment of hiPSC-derived ECs in ischemic heart. This article is protected by copyright. All rights reserved.

Background and Objective: miR-495 inhibition is known to enhance mesoderm differentiation, but its role in angiogenesis is not clear. The present study seeks to determine whether miR-495 mediates angiogenesis induced by human induced pluripotent stem cells (hiPSCs) in infarcted heart. Methods: The miR-495 in hiPSCs was knocked down via lentivirus. ECs were generated from hiPSCs (hiECs) and sorted by MACS using CD144 antibody (Fig. 1A). In vitro : the angiogenic potential of hiEC Anti-495 (hiEC Scr as control) was analyzed by tube formation and expression of angiogenic genes was detected by PCR array. In vivo : hiECs were transplanted into infarcted heart of immunodeficient mice (LAD ligation); after 4 weeks, hearts were harvested for immunostaining and genome PCR to identify implanted cells. Results: In vitro : The tube length of hiEC Anti-495 was increased (Fig. 1B) and multiple angiogenic genes (EFNB2, TIE2, IGF1, and JAG1) were upregulated in hiEC Anti-495 as compared to hiEC Scr (Fig. 1C). In vivo : Implantation of hiEC Anti-495 increased the isolectin-B4 + capillary density in the infarcted heart (Fig. 1D). hiEC Anti-495 integrated into mouse vascular networks, whereas the structural integration of hiEC Scr was seldom observed. Human DNA derived from hiEC Anti-495 in infarcted heart was higher as compared to hiEC Scr (Fig.1E). Conclusion: miR-495 represents a promising mediator for promoting angiogenic potential and functional integration of hiPSC-derived ECs for the treatment of infarcted heart.

Background and Objective.Exosomes secreted from mesenchymal stem cells (MSC) have demonstrated cardioprotective effects. This study examined the role of exosomes derived from MSC overexpressing CXCR4 for recovery of cardiac functions after myocardial infarction (MI).Methods. In vitro, exosomes from MSC transduced with lentiviral CXCR4 (ExoCR4) encoding a silencing sequence or null vector were isolated and characterized by transmission electron microscopy and dynamic light scattering. Gene expression was then analyzed by qPCR and Western blotting. Cytoprotective effects on cardiomyocytes were evaluated and effects of exosomes on angiogenesis analyzed.In vivo, an exosome-pretreated MSC-sheet was implanted into a region of scarred myocardium in a rat MI model. Angiogenesis, infarct size, and cardiac functions were then analyzed.Results. In vitro, ExoCR4significantly upregulatedIGF-1αand pAkt levels and downregulated active caspase 3 levelin cardiomyocytes. ExoCR4also enhanced VEGF expression and vessel formation. However, effects of ExoCR4were abolished by an Akt inhibitor or CXCR4 knockdown.In vivo, ExoCR4treated MSC-sheet implantation promoted cardiac functional restoration by increasing angiogenesis, reducing infarct size, and improving cardiac remodeling.Conclusions.This study reveals a novel role of exosomes derived from MSCCR4and highlights a new mechanism of intercellular mediation of stem cells for MI treatment.

MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSC miR-377) or knockdown microRNA-377 (MSC Anti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSC Null). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSC Anti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSC miR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSC Null-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.

Regenerative medicine offers hope for patients with myocardial infarction (MI). A good deal of clinical evidence shows that the transplantation of stem/progenitor cells from numerous sources can produce significant protective effects in the injured heart, such as enhanced perfusion and contractility. The therapeutic effects of transplanted cells depend on appropriate delivery into the damaged area of the heart. Thus, the method of cell delivery is an important aspect of stem/progenitor cell based therapy, because it affects the fate of transplanted cells and consequently determines the clinical outcome. It is still difficult to determine the optimal cell delivery strategy for MI treatment from the current preclinical and clinical studies, although cell injections have been widely used in the delivery of various types of progenitor cells in clinical trials. Additionally, cell patch engineering has emerged as one of the most promising new approaches. This review summarizes the methods, feasibility, safety, efficacy, and advancements of these two approaches from recent publications. The combined use of various delivery approaches with stem/ progenitor cell transplantation emerges as an important administration method in tissue engineering to promote cardiac regeneration.

BACKGROUND:Our previous studies indicated that MSC(CXCR4) improved cardiac function after myocardial infarction (MI). This study was aimed to investigate the specific role of MSC(CXCR4) in neovascularization of infarcted myocardium using a suicide gene approach. METHODS:MSCs were transduced with either lentivirus-null vector/GFP (MSC(Null) as control) or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC) differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSC(Null) or MSC(CXCR4) were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV) or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. RESULTS:The expression of VEGF-A and HIF-1α was significantly higher in MSC(CXCR4) as compared to MSC(Null) under hypoxia. Additionally, MSC(CXCR4) enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSC(CXCR4) under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor). In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSC(CXCR4) implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSC(CXCR4) were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. CONCLUSION:The transplanted MSC(CXCR4) enhanced neovascularization after MI by boosting release of angiogenic factors and increasing the potential of endothelial differentiation.

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP