Journal of Cellular and Molecular Medicine

Gene transfer into human CD34+ haematopoietic progenitor cells (HPC) and multi‐potent mesenchymal stromal cells (MSC) is an essential tool for numerous in vitro and in vivo applications including therapeutic strategies, such as tissue engineering and gene therapy. Virus based methods may be efficient, but bear risks like tumorigenesis and activation of immune responses. A safer alternative is non‐viral gene transfer, which is considered to be less efficient and accomplished with high cell toxicity. The truncated low affinity nerve growth factor receptor (ÄLNGFR) is a marker gene approved for human in vivo application. Human CD34+ HPC and human MSC were transfected with in vitro‐transcribed mRNA for ΔLNGFR using the method of nucleofection. Transfection efficiency and cell viability were compared to plasmid‐based nucleofection. Protein expression was assessed using flow cytometry over a time period of 10 days. Nucleofection of CD34+ HPC and MSC with mRNA resulted in significantly higher transfection efficiencies compared to plasmid transfection. Cell differentiation assays were performed after selecting ΔLNGFR positive cells using a fluorescent activating cell sorter. Neither cell differentiation of MSC into chondrocytes, adipocytes and osteoblasts, nor differentiation of HPC into burst forming unit erythroid (BFU‐E) colony forming unit‐granulocyte, erythrocyte, macrophage and megakaryocyte (CFU‐GEMM), and CFU‐granulocyte‐macrophage (GM) was reduced. mRNA based nucleofection is a powerful, highly efficient and non‐toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may be useful to transiently manipulate stem cell characteristics and thus combine principles of gene therapy and tissue engineering.

Osteoclast overactivation‐induced imbalance in bone remodelling leads to pathological bone destruction, which is a characteristic of many osteolytic diseases such as rheumatoid arthritis, osteoporosis, periprosthetic osteolysis and periodontitis. Natural compounds that suppress osteoclast formation and function have therapeutic potential for treating these diseases. Stachydrine (STA) is a bioactive alkaloid isolated from Leonurus heterophyllus Sweet and possesses antioxidant, anti‐inflammatory, anticancer and cardioprotective properties. However, its effects on osteoclast formation and function have been rarely described. In the present study, we found that STA suppressed receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL)‐induced osteoclast formation and bone resorption, and reduced osteoclast‐related gene expression in vitro. Mechanistically, STA inhibited RANKL‐induced activation of NF‐κB and Akt signalling, thus suppressing nuclear factor of activated T cells c1 induction and nuclear translocation. In addition, STA alleviated bone loss and reduced osteoclast number in a murine model of LPS‐induced inflammatory bone loss. STA also inhibited the activities of NF‐κB and NFATc1 in vivo. Together, these results suggest that STA effectively inhibits osteoclastogenesis both in vitro and in vivo and therefore is a potential option for treating osteoclast‐related diseases.

Inflammation and neuronal apoptosis aggravate the secondary damage after spinal cord injury (SCI). Rehmannioside A (Rea) is a bioactive herbal extract isolated from Rehmanniae radix with low toxicity and neuroprotection effects. Rea treatment inhibited the release of pro‐inflammatory mediators from microglial cells, and promoted M2 polarization in vitro, which in turn protected the co‐cultured neurons from apoptosis via suppression of the NF‐κB and MAPK signalling pathways. Furthermore, daily intraperitoneal injections of 80 mg/kg Rea into a rat model of SCI significantly improved the behavioural and histological indices, promoted M2 microglial polarization, alleviated neuronal apoptosis, and increased motor function recovery. Therefore, Rea is a promising therapeutic option for SCI and should be clinically explored.

Drug‐induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown.K_IR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (I_K1), are degraded in lysosomes. Amiodarone and dronedarone are classIIIantiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late‐endosome/lysosome system. Here we defined the potential interference inK_IR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibitedI_K1in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. InHK‐KWGFcells, both drugs dose‐ and time‐dependently increasedK_IR2.1 expression (2.0 ± 0.2‐fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3‐fold with dronedarone: 5 μM, 24 hrs) and late‐endosomal/lysosomalK_IR2.1 accumulation. IncreasedK_IR2.1 expression level was also observed in the presence of Na_v1.5 co‐expression. AugmentedK_IR2.1 protein levels and intracellular accumulation were also observed inCOS‐7,END‐2,MES‐1 andEPI‐7 cells. Both drugs had no effect on K_v11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3%P < 0.05 at −120 mV, 5 μM) enhancedI_KIR2.1upon 24‐hrs treatment, whereas dronedarone tended to increaseI_KIR2.1and it did not reach significance (43.8 ± 5.5%,P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhancedI_K1by inhibitingK_IR2.1 degradation.

Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia‐inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5′ regulatory region of human DMT1 (between −412 and −570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia‐inducible factor‐1 (HIF‐1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia‐inducible gene. We investigated the effects of hypoxia and hypoxia/re‐oxygenation on the expression of DMT1 and the content of HIF‐1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF‐1α, DMT1+IRE (iron response element) and DMT1−IRE proteins to chemical (CoCl₂) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF‐1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF‐1α could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1−IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF‐1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.

Introduction

The epicardium has recently been identified as an active and essential element of cardiac development. Recent reports have unveiled a variety of functions performed by the embryonic epicardium, as well as the cellular and molecular mechanisms regulating them. However, despite its developmental importance, a number of unsolved issues related to embryonic epicardial biology persist. In this review, we will summarize our current knowledge about (i) the ontogeny and evolution of the epicardium, including a discussion on the evolutionary origins of the proepicardium (the epicardial primordium), (ii) the nature of epicardial–myocardial interactions during development, known to be essential for myocardial growth and maturation, and (iii) the contribution of epicardially derived cells to the vascular and connective tissue of the heart. We will finish with a note on the relationships existing between the primordia of the viscera and their coelomic epithelial lining. We would like to suggest that at least a part of the properties of the embryonic epicardium are shared by many other coelomic cell types, such that the role of epicardium in cardiac development is a particular example of a more general mechanism for the contribution of coelomic and coelomic‐derived cells to the morphogenesis of organs such as the liver, kidneys, gonads or spleen.