Spheroids from adipose‐derived stem cells exhibit an miRNA profile of highly undifferentiated cells

Two‐dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three‐dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as “spheroids,” and we have previously demonstrated that spheroids from adipose‐derived stem cells (S‐ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S‐ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S‐ASCs. In this study, we investigate for the first time the miRNA expression pattern in S‐ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long‐term in vitro culture. The data reported in the current study suggest that S‐ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.     

Continuous exposure of nicotine and cotinine retards human primary pterygium cell proliferation and migration

Pterygium is a triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. Our previous meta-analysis found that cigarette smoking is associated with a reduced risk of pterygium. Yet, the biological effect of cigarette smoke components on pterygium has not been studied. Here we reported the proliferation and migration properties of human primary pterygium cells with continuous exposure to nicotine and cotinine. Human primary pterygium cells predominantly expressed the α5, β1, and γ subunits of the nicotinic acetylcholine receptor. Continuous exposure to the mixture of 0.15 μM nicotine and 2 μM cotinine retarded pterygium cell proliferation by 16.04% (P = 0.009) and hindered their migration by 11.93% ( P = 0.039), without affecting cell apoptosis. SNAIL and α-smooth muscle actin protein expression was significantly downregulated in pterygium cells treated with 0.15 μM nicotine-2 μM cotinine mixture by 1.33- ( P = 0.036) and 1.31-fold ( P = 0.001), respectively. Besides, the 0.15 μM nicotine-2 μM cotinine mixture also reduced matrix metalloproteinase (MMP)-1 and MMP-9 expressions in pterygium cells by 1.56- ( P = 0.043) and 1.27-fold ( P = 0.012), respectively. In summary, this study revealed that continuous exposure of nicotine and cotinine inhibited human primary pterygium cell proliferation and migration in vitro by reducing epithelial-to-mesenchymal transition and MMP protein expression, partially explaining the lower incidence of pterygium in cigarette smokers.     

Nrf2 dependent antiaging effect of milk-derived bioactive peptide in old fibroblasts

Prolonged passaging of primary fibroblast cells totally shapes the natural biological phenomena and leads to the appearance of features related to senescence. As a result, it is a good natural tool to delineate the molecular mechanism of cellular aging. The present investigation revealed the antiaging effect of milk-derived novel bioactive peptide (VLPVPQK). The peptide played an important role in downregulating apoptosis-related markers in late passages of cultured fibroblast cells. The peptide treatment to aged fibroblasts caused enhancement in cell migration, DNA integrity, and decrease in the lipid peroxidation, reactive oxygen species, nitric oxide production as well as pro-inflammatory cytokines, TNF-α and IL-6. Moreover, the peptide decreased the expression of apoptotic caspases, Bax, and senescence-associated β-galactosidase (SA-β-gal) proteins. The peptide pretreatment also enhanced the extracellular collagen protein and antiapoptotic, Bcl-xL. In addition, the peptide treatment reversed the senescence-related activity in fibroblasts by stimulating Nrf2 mediated antioxidative defense system and inhibiting the action of NFkB/p38MAPK signaling, similar to the commercially available inhibitor (SB203580) of p38MAPK. Thus, the peptide exhibits the antiaging effect in dermal fibroblast cells.     

Status of topoisomerase-2β protein in all-trans retinoic acid–treated human neuroblastoma (SK-N-SH) cells

Of the mammalian topoisomerase (Topo)-2 isozymes (α and β), Topo-2β protein has been reported to regulate neuronal development and differentiation. However, the status of Topo-2β in all-trans retinoic acid (ATRA)-treated human neuroblastoma (SK-N-SH) cells is not understood. More information about the effects of ATRA on SK-N-SH cells is needed to reveal the role of ATRA in the regulation of Topo-2β levels and spontaneous regression of SK-N-SH cells to predict the clinical activity. This study was proposed to investigate the status and role of Topo-2β protein in ATRA-induced survival and neuronal differentiation of SK-N-SH cells. Microscopic, sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitations and Western blot analysis were used to study and compare Topo-2β protein among 10 µM ATRA-treated SK-N-SH cells and controls at different time points. The level of Topo-2β protein increased in the initial days of treatment but markedly decreased upon induction of differentiation by ATRA in later stages. Upon ATRA treatment, SK-N-SH cells stretched, exhibited neurite extensions, and acquired a neuronal phenotype. Both treated and untreated SK-N-SH cells were able to migrate, occupy the scratched area, and completely recolonized 24 hours later. These results suggest an indirect role of Topo-2β protein in regulation of genes involved in cell migration and differentiation of ATRA-treated SK-N-SH cells. This study suggests that Topo-2β may be part of activation/repression of protein complexes activated by epigenetic modifying agents, differentiating signals, and inducible locus. However, detailed studies are needed to explore the ATRA-downstream genes leading to Topo-2β regulation and regulatory proteins of neuronal differentiation.     

The effect of VDR gene polymorphisms and vitamin D level on blood pressure,risk of preeclampsia,gestational age,and body mass index

We investigated the influence of vitamin D receptor (VDR) polymorphisms and vitamin D level on the blood pressure and the risk of preeclampsia. In a case-control study, 200 pregnant women, including 100 individuals with preeclampsia along with 100 healthy pregnant women, were studied for VDR FokI, TaqI, and BmsI polymorphisms and serum 25 (OH)-D level using polymerase chain reaction-restriction fragment length polymorphism method and commercial kit, respectively. The mean level of 25 (OH)-D in preeclamptic patients was significantly lower (16.6 ± 4.2 ng/mL, P < 0.001) compared with controls (19.6 ± 3.8 ng/mL). Among all women, a significantly higher systolic blood pressure and before-pregnancy body mass index and also lower gestational age were observed in the presence of 25 (OH)-D level < 20 ng/mL compared with the 20 to 30 ng/mL. A significantly higher frequency of VDR FokI C allele in preeclamptic patients (83%) than controls (74%) was associated with a 1.72-fold increased risk of preeclampsia. In all the studied individuals, the systolic and diastolic blood pressures were significantly higher in the presence of the FokI CC genotype compared with the TC and TT+TC genotypes. Neither VDR Taq1 nor VDR BmsI was associated with the risk of preeclampsia. The haplotype FokI C, TaqI C and BmsI A (CCA) compared with haplotype CTG increased the risk of preeclampsia by 1.4-fold (P = 0.33). Our study suggests an association between VDR FokI polymorphism and an insufficient serum level of 25 (OH)-D with the risk of preeclampsia and also the influence of insufficient 25 (OH)-D level and VDR FokI polymorphism on maternal factors, including blood pressure.     

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.     

Evaluation of combined effects of brief electrical stimulation and Schwann-like cells on sciatic nerve injury model

Severe nerve injuries can be treated with electrical stimulation and stem cell therapies, but little is known about the potential benefits of combining these two treatments. In an effort to investigate this combination, we conducted a study to evaluate the effectiveness of electrical stimulation and Schwann-like cell transplantation in female Wistar albino rats. Our study consisted of five groups of rats: a sham group, an injury group, an electrical stimulation group, a Schwann-like cell group, and a combination group. The experimental groups received electrical stimulation, Schwann-like cell transplantation, or both. The animals sciatic function index was evaluated during a 6-week recovery period, and nerve conduction velocity, wet muscle mass, and nerve tissues were also analyzed. The results of the study showed that all experimental groups had a faster functional recovery compared to the injury group, although the difference between groups was not statistically significant. Both the combination group and the Schwann-like cell transplantation group had a higher nerve conduction velocity compared to the other experimental groups. However, there was no significant difference between the combination and Schwann-like cell transplantation groups. Nonetheless, histological analysis showed a better axonal reorganization in the combination group. The study provides preliminary evidence of the potential benefits of combining electrical stimulation and Schwann-like cell transplantation in treating severe nerve injuries. However, further studies with larger sample sizes are needed to confirm these findings and optimize the treatment parameters.