Prolonged post-differentiation culture influences the expression and biophysics of Na + and Ca 2+ channels in induced pluripotent stem cell-derived ventricular-like cardiomyocytes Abstract Several studies have been reported in various domains from induction methods to utilities of somatic cell pluripotent reprogramming. However, one of the major struggles facing the research field of induced pluripotent stem cell (iPSC)-derived target cells is the lack of consistency in observations. This could be due to variety of reasons including varied culture periods post-differentiation. The cardiomyocytes (CMs) derived from iPSCs are commonly studied and proposed to be utilized in the comprehensive in vitro proarrhythmia initiative for drug safety screening. As the influence of varied culture periods on the electrophysiological properties of iPSC-CMs is not clearly known, using whole-cell patch clamp technique, we compared two groups of differentiated ventricular-like iPSC-CMs that are cultured for 10 to 15 days (D10–15) and more than 30 days (≥ D30) both under current and voltage clamps. The prolonged culture imparts increased excitability with high-frequency spontaneous action potentials, robust increase in the magnitude of peak Na+ current density, relatively shallow inactivation kinetics of Na+ channels, faster recovery from inactivation, and augmented Ca2+ current density. Quantitative real-time PCR studies of α-subunit transcripts showed enhanced mRNA expression of SCN1A, SCN5A Na+ channel subtypes, and CACNA1C, CACNA1G, and CACNA1I Ca2+ channel subtypes, in ≥ D30 group. Conclusively, the prolonged culture of differentiated iPSC-CMs affects the excitability, single-cell electrophysiological properties, and ion channel expressions. Therefore, following standard periods of culture across research studies while utilizing ventricular-like iPSC-CMs for in vitro health/disease modeling to study cellular functional mechanisms or test high-throughput drugs’ efficacy and toxicity becomes crucial.

Mouse model to study pulmonary intravascular macrophage recruitment and lung inflammation in acute necrotizing pancreatitis Abstract Patients suffering from severe acute pancreatitis (AP) can develop acute lung injury (ALI) with poor outcomes and the mechanisms involved remain incompletely understood. Pulmonary intravascular macrophages (PIMs), which are credited as promoters of ALI, are not constitutively present in humans and rodents; however, there is evidence of PIM recruitment in rodents during some pathological conditions, such as hepatic diseases. Therefore, this study assesses PIM recruitment in the lungs of a mouse model of acute necrotizing pancreatitis (ANP) induced with l-arginine monohydrochloride. Mice were euthanized after 24 h, 72 h and 120 h. Control mice received sham injections of saline. Pancreatic histopathological grading and plasma amylase were used to confirm the development of ANP in l-arginine-treated mice. Histopathological grading of lungs from the ANP mice at 72 h showed increased mononuclear phagocytes in alveolar septa, compared to that from the controls. Lungs from the ANP mice also showed increased numbers of CD68-immunopositive alveolar septal macrophages, suggestive of PIM recruitment, compared to those from the controls. Lungs from the ANP mice showed increased expression of IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1) and von Willebrand factor compared to those from the controls. The recruitment of CD68-positive septal macrophages was not observed in MCP-1 knockout mice with ANP at 72 h when compared to C57BL/6 wild-type mice. Taken together, we developed a mouse model of PIM recruitment dependent on MCP-1 that allows us to explore their roles in ANP-associated ALI.

The environmental obesogen bisphenol A increases macrophage self-renewal Abstract Self-renewal of macrophages is important for the healthy development and replenishment of tissue-resident macrophage pools. How this mechanism is controlled by endocrine signals is still largely unexplored. Here, we show that the endocrine disruptor bisphenol A (BPA) increases macrophage self-renewal. This effect was associated with phosphorylation of extracellular signal-regulated kinase (ERK) and a slight increase in the expression of liver X receptor alpha (LXRα). We found that LXRα inhibition induced, while LXRα activation impeded, macrophage self-renewal. LXRα signaling hence may protect from excessive macrophage expansion. Self-renewing macrophages, however, had negligible LXRα expression when compared with quiescent macrophages. Accordingly, tissue-resident macrophage pools, which are dominated by quiescent macrophages, were rich in LXRα-expressing macrophages. Overall, we show that BPA increases macrophage self-renewal and that this effect, at least in part, can be inhibited by increasing LXRα expression. Since BPA is accumulated in the adipose tissue, it has the potential to increase self-renewal of adipose tissue macrophages, leading to a condition that might negatively impact adipose tissue health.

Leucine attenuates muscle atrophy and autophagosome formation by activating PI3K/AKT/mTOR signaling pathway in rotator cuff tears Abstract Rotator cuff tears (RCTs), the most common tendon injury, are always accompanied by muscle atrophy, which is characterized by excessive protein degradation. Autophagy–lysosome systems are the crucial proteolytic pathways and are activated in atrophying muscle. Thus, inhibition of the autophagy–lysosome pathway might be an alternative way to minimize skeletal muscle atrophy. In this present study, combined with a tendon transection-induced rat model of massive rotator cuff tears, HE staining and transmission electron microscopy methods, we found leucine supplementation effectively prevented muscle atrophy, muscle injury and autophagosome formation. Utilizing immunoblotting, we discovered that leucine supplementation is able to inhibit the rise in autophagy-related protein expression (including LC3, Atrogin-1, MuRF1, Bnip3 and FoxO3) driven by tendon transection. The PI3K/AKT/mTOR pathway that was essential in autophagosome formation and autophagy was blocked in atrophying muscle and reactivated in the presence of leucine. Once administrated with LY294002 (PI3K inhibitor) and Rapamycin (mTOR inhibitor), leucine mediated by the anti-atrophic effects was nearly blunted. These results suggest that leucine potentially attenuates tendon transection-induced muscle atrophy through autophagy inhibition via activating the PI3K/AKT/mTOR pathway.

Interference of miR-943-3p with secreted frizzled-related proteins4 (SFRP4) in an asthma mouse model Abstract The aim of this study is to investigate the potential roles of miR-943-3p and its target gene secreted frizzled-related proteins4 (SFRP4) in allergic asthma and elucidate its underlying mechanism, which may prompt a new clue about developing novel treatments of this disease. An allergic asthma mouse model was generated by challenging with ovalbumin (OVA); lung pathological features of mice were viewed using H&E staining; thickness of subepithelial fibrosis and smooth muscle was measured using Masson’s trichrome staining. Inflammatory cells from bronchoalveolar lavage fluid (BALF) were counted based on Diff-Quik staining and morphometric analysis. Expressions of miR-943-3p, SFRP4 and Wnt signal pathway-associated proteins were detected using RT-PCR or immunoblotting, respectively. SFRP4 was downregulated in the bronchial biopsies of allergic asthma patients and represented a unique intersection between differentially expressed genes (DEGs) and genes in the Wnt signal pathway. Both miR-943-3p upregulation and SFRP4 downregulation were detected in allergic asthma patients and OVA-induced mice. Besides, OVA-induced mice possessed more inflammatory cells in BALF including macrophage (mac), eosinophil (eos), lymphocyte (lym) and neutrophil (neu), higher expression of collagen, β-catenin and c-Myc as well as thicker subepithelial fibrosis and smooth muscle in lung than control mice. In vivo delivery of miR-943-3p agomir worsened these symptoms, while both miR-943-3p antagomir and Ad-SFRP4 administration effectively alleviated this disease. Taken together, miR-943-3p accelerated the progression of airway inflammation and remodeling in allergic asthma via suppressing the activity of SFRP4 through Wnt signaling pathway in asthma patients and OVA-induced mice.

Distributions and relationships of chemically defined enteroendocrine cells in the rat gastric mucosa Abstract This paper provides quantitative data on the distributions of enteroendocrine cells (EEC), defined by the hormones they contain, patterns of colocalisation between hormones and EEC relations to nerve fibres in the rat gastric mucosa. The rat stomach has three mucosal types: non-glandular stratified squamous epithelium of the fundus and esophageal groove, a region of oxyntic glands in the corpus, and pyloric glands of the antrum and pylorus. Ghrelin and histamine were both contained in closed cells, not contacting the lumen, and were most numerous in the corpus. Gastrin cells were confined to the antrum, and 5-hydroxytryptamine (5-HT) and somatostatin cells were more frequent in the antrum than the corpus. Most somatostatin cells had basal processes that in the antrum commonly contacted gastrin cells. Peptide YY (PYY) cells were rare and mainly in the antrum. The only numerous colocalisations were 5-HT and histamine, PYY and gastrin and gastrin and histamine in the antrum, but each of these populations was small. Peptide-containing nerve fibres were found in the mucosa. One of the most common types was vasoactive intestinal peptide (VIP) fibres. High-resolution analysis showed that ghrelin cells were closely and selectively approached by VIP fibres. In contrast, gastrin cells were not selectively innervated by VIP or CGRP fibres. The study indicates that there are distinct populations of gastric EEC and selective innervation of ghrelin cells. It also shows that, in contrast to EEC of the small intestine, the majority of EEC within the stomach contained only a single hormone.

Npr2 null mutants show initial overshooting followed by reduction of spiral ganglion axon projections combined with near-normal cochleotopic projection Abstract Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus.

Systemic LPS induces toll-like receptor 3 (TLR3) expression and apoptosis in testicular mouse tissue Abstract It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon β (IFNβ) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.

The effect of sodium valproate on differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in two-dimensional culture and fibrin scaffold conditions Abstract Differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes is a complex phenomenon, and attempts to find an effective inducing agent are still ongoing. We studied the effect of fibrin scaffold and sodium valproate (VPA, as a histone deacetylase inhibitor) on the differentiation of human adipose-derived stem cells (hADSCs) into cardiomyocyte-like cells. The cells were cultured in culture flask (2D) and in fibrin scaffold (3D), fabricated of human plasma fibrinogen, with and without VPA (1 mM). QRT-PCR, Western blot, and immunochemistry assays were used to evaluate the expression of cardiac markers at gene and protein levels. High levels of CD44, CD90, CD73, and CD105 were expressed on the surface of hADSCs. Treated encapsulated hADSCs (3D) presented significantly higher mRNA expression of HAND1 (1.54-fold), HAND2 (1.59-fold), cTnI (1.76-fold), MLC2v (1.4-fold), Cx43 (1.38-fold), βMHC (1.34-fold), GATA4 (1.48-fold), and NKX2.5 (1.66-fold) in comparison to 2D conditions at four weeks after induction. The protein expressions of NKX2.5 (0.78 vs 0.65), cTnI (1.04 vs 0.81), and Cx43 (1.11 vs 1.08) were observed in the differentiated cells both in 3D and 2D groups, while control cells were absolutely negative for these proteins. The frequency of cTnI and Cx43-positive cells was significantly higher in 3D (24.2 ± 15 and 12 ± 3%) than 2D conditions (19.8 ± 3 and 10 ± 2%). Overall, the results showed that VPA can increase cardiomyogenesis in hADSCs and that fibrin scaffold enhances the inductive effect of VPA. Results of this study may improve cell-based protocols for implementation of more successful cardiac repair strategies.

AAV2/DJ-mediated alpha-synuclein overexpression in the rat substantia nigra as early stage model of Parkinson’s disease Abstract Parkinson’s disease (PD) is pathologically characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and alpha-synucleinopathy. We mimic the disease pathology with overexpression of either the human α-syn wildtype (α-syn-WT) or E46K mutant form (α-syn-E46K) in DA neurons of the SNpc in adult rats using AAV2/DJ as a viral vector for the first time. Transduction efficiency was compared to an equal virus titer expressing the green fluorescent protein (GFP). Motor skills of all animals were evaluated in the cylinder and amphetamine-induced rotation test over a total time period of 12 weeks. Additionally, stereological quantification of DA cells and striatal fiber density measurements were performed every 4 weeks after injection. Rats overexpressing α-syn-WT showed a progressive loss of DA neurons with 40% reduction after 12 weeks accompanied by a greater loss of striatal DA fibers. In contrast, α-syn-E46K led to this reduction after 4 weeks without further progress. Insoluble α-syn positive cytoplasmic inclusions were observed in both groups within DA neurons of the SNpc and VTA. In addition, both α-syn groups developed a characteristic worsening of the rotational behavior over time. However, only the α-syn-WT group reached statistically significant different values in the cylinder test. Summarizing these effects, we established a motor symptom animal model of PD by using AAV2/DJ in the brain for the first time. Thereby, overexpressing of α-syn-E46K mimicked a rather pre-symptomatic stage of the disease, while the α-syn-WT overexpressing animals imitated an early symptomatic stage of PD.