Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination.

Epithelial to mesenchymal transition (EMT) is a cellular process that converts epithelial cells to mesenchymal cells with migratory potential in developmental and pathological processes. Although originally considered a binary event, EMT in cancer progression involves intermediate states between a fully epithelial and a fully mesenchymal phenotype, which are characterized by distinct combinations of epithelial and mesenchymal markers. This phenomenon has been termed epithelial to mesenchymal plasticity (EMP), however, the intermediate states remain poorly described and it's unclear whether they exist during developmental EMT. Neural crest cells (NCC) are an embryonic progenitor cell population that gives rise to numerous cell types and tissues in vertebrates, and their formation and delamination is a classic example of developmental EMT. However, whether intermediate states also exist during NCC EMT and delamination remains unknown. Through single-cell RNA sequencing of mouse embryos, we identified intermediate NCC states based on their transcriptional signature and then spatially defined their locations in situ in the dorsolateral neuroepithelium. Our results illustrate the importance of cell cycle regulation and functional role for the intermediate stage marker Dlc1 in facilitating mammalian cranial NCC delamination and may provide new insights into mechanisms regulating pathological EMP.

Alcohol and vascular endothelial function: Biphasic effect highlights the importance of dose.

BACKGROUND: Alcohol (ethanol) consumption has different influences on arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Whereas endothelial cells play a critical role in maintaining arterial health, this study compared the effects of moderate and high alcohol concentrations on endothelial cell function. METHODS: Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or high-risk/heavy drinking (i.e., 50 mM) after which endothelial function was assessed. The effect of ethanol's primary metabolite acetaldehyde (10 and 25 µM) was also determined. RESULTS: Moderate ethanol exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, and increased homeostatic Notch signaling. In contrast, exposure to high-level ethanol (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and endothelial nitric oxide synthase activity were increased by both alcohol treatments, and to a greater extent in the 50 mM ethanol group. Acetaldehyde-elicited responses were generally the same as those of the high-level ethanol group. CONCLUSIONS: Ethanol has biphasic effects on several endothelial functions such that a moderate level maintains the endothelium in a nonactivated state, whereas high-level ethanol causes endothelial dysfunction, as does acetaldehyde. These data show the importance of dose when considering ethanol's effects on arterial endothelium, and could explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in some epidemiologic studies.

Caveolin-1 inhibition mediates the opposing effects of alcohol on γ-secretase activity in arterial endothelial and smooth muscle cells.

Notch is important to vessel homeostasis. We investigated the mechanistic role of caveolin-1 (Cav-1) in mediating the effects of alcohol (Ethanol/EtOH) on the γ-secretase proteolytic activity necessary for Notch signaling in vascular cells. Human coronary artery endothelial cells (HCAEC) were treated with EtOH (0-50 mM), Notch ligand delta-like ligand 4 (Dll4), and the γ-secretase inhibitor DAPT. EtOH stimulated Notch signaling in HCAEC as evidenced by increased Notch receptor (N1, N4) and target gene (hrt2, hrt3) mRNA levels with the most robust response achieved at 25 mM EtOH. Ethanol (25 mM) stimulated γ-secretase proteolytic activity, to the same extent as Dll4, in HCAEC membranes. Ethanol inhibited Cav-1 mRNA and protein levels in HCAEC. Caveolin-1 negatively regulated γ-secretase activity in HCAEC as Cav-1 knockdown stimulated it, while Cav-1 overexpression inhibited it. Moreover, Cav-1 overexpression blocked the stimulatory effect of EtOH on γ-secretase activity in HCAEC. Although EtOH also inhibited Cav-1 expression in human coronary artery smooth muscle cells (HCASMC), EtOH inhibited γ-secretase activity in HCASMC in contrast to its effect in HCAEC. The inhibitory effect of EtOH on γ-secretase in HCASMC was mimicked by Cav-1 knockdown and prevented by Cav-1 overexpression, suggesting that in these cells Cav-1 positively regulates γ-secretase activity. In conclusion, EtOH differentially regulates γ-secretase activity in arterial EC and SMC, being stimulatory and inhibitory, respectively. These effects are both mediated by caveolin-1 inhibition which itself has opposite effects on γ-secretase in the two cell types. This mechanism may underlie, in part, the effects of moderate drinking on atherosclerosis.

Rubus Capped Zinc Oxide Nanoparticles Induce Apoptosis in MCF-7 Breast Cancer Cells.

Rubus fairholmianus (RF) has widely been used to treat various ailments, including pain, diabetes, and cancer. Zinc oxide nanoparticles (ZnO NPs) have drawn attention in modern healthcare applications. Hence, we designed this study to synthesize zinc oxide (ZnO) nanoparticles using R. fairholmianus root extract to investigate its synergistic cytotoxic effect on MCF-7 cells and explore the possible cell death mechanism. ZnO NPs were synthesized via green synthesis using R. fairholmianus root extract, and the effect on MCF-7 cells was determined by looking at cellular morphology, proliferation, cytotoxicity, apoptosis, and reactive oxygen species (ROS). The results showed that cellular proliferation was reduced following treatment with R. fairholmianus capped zinc oxide nanoparticles (RFZnO NPs), while cytotoxicity and ROS were increased. There was also an increase in apoptosis as indicated by the significant increase in cytoplasmic cytochrome c and caspase 3/7 (markers of apoptosis), as well as increased levels of pro-apoptotic proteins (p53, Bax) and decreased levels of anti-apoptotic protein (Bcl-2). In conclusion, these results showed that RFZnO NPs induce apoptosis in breast cancer cells via a mitochondria-mediated caspase-dependent apoptotic pathway and suggest the use of acetone root extract of R. fairholmianus for the treatment of cancer-related ailments.

Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and notch-independent pathways.

BACKGROUND: Arterial endothelium plays a critical role in maintaining vessel homeostasis and preventing atherosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol (EtOH) consumption is associated with reduced atherosclerosis and stimulates Notch signaling in endothelial cells. The aim of this study was to determine whether EtOH protects the endothelium against serum amyloid A1 (SAA1)-induced activation/injury, and to determine whether this protection is exclusively Notch-dependent. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAEC) were stimulated or not with "pro-atherogenic" SAA1 (1 µM) in the absence or presence of EtOH (25 mM), the Notch ligand DLL4 (3 µg/ml), or the Notch inhibitor DAPT (20 µM). EtOH stimulated Notch signaling in HCAEC, as evidenced by increased expression of the Notch receptor and hrt target genes. Treatment with EtOH alone or stimulation of Notch signaling by DLL4 increased eNOS activity and enhanced HCAEC barrier function as assessed by trans-endothelial electrical resistance. Moreover, EtOH and DLL4 both inhibited SAA1-induced monolayer leakiness, cell adhesion molecule (ICAM, VCAM) expression, and monocyte adhesion. The effects of EtOH were Notch-dependent, as they were blocked with DAPT and by Notch receptor (N1, N4) knockdown. In contrast, EtOH's inhibition of SAA1-induced inflammatory cytokines (IL-6, IFN-γ) and reactive oxygen species (ROS) was Notch-independent, as these effects were unaffected by DAPT or by N1 and/or N4 knockdown. CONCLUSIONS: EtOH at moderate levels protects against SAA1-induced endothelial activation via both Notch-dependent and Notch-independent mechanisms. EtOH's maintenance of endothelium in a nonactivated state would be expected to preserve vessel homeostasis and protect against atherosclerosis development.

Levels of Cyclooxygenase 2, Interleukin-6, and Tumour Necrosis Factor-α in Fibroblast Cell Culture Models after Photobiomodulation at 660 nm.

Chemicals and signaling molecules released by injured cells at the beginning of wound healing prompt inflammation. In diabetes, prolonged inflammation is one of the probable causes for delayed wound healing. Increased levels of cyclooxygenase-2 (cox-2), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) are associated with the inflammatory response and in diabetes, and increased levels of these contribute to chronic wounds that do not heal. Rising levels of cox-2, IL-6, and TNF-α have also been associated with increased oxidative stress. Photobiomodulation (PBM) may impact wound healing processes by affecting the signaling pathways and molecules pertinent to tissue repair. In the present study, the effect of PBM (wavelength: 660 nm; energy density: 5 J/cm2) on levels of cox-2, IL-6, and TNF-α was determined in fibroblast cell culture models. Four WS1 models (normal, normal wounded, diabetic, and diabetic wounded) were irradiated at 660 nm, and the culture media was collected at 0, 24, and 48 h postirradiation. Cells that were not irradiated (0 J/cm2) served as the controls. The following parameters were determined postirradiation: cell morphology using light microscopy, cell viability using the Trypan Blue exclusion assay, and levels of the inflammatory markers cox-2, IL-6, and TNF-α were measured using ELISA. Cell migration increased in the wounded groups over the 48 h interval after PBM; viability improved postirradiation in the diabetic wounded groups at 0 and 24 h (P ≤ 0.05 and P ≤ 0.01, respectively); levels of cox-2 decreased in normal and diabetic wounded groups at 0 h (P ≤ 0.001) and increased in the diabetic and diabetic wounded groups at 48 h postirradiation (P ≤ 0.05 and P ≤ 0.01, respectively), while levels of IL-6 decreased in the normal (P ≤ 0.01), diabetic (P ≤ 0.05), and diabetic wounded (P ≤ 0.001) groups at 24 h and in the diabetic and diabetic wounded groups at 48 h (P ≤ 0.05) postirradiation. TNF-α was decreased in the normal wounded groups (P ≤ 0.05) at 48 h. Through its effect on decreased IL-6 levels in diabetic cell models, PBM at 660 nm may be successful at decreasing oxidative stress; however, the present study also found an increase in cox-2 levels at 48 h postirradiation.

In Vitro Wound Healing Potential of Photobiomodulation Is Possibly Mediated by Its Stimulatory Effect on AKT Expression in Adipose-Derived Stem Cells.

Increasing evidence suggests that adipose-derived stem cells (ADSCs) serve as a therapeutic approach for wound healing. The aim of this study was to determine the effect of photobiomodulation (PBM) on antioxidant enzymes in ADSCs. Four ADSC cell models, namely, normal, wounded, diabetic, and diabetic wounded, were irradiated with 660 nm (fluence of 5 J/cm2 and power density of 11.2 mW/cm2) or 830 nm (fluence of 5 J/cm2 and power density of 10.3 mW/cm2). Nonirradiated cells served as controls. Cell morphology and wound migration were determined using light microscopy. Cell viability was determined by the trypan blue exclusion assay. The enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of antioxidants (superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1)). AKT activation and FOXO1 levels were determined by immunofluorescence and western blotting. The gaps (wound) in PBM-treated wounded and diabetic wounded cell models closed faster than the controls. PBM treatment significantly increased antioxidant levels in all cell models. This reflects that oxidative stress is reduced on the counterpart of increased antioxidant levels. This might be due to the activation of the AKT signaling pathway as evidenced by the increased AKT signals via western blotting and immunofluorescence. This data suggests that PBM promotes wound healing by increasing antioxidant levels by activating AKT signaling.