No abstract

Background: Variants in the ring finger protein 213 ( RNF213 ) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. Methods: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. Results: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. Conclusions: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

There is a high incidence of failure and recurrence for chronic skin wounds following conventional therapies. To promote healing, the use of skin substitutes containing living cells as wound dressings has been proposed. The aim of this study was to produce a scaffold-free cell-based bilayered tissue-engineered skin substitute (TES) containing living fibroblasts and keratinocytes suitable for use as wound dressing, while considering production time, handling effort during the manufacturing process, and stability of the final product. The self-assembly method, which relies on the ability of mesenchymal cells to secrete and organize connective tissue sheet sustaining keratinocyte growth, was used to produce TESs. Three fibroblast-seeding densities were tested to produce tissue sheets. At day 17, keratinocytes were added onto 1 or 3 (reference method) stacked tissue sheets. Four days later, TESs were subjected either to 4, 10, or 17 days of culture at the air–liquid interface (A/L). All resulting TESs were comparable in terms of their histological aspect, protein expression profile and contractile behavior in vitro. However, signs of extracellular matrix (ECM) digestion that progressed over culture time were noted in TESs produced with only one fibroblast-derived tissue sheet. With lower fibroblast density, the ECM of TESs was almost completely digested after 10 days A/L and lost histological integrity after grafting in athymic mice. Increasing the fibroblast seeding density 5 to 10 times solved this problem. We conclude that the proposed method allows for a 25-day production of a living TES, which retains its histological characteristics in vitro for at least two weeks.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

No abstract

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.

The environment experienced by females can have long-lasting effects on offspring phenotype. The objective of this study was to determine if maternal stress-induced behaviour reprogramming in offspring is found in brook char and to test whether cortisol is the main mediator, by separating the potential effects of cortisol from that of other potential maternal factors. We exposed female brook trout (Salvelinus fontinalis) to different parallel treatments during the oogenesis period: undisturbed as controls (1) fed cortisol through food (2) or physically stressed by handling once a week (3). Additionally, we exposed half of the control eggs to a cortisol suspension before fertilisation (4). Cortisol consumption and handling did not elevate either maternal plasma or egg cortisol, although egg cortisol level was significantly increased when eggs were bathed in the suspension. We measured spatial learning and memory, boldness and neophobia in 6 month-old offspring and found no effects of treatments on learning, memory or behaviour. Our results suggest that the relationship between maternal stress, circulating and egg cortisol levels, other maternal factors, and behavioural reprogramming is context and species-specific.

Due to its superficial anatomical localization, the cornea is continuously subjected to injuries. Damages to the corneal epithelium trigger important changes in the composition of the extracellular matrix to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by membrane-bound integrins and ultimately lead to re-epithelialization of the injured epithelium through intracellular signalin. Among the many downstream targets of the integrin-activated signaling pathways, WNK1 is the kinase whose activity is the most strongly increased during corneal wound healing. We previously demonstrated that pharmacological inhibition of WNK1 prevents proper closure of wounded human tissue-engineered cornea in vitro. In the present study, we investigated the molecular mechanisms by which WNK1 contributes to corneal wound healing. By exploiting transcription factors microarrays, electrophoretic mobility-shift assay, and gene profiling analyses, we demonstrated that the DNA binding properties and expression of numerous transcription factors (TFs), including the well-known, ubiquitous TFs specific protein 1 (Sp1) and activator protein 1 (AP1), were reduced in hCECs upon WNK1 inhibition by WNK463. This process appears to be mediated at least in part by alteration in both the ubiquitination and glycosylation status of these TFs. These changes in TFs activity and expression impacted the transcription of several genes, including that encoding the α5 integrin subunit, a well-known target of both Sp1 and AP1. Gene profiling revealed that only a moderate number of genes in hCECs had their level of expression significantly altered in response to WNK463 exposition. Interestingly, analysis of the microarray data for these deregulated genes using the ingenuity pathway analysis software predicted that hCECs would stop migrating and proliferating but differentiate more when they are grown in the presence of the WNK1 inhibitor. These results demonstrate that WNK1 plays a critical function by orienting hCECs into the appropriate biological response during the process of corneal wound healing.