Tissue Barriers最新文献

Numerous signaling pathways are activated during hypoxia to facilitate angiogenesis, promoting interactions among endothelial cells and initiating downstream signaling cascades. Although the pivotal role of the nitric oxide (NO) response pathway is well-established, the involvement of arginine-specific metabolism and bioactive lipid mechanisms in 3D flow-activated in vitro models remains less understood. In this study, we explored the levels of arginine-specific metabolites and bioactive lipids in human coronary artery endothelial cells (HCAECs) under both transient and persistent hypoxia. We compared targeted metabolite levels between a 2D static culture model and a 3D microvessels-on-chip model. Notably, we observed robust regulation of NO metabolites in both transient and persistent hypoxic conditions. In the 2D model under transient hypoxia, metabolic readouts of bioactive lipids revealed increased oxidative stress markers, a phenomenon not observed in the 3D microvessels. Furthermore, we made a novel discovery that the responses of bioactive lipids were regulated by hypoxia inducible factor-1α (HIF-1α) in the 2D cell culture model and partially by HIF-1α and flow-induced shear stress in the 3D microvessels. Immunostaining confirmed the HIF-1α-induced regulation under both hypoxic conditions. Real-time oxygen measurements in the 3D microvessels using an oxygen probe validated that oxygen levels were maintained in the 3D model. Overall, our findings underscore the critical regulatory roles of HIF-1α and shear stress in NO metabolites and bioactive lipids in both 2D and 3D cell culture models.

Dengue virus (DENV) infections are commonly reported in the tropical and subtropical regions of the world. DENV is reported to exploit various strategies to cross the blood-brain barrier. The NS1 protein of DENV plays an important role in viral neuropathogenesis, resulting in endothelial hyperpermeability and cytokine-induced vascular leak. miRNAs are short non-coding RNAs that play an important role in post-transcriptional gene regulations. However, no comprehensive information about the involvement of miRNAs in DENV-NS1-mediated neuropathogenesis has been explored to date. We observed that DENV-NS1 significantly alters the cellular miRNome of human cerebral microvascular endothelial cells in a bystander fashion. Subsequent target prediction and pathway enrichment analysis indicated that these microRNAs and their corresponding target genes are involved in pathways associated with blood-brain barrier dysfunction such as "Adherens junction" and "Tight junction". Additionally, several miRNA-mRNA pairs were also found to be involved in cellular signaling pathways related to cytokine production, for instance, "Jak-STAT signaling pathway", "Chemokine signaling pathway", "IL-17 signaling pathway", "NF-κB signaling pathway", and "Viral protein interaction with cytokine and cytokine receptor". The dysregulated production of inflammatory cytokines is reported to compromise BBB permeability. This study is the first report to demonstrate that DENV-NS1-mediated miRNA perturbations are crucial in compromising endothelial barrier integrity. It also offers insights into potential therapeutic targets to mitigate DENV-NS1-induced vascular permeability and inflammation.

In vitro models are of great importance in advancing our understanding of human diseases, especially complex disorders with unknown etiologies like inflammatory bowel diseases (IBD). One of the key IBD features is the increased intestinal permeability. The disruption of the intestinal barrier can occur due to a destructive inflammatory response involving intestinal cell death. Alternatively, proteins that form tight junctions (TJ) fail to form function complexes and promote epithelial barrier disruption. The mechanisms behind this process are not fully understood. Thus, in vitro models that facilitate studying the intestinal barrier and its molecular components are of particular importance in the context of IBD. There are in vitro and ex vivo models that can be used to recapitulate some aspects of IBD. Among these are intestinal explants, crypts, and epithelial 3D-organoids. Here we describe some practical limitations of isolated crypts, gut tissue explants, and intestinal organoids as models in epithelial barrier biology, and TJ in particular. Our findings demonstrate that only 3D intestinal organoids formed from single cells are suitable to study barrier permeability in vitro, as primary crypt-derived organoids do not retain epithelial integrity due to cell death. Importantly, 3D organoids raised in culture conditions may fail to recapitulate inflammatory and barrier phenotypes of the source mouse model. To study the features of the inflamed epithelium, ex vivo intestinal explants and crypts were employed. We show here that isolated crypts do not preserve native TJ structure in a long-term experimental setting and tend to disintegrate in the unsupported culture environment. However, intestinal explants were stable in culture conditions for about 24 hours and demonstrated their applicability for short-term living tissue imaging and fluorescence recovery after photobleaching (FRAP). Thus, a combination of 3D organoids and intestinal explants provides a more accurate experimental platform to understand the intestinal epithelial barrier.

Traumatic penumbra (TP) is a secondary injury area located around the core area of traumatic brain injury after brain trauma, and is an important factor affecting the outcome of traumatic brain injury (TBI). The main pathological change caused by TP is brain edema, including (cellular brain edema and vascular brain edema). The formation and development of brain edema in the TP area are closely related to the blood-brain barrier (BBB) and vascular endothelial growth factor (VEGF). VEGF is a vascular permeability factor that can promote angiogenesis and increase BBB permeability, and there is a debate on the pros and cons of its role in early TBI. Therefore, in the early stage of TBI, when using the VEGF inhibitor bevacizumab to treat TP area brain edema, the timing of bevacizumab administration is particularly important, and there are currently no relevant literature reports. This article explores the treatment time window and optimal treatment time point of bevacizumab in the treatment of cerebral edema in the TP area by administering the same dose of bevacizumab at different time points after brain injury in rats. The results showed that there was traumatic brain edema in TP area, BBB structure and function were damaged, VEGF expression and angiogenesis were increased. Compared with TBI + NS Group, after Bevacizumab treatment, brain edema in TP area was alleviated, BBB structure and function were improved, VEGF expression and angiogenesis were decreased in each treatment group, and the effect of TBI + Bevacizumab 1 h group was the most significant. Bevacizumab can be used as a targeted therapy for traumatic brain edema. The therapeutic time window of bevacizumab for traumatic brain edema is within 12 hours after TBI, and 1 h is the optimal therapeutic time point.

The incidence of kidney disease from acute and chronic conditions continues to escalate worldwide. Interventions to replace renal function after organ failure remain limited to dialysis or transplantation, as human kidneys exhibit a limited capacity to repair damaged cells or regenerate new ones. In contrast, animals ranging from flies to fishes and even some mammals like the spiny mouse exhibit innate abilities to regenerate their kidney cells following injury. Now, a recent study has illuminated how the Mexican salamander, Ambystoma mexicanum, most commonly known as the axolotl, possesses a kidney with remarkable similarity to humans, which can robustly regenerate following acute chemical damage. These discoveries position the axolotl as a new model that can be used to advance our understanding about the fundamental mechanisms of kidney regeneration.

Background: A number of peptide incretin receptor agonists (IRAs) show promise as therapeutics for Alzheimer's disease (AD) and Parkinson's disease (PD). Transport across the blood-brain barrier (BBB) is one way for IRAs to act directly within the brain. To determine which IRAs are high priority candidates for treating these disorders, we have studied their brain uptake pharmacokinetics.

Methods: We quantitatively measure the ability of four IRAs to cross the BBB. We injected adult male CD-1 mice intravenously with ¹²⁵I- or ¹⁴C-labeled albiglutide, dulaglutide, DA5-CH, or tirzepatide and used multiple-time regression analyses to measure brain kinetics up to 1 hour. For those IRAs failing to enter the brain 1 h after intravenous injection, we also investigated their ability to enter over a longer time frame (i.e., 6 h).

Results: Albiglutide and dulaglutide had the fastest brain uptake rates within 1 hour. DA5-CH appears to enter the brain rapidly, reaching equilibrium quickly. Tirzepatide does not appear to cross the BBB within 1 h after iv injection but like albumin, did so slowly over 6 h, presumably via the extracellular pathways.

Conclusions: We find that IRAs can cross the BBB by two separate processes; one that is fast and one that is slow. Three of the four IRAs investigated here have fast rates of transport and should be taken into consideration for testing as AD and PD therapeutics as they would have the ability to act quickly and directly on the brain as a whole.

Cilia are hair-like structures found on the surface of nearly all vertebrate cell types where they have central roles in regulating development and orchestrating physiological events. There is growing interest in understanding the mechanisms of ciliogenesis due to the profound consequences that follow from the absence of proper ciliary function, which include diseases that affect the renal, respiratory, reproductive, nervous, visual, and digestive systems, among others. Now, a recent report has discerned new roles for the transcription factor estrogen-related receptor gamma a (esrrγa) in ciliated cell ontogeny within the embryonic zebrafish kidney and other tissues. Further, the team of researchers discovered that genetic ablation of murine homolog ERRγ in adult kidney epithelial cells led to shortened cilia, which precedes cystogenesis. These intriguing findings expand our fundamental understanding of the pathological basis of cilia defects, which is relevant for identifying future therapeutic targets for ciliopathies.

Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm², indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated liquid handling and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine's efficacy, toxicity, and permeability under near-physiological conditions.