In Vitro Methods for Measuring the Permeability of Cell Monolayers

Cell monolayers, including endothelial and epithelial cells, play crucial roles in regulating the transport of biomolecules to underlying tissues and structures via intercellular junctions. Moreover, the monolayers form a semipermeable barrier across which leukocyte transmigration is tightly regulated. The inflammatory cytokines can disrupt the epithelial and endothelial permeability, thus the reduced barrier integrity is a hallmark of epithelial and endothelial dysfunction related with numerous pathological conditions, including cancer-related inflammation. Therefore, the assessment of barrier function is critical in in vitro models of barrier-forming tissues. This review summarizes the commercially available in vitro systems used to measure the permeability of cellular monolayers. The presented techniques are separated in two large groups: macromolecular tracer flux assays, and electrical impedance measurement-based permeability assays. The presented techniques are briefly described and compared.

PAF Physiology in Target Organ Systems—A Deep Dive to Understand the PAF Mystery in Pathogenesis of Disease

The purpose of this literature review is to gain an overview of the role of platelet-activating factor (PAF) within each of the body systems and how it contributes to normal and pathophysiological states. The review showed that there are multiple functions of PAF that are common to several body systems; however, there is little evidence to explain why PAF has this affect across multiple systems. Interestingly, there seems to be conflicting research as to whether PAF is an overall protective or pathogenic pathway. Within this research, it was found that there are different pathways depending on the specific body system, as well as between body systems. However, one universal function reported in the literature is of PAF as a pro-inflammatory molecule. Overall, this review identified five major functions of PAF: vasoconstriction, increased inflammation, vascular remodeling, increased edema, and endothelial permeability.

Leukotriene D4 Upregulates Oxidized Low-Density Lipoprotein Receptor 1 and CD36 to Enhance Oxidized LDL Uptake and Phagocytosis in Macrophages Through Cysteinyl Leukotriene Receptor 1

Endothelial permeability, leukocyte attachment, and unregulated oxidized LDL (oxLDL) uptake by macrophages leading to the formation of foam cells are all vital in the initiation and progression of atherosclerosis. During inflammation, several inflammatory mediators regulate this process through the expression of distinct oxLDL binding cell surface receptors on macrophages. We have previously shown that Leukotriene D4 (LTD4) promotes endothelial dysfunction, increasing endothelial permeability and enhancing TNFα-mediated attachment of monocytes to endothelium, which hints at its possible role in atherosclerosis. Here we analyzed the effect of LTD4 on macrophage function. Macrophages mainly express CysLT1R and flux calcium in response to LTD4. Further, LTD4 potentiates phagocytosis in macrophages as revealed by the uptake of zymosan particles. Notably, LTD4 augmented macrophage phagocytosis and oxLDL uptake which is sensitive to MK-571 [Montelukast (MK)], a CysLT1R-specific antagonist. Mechanistically, LTD4 upregulated two receptors central to foam cell formation, oxidized low-density lipoprotein receptor-1 (OLR1/LOX-1), and CD36 in a time and dose-dependent manner. Finally, LTD4 enhanced the secretion of chemokines MCP-1 and MIP1β. Our results suggest that LTD4 contributes to atherosclerosis either through driving foam cell formation or recruitment of immune cells or both. CysLT1R antagonists are safely being used in the treatment of asthma, and the findings from the current study suggest that these can be re-purposed for the treatment of atherosclerosis.

Resolvin D1 inhibits endothelial permeability and mitochondrial damage following cardiac ischemia reperfusion in diabetic mice

Purpose Novel strategies for preventing myocardial ischemia reperfusion injury (MIRI) in a diabetic heart are urgently needed. Resolvin D1 (RvD1) plays important therapeutic roles in inflammatory diseases. However, the therapeutic role of RvD1 in diabetic MIRI is still unknown. Methods Diabetic mice were established with a high-fat diet and streptozotocin (STZ). The mice were pretreated with RvD1 via intraperitoneal injection for 3 days, followed by MIRI. To evaluate the effects of RvD1 on chronic cardiac remodelling, RvD1 was administered for another 2 weeks after MIRI. The effects of RvD1 following MIRI were measured, including the severity of infarct size, regional inflammation, cardiac function, and permeability of cultured endothelial monolayers. Mitochondrial reactive oxygen species (MitoROS) and mitochondrial membrane potential (MMP) were determined using MitoSOX and JC-1. Results RvD1 pretreatment significantly reduced infarct size and the Evans blue content in diabetic injured hearts, which was associated with improved endothelial permeability. At 2 weeks after MIRI, RvD1 treatment partially improved cardiac performance and reduced cardiac fibrosis in diabetic MIRI mice. In vitro, RvD1 attenuated endothelial leakage induced by hypoxia-reoxygenation, H2O2, and lipopolysaccharide (LPS) under high glucose (HG) conditions. Meanwhile, RvD1 remarkably protected endothelial cells from H2O2-induced mitochondrial damage, as evidenced by increased MMP and decreased MitoROS, which was associated with the preservation of VE-cadherin. Conclusion RvD1 alleviates MIRI-induced endothelial permeability and mitochondrial damage injuries in diabetic hearts. Therefore, RvD1 could be a potential therapeutic target for MIRI in diabetes.

Frizzled-4 regulates β-catenin in endothelial cells exposed to disturbed flow via an atypical Wnt pathway leading to proinflammatory activation and increased permeability

Objective: Endothelial cells are regulated by hemodynamic wall shear stress and multidirectional shear stress is known to promote endothelial dysfunction, although the molecular mechanisms are poorly defined. Wnt pathways play an important role in non-vascular mechanoresponsive cells. Here we investigated their role in endothelial mechanosignalling and endothelial dysfunction. Approach & Results: Human aortic endothelial cells were exposed to shear stress using an orbital shaker. The expression of Frizzled-4 receptors was significantly increased in endothelial cells exposed to low magnitude multidirectional flow (LMMF) relative to high magnitude uniaxial flow (HMUF). Increased expression was also detected in regions of the murine aortic arch exposed to LMMF. The increased Frizzled-4 expression in cultured cells was abrogated following knockdown of R-spondin-3 (RSPO-3) using RNA interference. LMMF also increased the stabilisation and nuclear localisation of β-catenin, an effect that was dependent on Frizzled-4 and RSPO-3. Inhibition of β-catenin using a small molecule inhibitor (iCRT5), or knockdown of Frizzled-4 or R-spondin-3 resulted in a significant reduction of pro-inflammatory gene expression in endothelial cells exposed to LMMF. Stabilisation of the β-catenin destruction complex using IWR-1 under LMMF also reduced pro-inflammatory gene expression, as did inhibition of Wnt5a signalling. Interestingly, inhibition of the canonical Wnt pathway had no effect. Inhibition of β-catenin signalling also reduced endothelial permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. Conclusions: These data suggest the presence of an atypical Wnt-β-catenin pathway in endothelial cells that promotes inflammatory activation and barrier disruption in response to LMMF.