miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Force-induced changes of α-catenin conformation stabilize vascular junctions independent of vinculin

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin binds F-actin only weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. This epitope, we found to become exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest, that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin, which support constitutive strong binding to actin.

Endothelial-Mesenchymal Transition in Cardiovascular Disease

Endothelial-to-mesenchymal transition is a dynamic process in which endothelial cells suppress constituent endothelial properties and take on mesenchymal cell behaviors. To begin the process, endothelial cells loosen their cell-cell junctions, degrade the basement membrane, and migrate out into the perivascular surroundings. These initial endothelial behaviors reflect a transient modulation of cellular phenotype, that is, a phenotypic modulation, that is sometimes referred to as partial endothelial-to-mesenchymal transition. Loosening of endothelial junctions and migration are also seen in inflammatory and angiogenic settings such that endothelial cells initiating endothelial-to-mesenchymal transition have overlapping behaviors and gene expression with endothelial cells responding to inflammatory signals or sprouting to form new blood vessels. Reduced endothelial junctions increase permeability, which facilitates leukocyte trafficking, whereas endothelial migration precedes angiogenic sprouting and neovascularization; both endothelial barriers and quiescence are restored as inflammatory and angiogenic stimuli subside. Complete endothelial-to-mesenchymal transition proceeds beyond phenotypic modulation such that mesenchymal characteristics become prominent and endothelial functions diminish. In proadaptive, regenerative settings the new mesenchymal cells produce extracellular matrix and contribute to tissue integrity whereas in maladaptive, pathologic settings the new mesenchymal cells become fibrotic, overproducing matrix to cause tissue stiffness, which eventually impacts function. Here we will review what is known about how TGF (transforming growth factor) β influences this continuum from junctional loosening to cellular migration and its relevance to cardiovascular diseases.

Recent Advances in Tumor Targeting via EPR Effect for Cancer Treatment

Cancer causes the second-highest rate of death world-wide. A major shortcoming inherent in most of anticancer drugs is their lack of tumor selectivity. Nanodrugs for cancer therapy administered intravenously escape renal clearance, are unable to penetrate through tight endothelial junctions of normal blood vessels and remain at a high level in plasma. Over time, the concentration of nanodrugs builds up in tumors due to the EPR effect, reaching several times higher than that of plasma due to the lack of lymphatic drainage. This review will address in detail the progress and prospects of tumor-targeting via EPR effect for cancer therapy.

High Content Analysis uncovers functionally relevant metastable phenotypic states in human Endothelial Cells monolayers

SummaryEndothelial cells (EC) present distinct cell properties in different tissues. Heterogeneity within the same vascular bed has been proposed to underpin emergent behaviours in EC monolayers. Quantification and functional relevance of EC phenotypic variance are challenging to assess. Here we developed an EC profiling tool (ECPT) to uniquely enable single EC profiling within a monolayer providing spatial and relational information regarding cell proliferation, inter-endothelial Junctions and NOTCH activation. We used ECPT to characterise differential phenotypes in arterial, venous and microvascular EC populations. Our analysis highlighted extensive heterogeneity within individual monolayers and revealed VEGF-modulable metastability of NOTCH signalling which in turn regulates inter-endothelial junction’s stability. We suggest that accounting for adaptive emerging endothelial behaviours is necessary to develop revascularisation strategies for regenerative medicine and to design more effective EC-targeting drugs for cardiovascular diseases and cancer.