Molecular structure and function of biological barriers

Biological barriers are indispensable for the integrity and function of many vertebrate organs. The barrier function is based on intercellular protein complexes of the plasma membrane which form paracellular diffusion barriers and separate internal and external fluid compartments, an indispensable prerequisite for every organ development and function. The review summarizes key characteristics and molecular structure of intercellular junctions (tight junctions and adherens junctions) responsible for cellular barrier formation. One of the most important such cellular barriers is the blood-brain barrier (BBB) which forms an active interface between the circulation and neural tissue. Its principal cellular components are cerebral endothelial cells, pericytes and astrocytes, whose finely tuned interactions are needed for a proper function. The review highlights the most important functions of the BBB including some novel regulatory aspects as well

The Blood-Brain Barrier and Its Intercellular Junctions in Age-Related Brain Disorders

With age, our cognitive skills and abilities decline. Maybe starting as an annoyance, this decline can become a major impediment to normal daily life. Recent research shows that the neurodegenerative disorders responsible for age associated cognitive dysfunction are mechanistically linked to the state of the microvasculature in the brain. When the microvasculature does not function properly, ischemia, hypoxia, oxidative stress and related pathologic processes ensue, further damaging vascular and neural function. One of the most important and specialized functions of the brain microvasculature is the blood-brain barrier (BBB), which controls the movement of molecules between blood circulation and the brain parenchyma. In this review, we are focusing on tight junctions (TJs), the multiprotein complexes that play an important role in establishing and maintaining barrier function. After a short introduction of the cell types that modulate barrier function via intercellular communication, we examine how age, age related pathologies and the aging of the immune system affects TJs. Then, we review how the TJs are affected in age associated neurodegenerative disorders: Alzheimer's disease and Parkinson's disease. Lastly, we summarize the TJ aspects of Huntington's disease and schizophrenia. Barrier dysfunction appears to be a common denominator in neurological disorders, warranting detailed research into the molecular mechanisms behind it. Learning the commonalities and differences in the pathomechanism of the BBB injury in different neurological disorders will predictably lead to development of new therapeutics that improve our life as we age

De-adhesion dynamics of melanoma cells from brain endothelial layer

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers

Cerebral pericytes and endothelial cells communicate through inflammasome-dependent signals

By upregulation of cell adhesion molecules and secretion of proinflammatory cytokines, cells of the neurovascular unit, including pericytes and endothelial cells, actively participate in neuroinflammatory reactions. As previously shown, both cell types can activate inflammasomes, cerebral endothelial cells (CECs) through the canonical pathway, while pericytes only through the noncanonical pathway. Using complex in vitro models, we demonstrate here that the noncanonical inflammasome pathway can be induced in CECs as well, leading to a further increase in the secretion of active interleukin-1β over that observed in response to activation of the canonical pathway. In parallel, a more pronounced disruption of tight junctions takes place. We also show that CECs respond to inflammatory stimuli coming from both the apical/blood and the basolateral/brain directions. As a result, CECs can detect factors secreted by pericytes in which the noncanonical inflammasome pathway is activated and respond with inflammatory activation and impairment of the barrier properties. In addition, upon sensing inflammatory signals, CECs release inflammatory factors toward both the blood and the brain sides. Consequently, CECs activate pericytes by upregulating their expression of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome-forming pattern recognition receptor. In conclusion, cerebral pericytes and endothelial cells mutually activate each other in inflammation

Rho-Kinase Inhibition Ameliorates Dasatinib-Induced Endothelial Dysfunction and Pulmonary Hypertension

The mutti-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs). The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo, presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects

Paracellular and transcellular migration of metastatic cells through the cerebral endothelium

Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases. Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood-brain barrier. By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps. We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia-like membrane protrusions towards the tumour cells. Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route. On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells. When co-culturing melanoma cells with cerebral endothelial cells, we observed N-cadherin enrichment at melanoma-melanoma and melanoma-endothelial cell borders. However, for breast cancer cells N-cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo. Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells