VE-Cadherin and Vesicles Differentially Regulate Lymphatic Vascular Permeability to Solutes of Various Sizes

Lymphatic vascular permeability prevents lymph leakage that is associated with lymphedema, lymphatic malformations, obesity, and inflammation. However, the molecular control of lymphatic permeability remains poorly understood. Recent studies have suggested that adherens junctions and vesicle transport may be involved in regulating lymphatic vessel permeability. To determine the contribution of each transport pathway, we utilized an ex vivo permeability assay to directly measure the solute flux of various molecular weight solutes across a range of pressures in intact murine collecting lymphatic vessels. Pharmacological and biological tools were used to probe the relative contributions of vesicles and junction proteins in the lymphatic vasculature. We show that the permeability of collecting lymphatic vessels is inversely related to the solute molecular weight. Further, our data reveal that vesicles selectively transport BSA, as an inhibitor of vesicle formation significantly decreased the permeability to BSA (∼60% decrease, n = 8, P = 0.02), but not to 3 kDa dextran (n = 7, P = 0.41), α-lactalbumin (n = 5, P = 0.26) or 70 kDa dextran (n = 8, P = 0.13). In contrast, disruption of VE-cadherin binding with a function blocking antibody significantly increased lymphatic vessel permeability to both 3 kDa dextran (5.7-fold increase, n = 5, P < 0.0001) and BSA (5.8-fold increase, n = 5, P < 0.0001). Thus, in the lymphatic vasculature, adherens junctions did not exhibit selectivity for any of the solutes tested here, whereas vesicles specifically transport BSA. Overall, the findings suggest that disease states that disrupt VE-cadherin localization or expression will cause significant leakage of solutes and fluid from the lymphatic vasculature.

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies

Endothelial cells (ECs) lining the blood vessels are important players in many biological phenomena but are crucial in hypoxia-dependent diseases where their deregulation contributes to pathology. On the other hand, processes mediated by ECs, such as angiogenesis, vessel permeability, interactions with cells and factors circulating in the blood, maintain homeostasis of the organism. Understanding the diversity and heterogeneity of ECs in different tissues and during various biological processes is crucial in biomedical research to properly develop our knowledge on many diseases, including cancer. Here, we review the most important aspects related to ECs’ heterogeneity and list the available in vitro tools to study different angiogenesis-related pathologies. We focus on the relationship between functions of ECs and their organo-specificity but also point to how the microenvironment, mainly hypoxia, shapes their activity. We believe that taking into account the specific features of ECs that are relevant to the object of the study (organ or disease state), especially in a simplified in vitro setting, is important to truly depict the biology of endothelium and its consequences. This is possible in many instances with the use of proper in vitro tools as alternative methods to animal testing.

In vitro and in vivo testing of a novel local nicardipine delivery system to the brain: a preclinical study

OBJECTIVEThe management of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains a highly demanding challenge in critical care medicine. Despite all efforts, the calcium channel antagonist nimodipine remains the only drug approved for improving outcomes after aSAH. However, in its current form of application, it provides less than optimal efficacy and causes dose-limiting hypotension in a substantial number of patients. Here, the authors tested in vitro the release dynamics of a novel formulation of the calcium channel blocker nicardipine and in vivo local tolerance and tissue reaction using a chronic cranial window model in mice.METHODSTo characterize the release kinetics in vitro, dissolution experiments were performed using artificial cerebrospinal fluid over a time period of 21 days. The excipients used in this formulation (NicaPlant) for sustained nicardipine release are a mixture of two completely degradable polymers. A chronic cranial window in C57BL/6 mice was prepared, and NicaPlant slices were placed in proximity to the exposed cerebral vasculature. Epifluorescence video microscopy was performed right after implantation and on days 3 and 7 after surgery. Vessel diameter of the arteries and veins, vessel permeability, vessel configuration, and leukocyte–endothelial cell interaction were quantified by computer-assisted analysis. Immunofluorescence staining was performed to analyze inflammatory reactions and neuronal alterations.RESULTSIn vitro the nicardipine release profile showed an almost linear curve with about 80% release at day 15 and full release at day 21. In vivo epifluorescence video microscopy showed a significantly higher arterial vessel diameter in the NicaPlant group due to vessel dilatation (21.6 ± 2.6 µm vs 17.8 ± 1.5 µm in controls, p < 0.01) confirming vasoactivity of the implant, whereas the venous diameter was not affected. Vessel dilatation did not have any influence on the vessel permeability measured by contrast extravasation of the fluorescent dye in epifluorescence microscopy. Further, an increased leukocyte–endothelial cell interaction due to the implant could not be detected. Histological analysis did not show any microglial activation or accumulation. No structural neuronal changes were observed.CONCLUSIONSNicaPlant provides continuous in vitro release of nicardipine over a 3-week observation period. In vivo testing confirmed vasoactivity and lack of toxicity. The local application of this novel nicardipine delivery system to the subarachnoid space is a promising tool to improve patient outcomes while avoiding systemic side effects.

Modulating Sphingosine-1-Phosphate receptors to improve chemotherapy delivery to Ewing sarcoma

ABSTRACTTumor vasculature is innately dysfunctional. Poorly functional tumor vessels inefficiently deliver chemotherapy to tumor cells; vessel hyper-permeability promotes chemotherapy delivery primarily to a tumor’s periphery. Here we identify a method for enhancing chemotherapy delivery and efficacy in Ewing sarcoma (ES) in mice by modulating tumor vessel permeability. Vessel permeability is partially controlled by the G protein-coupled Sphinosine-1-phosphate receptors 1 and 2 (S1PR1 and S1PR2) on endothelial cells. S1PR1 promotes endothelial cell junction integrity while S1PR2 destabilizes it. We hypothesize that an imbalance of S1PR1:S1PR2 is partially responsible for the dysfunctional vascular phenotype characteristic of ES and that by altering the balance in favor of S1PR1, ES vessel hyper-permeability can be reversed. In this study, we demonstrate that pharmacologic activation of S1PR1 by SEW2871 or inhibition of S1PR2 by JTE-013 caused more organized, mature, and functional tumor vessels. Importantly, S1PR1 activation or S1PR2 inhibition improved chemotherapy delivery to the tumor and anti-tumor efficacy. Our data suggests that pharmacologic targeting of S1PR1 and S1PR2 may be a useful adjuvant to standard chemotherapy for ES patients.NOVELTY AND IMPACTThis study demonstrates that Sphingosine-1-Phosphate (S1P) receptors are potential novel targets for tumor vasculature remodeling and adjuvant therapy for the treatment of Ewing Sarcoma. Unlike receptor tyrosine kinases that have already been extensively evaluated for use as vascular normalizing agents in oncology, S1P receptors are G protein-coupled receptors, which have not been well studied in tumor endothelium. Pharmacologic activators and inhibitors of S1P receptors are currently in clinical trials for treatment of auto-immune and cardiovascular diseases, indicating potential for clinical translation of this work.