ABSTRACT
During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.
Subject(s)
Adherens Junctions/metabolism , Endothelium, Vascular/physiology , Histamine/metabolism , Inflammation/pathology , Leukocytes/physiology , Lipopolysaccharides/metabolism , Neutrophils/physiology , Capillary Permeability , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Movement , Cells, Cultured , Cytokines/metabolism , Cytoskeleton/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Transendothelial and Transepithelial MigrationABSTRACT
Thrombospondin 4 (TSP-4) expression is induced in the heart and vasculature under pathological conditions, including myocardial infarction, myocardial pressure overload, and hypertension. TSP-4 is linked to remodelling processes, where it may affect extracellular matrix protein organization. In previous work, we studied the role of TSP-4 in small arteries during hypertension using Ang II-treated Thrombospondin 4 knockout (Thbs4-/-) mice. We reported increased heart weight, as well as the occurrence of aortic aneurysms in the Ang II-treated Thbs4-/- animals. In the present study, we further characterized the hearts and aortas from these animals. Hypertrophy of cardiomyocytes, together with perivascular fibrosis and inflammation was observed in the Ang II-treated Thbs4-/- hearts. In the aortas, an increase in the aortic wall cross-sectional area (CSA) and wall thickness of the Ang II-treated Thbs4-/- mice was found. More detailed investigation of the Ang II-treated Thbs4-/- aortas also revealed the appearance of aortic dissections in the outer medial layer of the arteries, as well as pronounced inflammation. No differences were found in several other extracellular matrix-related parameters, such as number of elastin breaks or stress-strain relationships. However, at the ultrastructural level, collagen fibers showed alterations in diameter in the media and adventitia of the Ang II-treated Thbs4-/- mice, in the area prone to dissection. In conclusion, we identified TSP-4 as an important protein in the development of cardiac hypertrophy and aortic dissections in Ang II-induced hypertension.
