During immune surveillance and inflammation, leukocytes exit the vasculature through transient openings in the endothelium without causing plasma leakage. However, the exact mechanisms behind this intriguing phenomenon are still unknown. Here we report that maintenance of endothelial barrier integrity during leukocyte diapedesis requires local endothelial RhoA cycling. Endothelial RhoA depletion in vitro or Rho inhibition in vivo provokes neutrophil-induced vascular leakage that manifests during the physical movement of neutrophils through the endothelial layer. Local RhoA activation initiates the formation of contractile F-actin structures that surround emigrating neutrophils. These structures that surround neutrophil-induced endothelial pores prevent plasma leakage through actomyosin-based pore confinement. Mechanistically, we found that the initiation of RhoA activity involves ICAM-1 and the Rho GEFs Ect2 and LARG. In addition, regulation of actomyosin-based endothelial pore confinement involves ROCK2b, but not ROCK1. Thus, endothelial cells assemble RhoA-controlled contractile F-actin structures around endothelial pores that prevent vascular leakage during leukocyte extravasation.

ObjectivesThrombotic and microvascular complications are frequently seen in deceased COVID‐19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation‐induced endothelial activation remains highly contentious.MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial–endothelial cell barrier.ResultsWe show that primary human endothelial cells express very low levels of the SARS‐CoV‐2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS‐CoV‐2. We also show that SARS‐CoV‐2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co‐culture model endothelial cells are not infected with SARS‐CoV‐2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM‐1 expression and releasing pro‐inflammatory cytokines.ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS‐CoV‐2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS‐CoV‐2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS‐CoV‐2 pathogenesis by sensing adjacent infection and mounting a pro‐inflammatory response to SARS‐CoV‐2.

Blood and lymphatic vessels are lined by endothelial cells which constantly interact with their luminal and abluminal extracellular environments. These interactions confer physical forces on the endothelium, such as shear stress, stretch and stiffness, to mediate biological responses. These physical forces are often altered during disease, driving abnormal endothelial cell behavior and pathology. Therefore, it is critical that we understand the mechanisms by which endothelial cells respond to physical forces. Traditionally, endothelial cells in culture are grown in the absence of flow on stiff substrates such as plastic or glass. These cells are not subjected to the physical forces that endothelial cells endure in vivo, thus the results of these experiments often do not mimic those observed in the body. The field of vascular biology now realize that an intricate analysis of endothelial signaling mechanisms requires complex in vitro systems to mimic in vivo conditions. Here, we will review what is known about the mechanical forces that guide endothelial cell behavior and then discuss the advancements in endothelial cell culture models designed to better mimic the in vivo vascular microenvironment. A wider application of these technologies will provide more biologically relevant information from cultured cells which will be reproducible to conditions found in the body.

Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens. It serves as a protective response that involves leukocytes, blood vessels and molecular mediators with the purpose to eliminate the initial cause of cell injury and to initiate tissue repair. Inflammation is tightly regulated by the body and is associated with transient crossing of leukocytes through the blood vessel wall, a process called transendothelial migration (TEM) or diapedesis. TEM is a close collaboration between leukocytes on one hand and the endothelium on the other. Limiting vascular leakage during TEM but also when the leukocyte has crossed the endothelium is essential for maintaining vascular homeostasis. Although many details have been uncovered during the recent years, the molecular mechanisms from the vascular part that drive TEM still shows significant gaps in our understanding. This review will focus on the local signals that are induced in the endothelium that regulate leukocyte TEM and simultaneous preservation of endothelial barrier function.