Background: Loss of Krev-interaction trapped 1 (KRIT1) drives the activation of endothelial cells. Results: Blocking vascular endothelial growth factor (VEGF) signaling reverses the endothelial cell changes after KRIT1 depletion and permeability in KRIT1-deficient mice. Conclusion: Increased VEGF signaling downstream of KRIT1 loss alters endothelial cell behavior and is responsible for permeability in vivo. Significance: These studies describe a novel regulation of endothelial barrier function by KRIT1.

Activated neutrophils interacting with the vessel wall can alter vascular permeability to macromolecules such as albumin via release of various secretion products that induce changes in the endothelial monolayer. In the current work we used cremaster microvessels of anesthetized mice to show that, in addition to this paracrine mechanism, leukocyte ligation of endothelial ICAM-1 directly activates endothelial cell (EC) signaling, altering EC permeability to albumin [i.e., solute permeability (P(s))]. We show that antibody cross-linking of surface ICAM-1 in intact microvessels is sufficient to increase P(s) even in the absence of interacting leukocytes. Unstimulated arterioles do not support leukocyte-EC interactions, but despite this, antibody ligation of ICAM-1 in these vessels induced a twofold increase in P(s). Similarly, in venules that were depleted of interacting neutrophils, P(s) was decreased to below resting levels and was restored by ligation of ICAM-1. Use of function-blocking antibodies to separately block leukocyte rolling or adhesion under unstimulated or TNF-α-activated conditions established that both rolling and adhered leukocytes contribute to P(s) regulation in situ. Both rolling and adhesion activated EC-dependent signaling mechanisms that increased P(s). ICAM-1 ligation with primary antibody alone or primary followed by secondary antibodies showed that regulation of P(s) is directly dependent on the degree of ICAM-1 clustering. Under physiological versus inflamed conditions, respectively, this ICAM-1 clustering-dependent regulation of P(s) switches from PKC dependent and Src independent to Src dependent and PKC independent. This study thus identifies a new mechanism by which antiadhesion treatment may constitute a potential therapy for tissue edema.

In microvessels, acute inflammation is typified by an increase in leukocyte-endothelial cell interactions, culminating in leukocyte transmigration into the tissue, and increased permeability to water and solutes, resulting in tissue edema. The goal of this study was to establish a method to quantify solute permeability (P(s)) changes in microvessels in intact predominantly blood-perfused networks in which leukocyte transmigratory behavior could be precisely described using established paradigms. We used intravital confocal microscopy to measure solute (BSA) flux across microvessel walls, hence P(s). A quantitative fluorescence approach (Huxley VH, Curry FE, and Adamson RH. Am J Physiol Heart Circ Physiol 252: H188-H197, 1987) was adapted to the imaged confocal tissue slice in which the fluorescent source volume and source surface area of the microvessel were restricted to the region of vessel that was contained within the imaged confocal tissue section. P(s) measurements were made in intact cremaster muscle microvasculature of anesthetized mice and compared with measurements of P(s) made in isolated rat skeletal muscle microvessels. Mouse arteriolar P(s) was 9.9 +/- 1.1 x 10(-7) cm/s (n = 16), which was not different from 8.4 +/- 1.3 x 10(-7) cm/s (n = 6) in rat arterioles. Values in venules were significantly (P < 0.05) higher: 44.4 +/- 7.9 x 10(-7) cm/s (n = 14) in mice and 25.0 +/- 3.7 x 10(-7) cm/s in rats. Convective coupling was estimated to contribute <10% to the measured P(s) in both microvessel types and both animal models. We conclude that this approach provides an appropriate quantification of P(s) in the intact microvasculature and that arteriolar P(s), while lower than in venules, is nevertheless consistent with arterioles being a significant source of interstitial protein.

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells (HUVECs) enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable to TNF-a treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured trans-endothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa MW) across the monolayer. Removal of EGL components decreased TEER, but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Endothelial cells (EC) respond to mechanical forces such as shear stress in a variety of ways, one of which is cytoskeletal realignment in the direction of flow. Our earlier studies implicated the extracellular matrix protein fibronectin in mechanosensory signaling to ECs in intact arterioles, via a signaling pathway dependent on the heparin-binding region of the first type III repeat of fibrillar fibronectin (FNIII1H). Here we test the hypothesis that FNIII1H is required for EC stress fiber realignment under flow. Human umbilical vein ECs (HUVECs) exposed to defined flow conditions were used as a well-characterized model of this stress fiber alignment response. Our results directly implicate FNIII1H in realignment of stress fibers in HUVECs and, importantly, show that the matricryptic heparin-binding RWRPK sequence located in FNIII1 is required for the response. Furthermore, we show that flow-mediated stress fiber realignment in ECs adhered via αβ-integrin-specific ligands does not occur in the absence of FHIII1H, whereas, in contrast, αβ-integrin-mediated stress fiber realignment under flow does not require FNIII1H. Our findings thus indicate that there are two separate mechanosignaling pathways mediating the alignment of stress fibers after exposure of ECs to flow, one dependent on αβ-integrins and one dependent on FNIII1H. This study strongly supports the conclusion that the RWRPK region of FNIII1H may have broad capability as a mechanosensory signaling site.

Changes in the stability of endothelial cell‐cell contacts are commonly associated with chronic and acute vascular inflammation. However, it remains unclear how changes in endothelial cell‐cell contact impact the progress of vascular inflammation. KRIT1‐deficient animals lack the adherens junction accessory protein KRIT1, and exhibit a loss of cell‐cell contact stability. Krit1+/‐ mice exhibit increased baseline microvessel permeability, but maintain normal leukocyte responses. Stimulation of Krit1+/‐ mice with various inflammatory mediators enhanced edema formation but not leukocyte activation. Krit1+/‐ mice exhibited relatively normal leukocyte responses to TNF‐α, IL‐1β, and histamine, but, notably, Krit1+/‐ vessels did not become more permeable following TNF‐a treatment, contrary to what was seen in littermate controls. Though TNF‐α signaling in KRIT1 deficient endothelial cells appeared normal, these cells displayed increased oxidative stress, and treatment with TNF‐α failed to enhance reactive oxygen species (ROS) levels. In vivo, targeted inhibition of endothelial ROS was sufficient to restore microvascular permeability in Krit1+/‐ mice to WT levels. However, antioxidant treatment of TNF‐α‐treated WT and Krit1+/‐ animals reversed only venular permeability. KRIT1 depletion‐induced oxidative stress could be reversed by treatment with an NADPH‐oxidase inhibitor, suggesting that TNF‐α driven induction of NADPH oxidase may be inoperative in the absence of KRIT1. Thus, destabilization of endothelial adherens junctions following depletion of KRIT1 plays a vascular bed‐dependent role in modifying the vascular response to inflammation.

Disruption of endothelial cell‐cell contact is a key event in many cardiovascular diseases, and a characteristic of pathologically activated vascular endothelium. The CCM family of proteins (KRIT1, PDCD10, and CCM2) are critical regulators of endothelial cell‐cell contact and vascular homeostasis. Here, we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1‐depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β‐catenin signaling and up‐regulates VEGF‐A protein expression. In KRIT1‐depleted cells, increased VEGF‐A levels led to increased VEGFR2 activation and subsequent alteration of cytoskeletal organization, migration, and barrier function, and to in vivo endothelial permeability in KRIT1 deficient animals. VEGFR2 activation also increases β‐catenin phosphorylation, but is only partially responsible for KRIT1 depletion‐dependent disruption of cell‐cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1 deficient cells and microvessel permeability in Krit1+/‐mice, however VEGF signaling is not the primary cause of disrupted endothelial cell‐cell contacts in the absence of KRIT1.Sources of Funding:AHA 14PRE20380009 to Peter DiStefanoNIH HL117885‐01 to Angela Glading