Contribution of protein-protein interactions to the endothelial-barrier-stabilizing function of KRIT1.

Krev-interaction trapped protein 1 (KRIT1) is an endothelial scaffold protein that promotes adherens junction (AJ) stability. The precise mechanism by which KRIT1 promotes barrier stabilization is unclear. We tested the ability of a panel of KRIT1 constructs containing mutations that inhibit Rap1 binding, ICAP1α binding, disrupt KRIT1's phosphotyrosine-binding (PTB) domain, or direct KRIT1 to the plasma membrane, either alone or in combination, to restore barrier function in KRIT1-deficient endothelial cells. We found that ablating the 192NPAY195 motif or disrupting the PTB domain was sufficient to restore AJ protein localization and barrier function to control levels, irrespective of the junctional localization of KRIT1 or Rap1 binding. The ability of our KRIT1 constructs to rescue AJ and barrier function in KRIT1-depleted endothelial cells correlated with decreased ß1 integrin activity and maintenance of cortical actin fibers. Taken together, our findings indicate that Rap1 binding, ICAP1α binding and junctional localization are not required for the ability of KRIT1 to stabilize endothelial contacts, and suggest that the ability of KRIT1 to limit integrin activity could be involved in barrier stabilization.

Protein kinase Cα regulates the nucleocytoplasmic shuttling of KRIT1.

KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.

VEGF signalling enhances lesion burden in KRIT1 deficient mice.

The exact molecular mechanisms underlying CCM pathogenesis remain a complicated and controversial topic. Our previous work illustrated an important VEGF signalling loop in KRIT1 depleted endothelial cells. As VEGF is a major mediator of many vascular pathologies, we asked whether the increased VEGF signalling downstream of KRIT1 depletion was involved in CCM formation. Using an inducible KRIT1 endothelial-specific knockout mouse that models CCM, we show that VEGFR2 activation plays a role in CCM pathogenesis in mice. Inhibition of VEGFR2 using a specific inhibitor, SU5416, significantly decreased the number of lesions formed and slightly lowered the average lesion size. Notably, VEGFR2 inhibition also decreased the appearance of lesion haemorrhage as denoted by the presence of free iron in adjacent tissues. The presence of free iron correlated with increased microvessel permeability in both skeletal muscle and brain, which was completely reversed by SU5416 treatment. Finally, we show that VEGFR2 activation is a common downstream consequence of KRIT1, CCM2 and CCM3 loss of function, though the mechanism by which VEGFR2 activation occurs likely varies. Thus, our study clearly shows that VEGFR2 activation downstream of KRIT1 depletion enhances the severity of CCM formation in mice, and suggests that targeting VEGF signalling may be a potential future therapy for CCM.

KRIT1 protein depletion modifies endothelial cell behavior via increased vascular endothelial growth factor (VEGF) signaling.

Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), 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 ß-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (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 ß-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 likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.

Decreased Krev interaction-trapped 1 expression leads to increased vascular permeability and modifies inflammatory responses in vivo.

OBJECTIVE: The regulation of vascular permeability, leukocyte trafficking, and the integrity of endothelial cell-cell contacts are closely linked by a complex mechanism of interregulation. Here, we investigate the role of Krev interaction-trapped 1 (KRIT1), an adherens junction accessory protein required for cell-cell junction stability, in regulating these vascular functions. METHODS AND RESULTS: Krit1(+/-) mice exhibited an enhanced edematous response to the complex inflammatory stimuli found in the passive K/BxN model of inflammatory arthritis and the murine air pouch model, yet leukocyte infiltration was unchanged. Correspondingly, reduced KRIT1 expression increased baseline arteriole and venule permeability 2-fold over that of wild-type littermates, as measured by intravital microscopy of the intact cremaster muscle vascular network, but this increase was not accompanied by increased leukocyte extravasation or activation. Direct stimulation with tumor necrosis factor-α induced increased permeability in wild-type mice, but surprisingly no increase over baseline levels was observed in Krit1(+/-) mice, despite extensive leukocyte activation. Finally, adoptive transfer of Krit1(+/-) bone marrow failed to increase permeability in wild-type mice. However, reduced expression of KRIT1 in the hematopoietic lineage dampened the differences observed in baseline permeability. CONCLUSIONS: Taken together, our data indicate an integral role for KRIT1 in microvessel homeostasis and the vascular response to inflammation.

KRIT1-mediated regulation of neutrophil adhesion and motility.

Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though ß1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.

KRIT1: A Traffic Warden at the Busy Crossroads Between Redox Signaling and the Pathogenesis of Cerebral Cavernous Malformation Disease.

Significance: KRIT1 (Krev interaction trapped 1) is a scaffolding protein that plays a critical role in vascular morphogenesis and homeostasis. Its loss-of-function has been unequivocally associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease of genetic origin characterized by defective endothelial cell-cell adhesion and ensuing structural alterations and hyperpermeability in brain capillaries. KRIT1 contributes to the maintenance of endothelial barrier function by stabilizing the integrity of adherens junctions and inhibiting the formation of actin stress fibers. Recent Advances: Among the multiple regulatory mechanisms proposed so far, significant evidence accumulated over the past decade has clearly shown that the role of KRIT1 in the stability of endothelial barriers, including the blood-brain barrier, is largely based on its involvement in the complex machinery governing cellular redox homeostasis and responses to oxidative stress and inflammation. KRIT1 loss-of-function has, indeed, been demonstrated to cause an impairment of major redox-sensitive mechanisms involved in spatiotemporal regulation of cell adhesion and signaling, which ultimately leads to decreased cell-cell junction stability and enhanced sensitivity to oxidative stress and inflammation. Critical Issues: This review explores the redox mechanisms that influence endothelial cell adhesion and barrier function, focusing on the role of KRIT1 in such mechanisms. We propose that this supports a novel model wherein redox signaling forms the common link between the various pathogenetic mechanisms and therapeutic approaches hitherto associated with CCM disease. Future Directions: A comprehensive characterization of the role of KRIT1 in redox control of endothelial barrier physiology and defense against oxy-inflammatory insults will provide valuable insights into the development of precision medicine strategies. Antioxid. Redox Signal. 38, 496-528.

Is Location Everything? Regulation of the Endothelial CCM Signaling Complex.

Recent advances have steadily increased the number of proteins and pathways known to be involved in the development of cerebral cavernous malformation (CCM). Our ability to synthesize this information into a cohesive and accurate signaling model is limited, however, by significant gaps in our knowledge of how the core CCM proteins, whose loss of function drives development of CCM, are regulated. Here, we review what is known about the regulation of the three core CCM proteins, the scaffolds KRIT1, CCM2, and CCM3, with an emphasis on binding interactions and subcellular location, which frequently control scaffolding protein function. We highlight recent work that challenges the current model of CCM complex signaling and provide recommendations for future studies needed to address the large number of outstanding questions.

Measurement of Endothelial Barrier Function in Mouse Models of Cerebral Cavernous Malformations Using Intravital Microscopy.

Vascular permeability is a major function of the microvasculature that is regulated by multiple factors including blood pressure, blood viscosity, and endothelial barrier function. Intravital microscopy has been used to directly assess vascular permeability in vivo, which allows for the accurate measurement of endothelial barrier function in a truly physiological hemodynamic context. Here, we describe the procedure for measuring endothelial barrier function in mouse models of cerebral cavernous malformations, including micropipette preparation, anesthesia, tracheotomy, jugular catheterization, cremaster dissection, imaging, and data analysis. These animals exhibit an increase in microvessel permeability and abnormal vessel morphology, which require special consideration.

Isolation of Cerebral Endothelial Cells from CCM1/KRIT1 Null Mouse Brain.

Cerebral cavernous malformation (CCM) is driven by changes in the cerebral microvascular endothelial cell population. Mouse models of CCM have successfully recapitulated the disease in vivo; however, dissection of the disease pathogenesis and molecular mechanism is challenging in vivo due to limited access to the involved tissue in live animals. Therefore, in vitro tissue culture models are required. This protocol is designed to facilitate the isolation of cerebral microvascular endothelial cells from whole murine brain tissue. The protocol utilizes papain for a shorter, single digestion step to maximize cell recovery and viability. Using this technique, we are able to isolate cells from a murine CCM model in which the absence of CCM proteins is driven by Cre-mediated recombination at birth, and results in CCM-like vascular malformations in adult animals.

Microvascular Mimetics for the Study of Leukocyte-Endothelial Interactions.

INTRODUCTION: The pathophysiological increase in microvascular permeability plays a well-known role in the onset and progression of diseases like sepsis and atherosclerosis. However, how interactions between neutrophils and the endothelium alter vessel permeability is often debated. METHODS: In this study, we introduce a microfluidic, silicon-membrane enabled vascular mimetic (µSiM-MVM) for investigating the role of neutrophils in inflammation-associated microvascular permeability. In utilizing optically transparent silicon nanomembrane technology, we build on previous microvascular models by enabling in situ observations of neutrophil-endothelium interactions. To evaluate the effects of neutrophil transmigration on microvascular model permeability, we established and validated electrical (transendothelial electrical resistance and impedance) and small molecule permeability assays that allow for the in situ quantification of temporal changes in endothelium junctional integrity. RESULTS: Analysis of neutrophil-expressed ß1 integrins revealed a prominent role of neutrophil transmigration and basement membrane interactions in increased microvascular permeability. By utilizing blocking antibodies specific to the ß1 subunit, we found that the observed increase in microvascular permeability due to neutrophil transmigration is constrained when neutrophil-basement membrane interactions are blocked. Having demonstrated the value of in situ measurements of small molecule permeability, we then developed and validated a quantitative framework that can be used to interpret barrier permeability for comparisons to conventional Transwell™ values. CONCLUSIONS: Overall, our results demonstrate the potential of the µSiM-MVM in elucidating mechanisms involved in the pathogenesis of inflammatory disease, and provide evidence for a role for neutrophils in inflammation-associated endothelial barrier disruption.

Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium.

The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF-α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin.

Cerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity. This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies.

Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier.

The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.

Control of vascular permeability by adhesion molecules.

Vascular permeability is a vital function of the circulatory system that is regulated in large part by the limited flux of solutes, water, and cells through the endothelial cell layer. One major pathway through this barrier is via the inter-endothelial junction, which is driven by the regulation of cadherin-based adhesions. The endothelium also forms attachments with surrounding proteins and cells via 2 classes of adhesion molecules, the integrins and IgCAMs. Integrins and IgCAMs propagate activation of multiple downstream signals that potentially impact cadherin adhesion. Here we discuss the known contributions of integrin and IgCAM signaling to the regulation of cadherin adhesion stability, endothelial barrier function, and vascular permeability. Emphasis is placed on known and prospective crosstalk signaling mechanisms between integrins, the IgCAMs- ICAM-1 and PECAM-1, and inter-endothelial cadherin adhesions, as potential strategic signaling nodes for multipartite regulation of cadherin adhesion.

Rap1 and its effector KRIT1/CCM1 regulate beta-catenin signaling.

KRIT1, also called CCM1, is a member of a multiprotein complex that contains the products of the CCM2 and PDCD10 (also known as CCM3) loci. Heterozygous loss of any of the genes that encode these proteins leads to cerebral cavernous malformations (CCM), which are vascular lesions that are found in around 0.5% of humans. KRIT1 mediates the stabilization of beta-catenin-containing endothelial cell-cell junctions downstream of the Rap1 GTPase. Here, we report that Rap1 and KRIT1 are negative regulators of canonical beta-catenin signaling in mice and that hemizygous Krit1 deficiency exacerbates beta-catenin-driven pathologies. Depletion of endothelial KRIT1 caused beta-catenin to dissociate from vascular endothelial (VE)-cadherin and to accumulate in the nucleus with consequent increases in beta-catenin-dependent transcription. Activation of Rap1 inhibited beta-catenin-dependent transcription in confluent endothelial cells; this effect required the presence of intact cell-cell junctions and KRIT1. These effects of KRIT1 were not limited to endothelial cells; the KRIT1 protein was expressed widely and its depletion increased beta-catenin signaling in epithelial cells. Moreover, a reduction in KRIT1 expression also increased beta-catenin signaling in vivo. Hemizygous deficiency of Krit1 resulted in a ~1.5-fold increase in intestinal polyps in the Apc(Min/+) mouse, which was associated with increased beta-catenin-driven transcription. Thus, KRIT1 regulates beta-catenin signaling, and Krit1(+/-) mice are more susceptible to beta-catenin-driven intestinal adenomas.