Angela J. Glading

KRIT1 plays a key role in regulating the barrier function of endothelial cells, where it localizes to the adherens junctions, cytoplasm, and nucleus. However, how subcellular localization may regulate KRIT1 remains unclear. Here, we investigate what effect nuclear localization has on its ability to stabilize the endothelial barrier. We generated a KRIT1 mutant lacking the endogenous nuclear localization signal (NLS) and a nuclear‐targeted KRIT1 isoform created by attaching the NLS of simian virus 40 and expressed these constructs in cells depleted of endogenous KRIT1. After confirming the relative non‐nuclear (KRIT1 ^ΔNLS ) and nuclear (KRIT1 ^NLS+ ) enrichment of these constructs using confocal microscopy and cellular fractionation, we assessed whether these constructs were able to functionally rescue phenotypes characteristic of KRIT1‐depleted cells. Our results showed that nuclear localized KRIT1 remains fully functional. However, the KRIT1 ^ΔNLS construct failed to rescue the KRIT1 depletion phenotype. KRIT1 ^ΔNLS also disrupted binding to ICAP1α, as shown by co‐immunoprecipitation. To determine whether the loss of function was due to loss of ICAP1α‐mediated conformational change or altered localization, we incorporated two mutants known to disrupt the N‐ to C‐terminal interaction in KRIT1, ¹⁹² NPXY ¹⁹⁵ → APAA or W688A into our KRIT1 ^ΔNLS and KRIT1 ^NLS+ constructs. The presence of these additional mutations had unexpected effects on nuclear localization of KRIT1 ^ΔNLS and KRIT1 ^NLS+ but were able to restore barrier‐stabilizing function to KRIT1 ^ΔNLS . Overall, our findings provide key insights into the role of ICAP1α binding and nuclear localization in the regulation of KRIT1 and raise new questions regarding potentially novel functions of KRIT1 in the nucleus.

Cerebral cavernous malformations (CCM) are angiographically occult vascular anomalies of the brain, characterized by dilated capillaries, increased vascular permeability, and loss of endothelial junctional protein complexes. Loss-of-function mutations in one of the three genes, namely KRIT1/CCM1, CCM2, and PDCD10/CCM3, have been associated with the disease pathogenesis, although the contribution of other genetic determinants besides CCM genes has been recently identified. Despite recent advances in understanding the molecular mechanism of the disease, the current lack of therapies and its unpredictable clinical behavior represent a significant challenge in the identification of diagnostic biomarkers. ADGRL4/ETLD1 (epidermal growth factor, latrophilin and seven transmembrane domain-containing protein 1), a G-protein coupled receptor (GPCR) protein is a known biomarker of angiogenesis and inflammation, and it has been suggested to be a key therapeutic target for stroke and high-grade gliomas. However, the relevance of ELTD1 in CCM pathogenesis remains unexplored. Through different analyses, including whole RNA transcriptome, immunohistochemistry, and real-time PCR, conducted in human CCM patients, cellular and animal models, herein we show the association between CCM disease and ELTD1. The whole RNA transcriptome approaches demonstrated ELTD1 is differentially expressed, and gene expression analysis revealed it is significantly upregulated in surgically resected tissue and plasma samples from CCMs, and clinical correlation analysis showed that increased ELTD1 associates with the presence of Focal Neurological Deficits in patients. Immuno-expression showed a strong ELTD1 immunoreactivity in endothelial cells lining affected lesions. According to these elevated levels of ELTD1 in human patients, we also showed a robust increase in the expression level of ELTD1 in cellular and animal models of CCM disease. Taken together, our results demonstrate for the first time ELTD1 involvement in CCM pathogenesis, and its tight link with CCM genes. Thanks to this putative new prognostic biomarker, future clinical studies in larger patient cohorts will aim at improving the CCM disease management and risk stratification in patients, as well as placing the basis for targeted therapeutic strategies in CCM.

KRIT1 is a 75 kDa scaffolding protein which regulates endothelial cell phenotype by limiting the response to inflammatory stimuli and maintaining a quiescent and stable endothelial barrier. Loss-of-function mutations in KRIT1 lead to the development of cerebral cavernous malformations (CCM), a disease marked by the formation of abnormal blood vessels which exhibit a loss of barrier function, increased endothelial proliferation, and altered gene expression. While many advances have been made in our understanding of how KRIT1, and the functionally related proteins CCM2 and PDCD10, contribute to the regulation of blood vessels and the vascular barrier, some important open questions remain. In addition, KRIT1 is widely expressed and KRIT1 and the other CCM proteins have been shown to play important roles in non-endothelial cell types and tissues, which may or may not be related to their role as pathogenic originators of CCM. In this review, we discuss some of the unsettled questions regarding the role of KRIT1 in vascular physiology and discuss recent advances that suggest this ubiquitously expressed protein may have a role beyond the endothelial cell.

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 Ly6G^{tm2621(cre)Arte} Krit1^flox/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 β1 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 Krit1^PMNKO 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.

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.

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 β1 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.

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.

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.

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.