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.

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.

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.

A C. elegans model of electronic cigarette use: Physiological effects of e-liquids in nematodes.

BACKGROUND: Electronic cigarettes (e-cigs) have recently become very popular particularly among the younger generation. These nicotine delivery devices are viewed as a preferable alternative to more conventional forms of tobacco use and are thought to reduce the risk of chronic obstructive pulmonary disease, the third leading cause of death worldwide. However, there is very little data available on the consequences of e-cig use, though recently nicotine-independent inflammatory responses have been reported. The genetic model organism Caenorhabditis elegans is a soil nematode whose cell biology is remarkably well conserved with mammals. Here, we used C. elegans to test the physiologic effects of e-liquids used to refill e-cigs. METHODS: Larval worms were exposed from hatching onwards to low concentrations (0.2 %) of e-liquids, distilled e-liquid vapor, propylene glycol (PG), or M9 buffer as a negative control. E-liquids tested included grape, menthol, and V2 Red "classic tobacco" flavors. Nicotine (48 ppm) was tested as a second level variable. Stereotypical physiological outputs were then measured, including developmental rate, fecundity, locomotion, lifespan, and the induction of canonical stress signaling pathways. RESULTS: A small but significant impairment of developmental rate and brood size was observed for PG and V2 Red treated worms compared to the negative control. Worms treated with e-liquids containing nicotine fared significantly worse than those that did not, but vaporization did not increase toxicity. Finally, both PG and V2 Red e-liquid induced an oxidative stress response in the absence of nicotine. CONCLUSIONS: PG exposure is sufficient to induce an oxidative stress response in nematodes, while nicotine is not. Both PG and nicotine independently influence physiologic measures of health and viability. The e-liquid flavorings did not significantly impact outcomes and there was no evidence for vaporization altering toxicity. These data suggest that the major physiologically significant component of e-liquids besides nicotine is likely the common solvent PG. We conclude that C. elegans are an appropriate model to rapidly assess parameters that may contribute to the basic cell biological effects of e-cigs.