Gwennan André-Grégoire

Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1

AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.

Pharmacological targeting of apelin impairs glioblastoma growth

Glioblastomas are aggressive brain tumours that contain a subpopulation of highly plastic self-renewing cancer cells. Harford-Wright et al. show that the vasoactive peptide apelin, secreted by brain endothelial cells, regulates glioblastoma patient-derived cells with stem-like properties. Pharmacological blockade of apelin hampers glioblastoma cell expansion and improves survival in xenografted mice.

Spitting out the demons: Extracellular vesicles in glioblastoma.

ABSTRACT Discovered decades ago, extracellular vesicles (EVs) emerge as dedicated organelles, able to deliver protected, specific cellular cues throughout the organism. While virtually every cell can release EVs, cancer cells co-opted this feature and efficiently unleashed them both in the tumor microenvironment and toward healthy tissues. This might contribute to tumor aggressiveness and spreading. Cancer-derived EVs that contain DNA, mRNA, miRNA, and packed and transmembrane proteins can operate locally or at distance. This review will focus on the high-grade brain tumor (i.e. glioblastoma)-derived EVs, discussing recent reports on i) their phenotype and content, ii) their putative functions, and iii) their clinical potential for improving diagnosis and therapeutics.

The E3 ubiquitin ligase MARCH3 controls the endothelial barrier

Cell–cell contacts coordinate the endothelial barrier function in response to external cues. To identify new mediators involved in cytokine‐promoted endothelial permeability, we screened a siRNA library targeting E3 ubiquitin ligases. Here, we report that silencing of the late endosome/lysosomal membrane‐associated RING‐CH‐3 (MARCH3) enzyme protects the endothelial barrier. Furthermore, transcriptome analysis unmasked the upregulation of the tight junction‐encoding gene occludin (OCLN) in MARCH3‐depleted cells. Indeed, MARCH3 silencing results in the strengthening of cell‐cell contacts, as evidenced by the accumulation of junctional proteins. From a molecular standpoint, the FoxO1 forkhead transcription repressor was inactivated in the absence of MARCH3. This provides a possible molecular link between MARCH3 and the signaling pathway involved in regulating the expression of junctional proteins and barrier integrity.

Targeting of Rac1 prevents bronchoconstriction and airway hyperresponsiveness

Background The molecular mechanisms responsible for airway smooth muscle cells’ (aSMCs) contraction and proliferation in airway hyperresponsiveness (AHR) associated with asthma are still largely unknown. The small GTPases of the Rho family (RhoA, Rac1, and Cdc42) play a central role in SMC functions including migration, proliferation, and contraction. Objective The objective of this study was to identify the role of Rac1 in aSMC contraction and to investigate its involvement in AHR associated with allergic asthma. Methods To define the role of Rac1 in aSMC, ex and in vitro analyses of bronchial reactivity were performed on bronchi from smooth muscle (SM)‐specific Rac1 knockout mice and human individuals. In addition, this murine model was exposed to allergens (ovalbumin or house dust mite extract) to decipher in vivo the implication of Rac1 in AHR. Results The specific SMC deletion or pharmacological inhibition of Rac1 in mice prevented the bronchoconstrictor response to methacholine. In human bronchi, a similar role of Rac1 was observed during bronchoconstriction. We further demonstrated that Rac1 activation is responsible for bronchoconstrictor‐induced increase in intracellular Ca2+ concentration and contraction both in murine and in human bronchial aSMCs, through its association with phospholipase C &bgr;2 and the stimulation of inositol 1,4,5‐trisphosphate production. In vivo, Rac1 deletion in SMCs or pharmacological Rac1 inhibition by nebulization of NSC23766 prevented AHR in murine models of allergic asthma. Moreover, nebulization of NSC23766 decreased eosinophil and neutrophil populations in bronchoalveolar lavages from mice with asthma. Conclusions Our data reveal an unexpected and essential role of Rac1 in the regulation of intracellular Ca2+ and contraction of aSMCs, and the development of AHR. Rac1 thus appears as an attractive therapeutic target in asthma, with a combined beneficial action on both bronchoconstriction and pulmonary inflammation. Graphical abstract Figure. No Caption available.

Inhibition of mTOR in head and neck cancer cells alters endothelial cell morphology in a paracrine fashion

Head and neck squamous cell carcinoma (HNSCC) represent aggressive classes of tumors with a high mortality rate. The mammalian target of rapamycin (mTOR) pathway is instrumental in their initiation and expansion. Although results from pre‐clinical models promise mTOR targeting as a potent novel therapeutic approach, its impact on the tumor microenvironment, such as endothelial cells is only scarcely investigated. Here, we first confirmed the effects of mTOR pharmacological inhibition on cell viability, clonogenicity, and proliferation in HNSCC human cell lines, HN26, and HN30. While Everolimus and Torin1 potently blunted mTOR‐based proliferation of HN26 and HN30 lines, endothelial cells were left intact. To further explore the possibility of a paracrine bystander action of HNSCC‐treated cells on endothelial cells, conditioned medium from Everolimus‐ and Torin1‐challenged HN26 and HN30 cells were collected and applied to naive human endothelial cells. Although endothelial cell viability was again not modified, morphology and mobility were changed. Indeed, spreading of endothelial cells was altered upon challenge with mTOR‐pretreated tumor conditioned‐media, as measured via cell impedance and imagery. Interestingly, this was associated with an augmentation of focal adhesion kinase (FAK) active phosphorylation and enhanced migratory behavior. From a molecular standpoint, the production of vascular endothelial growth factor was elevated in treated HNSCC cells and might contribute to FAK phosphorylation. Although mTOR inhibition in tumor cells did hinder their growth, it also favors the release of factors that subsequently enable endothelial cell migration. Further studies will address how this paracrine action may affect tumor‐driven angiogenesis upon pharmacological treatments.

0539 : Cellular Rac1 functions are dependent of the origin of smooth muscle cells

Introduction Recently, we demonstrated that the GTPase Rac1 is involved in the control of arterial pressure by modulating vascular smooth muscle cells (vSMC) contraction. Rac1 promotes vSMC relaxation by positively regulating cyclic GMP-dependent signaling. In constrat, we observed a contractile action of Rac1 in non-vascular SMC. The objective of this study was thus to decipher the molecular mechanism explaining the different Rac1 effects depending on the type of SMC. Methods and results EHT1864, a selective Rac1 inhibitor, had no effect on vasoconstriction but dramatically decreased carbachol (Cch)-induced contraction of bronchial rings. In primary cell culture, measurements of intracellular free [Ca 2+ ] by a fluorescent Ca 2+ probe showed that Cchinduced rise in cytosolic [Ca 2+ ] was prevented by EHT1864 in airways SMC (aSMC) but not in vSMC. In addition, Rac1 inhibition prevented IP3 formation in Cch-stimulated aSMC, indicating an essential and specific role of Rac1 in phospholipase C/IP3/Ca 2+ signaling in aSMC. By RT-PCR and western blot analysis, we observed that PLCβ2 is the most expressed isoform in aSMC whereas PLCg is the predominant isoform in vSMC. Coimmunoprecipitation experiments indicated that muscarinic receptor stimulation triggers the formation of a protein complex associating Rac1 with PLCβ2 in aSMC. In contrast, no interaction Rac1/PLCγ€ was detected in vSMC stimulated with vasoconstrictors. Conclusion Our results demonstrated for the first time that stimulation of Rac1 activity lead to a rise of Ca 2+ concentration, through a mechanism specific to SMC expressing PLCβ2 isoform. We unveiled that PLCβ2 requires association with Rac1 to initiate its enzymatic activity and promote SMC contraction. The author hereby declares no conflict of interest