Implication of Rac1 GTPase in molecular and cellular mitochondrial functions.

Rac1 is a member of the Rho GTPase family which plays major roles in cell mobility, polarity and migration, as a fundamental regulator of actin cytoskeleton. Signal transduction by Rac1 occurs through interaction with multiple effector proteins, and its activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The small protein is mainly anchored to the inner side of the plasma membrane but it can be found in endocellular compartments, notably endosomes and cell nuclei. The protein localizes also into mitochondria where it contributes to the regulation of mitochondrial dynamics, including both mitobiogenesis and mitophagy, in addition to signaling processes via different protein partners, such as the proapoptotic protein Bcl-2 and chaperone sigma-1 receptor (σ-1R). The mitochondrial form of Rac1 (mtRac1) has been understudied thus far, but it is as essential as the nuclear or plasma membrane forms, via its implication in regulation of oxidative stress and DNA damages. Rac1 is subject to diverse post-translational modifications, notably to a geranylgeranylation which contributes importantly to its mitochondrial import and its anchorage to mitochondrial membranes. In addition, Rac1 contributes to the mitochondrial translocation of other proteins, such as p53. The mitochondrial localization and functions of Rac1 are discussed here, notably in the context of human diseases such as cancers. Inhibitors of Rac1 have been identified (NSC-23766, EHT-1864) and some are being developed for the treatment of cancer (MBQ-167) or central nervous system diseases (JK-50561). Their effects on mtRac1 warrant further investigations. An overview of mtRac1 is provided here.

Allergic Sensitization Driving Immune Phenotyping and Disease Severity in a Mouse Model of Asthma.

PURPOSE: Asthma is a frequent chronic inflammatory bronchial disease affecting more than 300 million patients worldwide, 70% of whom are secondary to allergy. The diversity of asthmatic endotypes contributes to their complexity. The inter-relationship between allergen and other exposure and the airway microbiome adds to the phenotypic diversity and defines the natural course of asthma. Here, we compared the mouse models of house dust mite (HDM)-induced allergic asthma. Allergic sensitization was performed via various routes and associated with outcomes. METHODS: Mice were sensitized with HDM via the oral, nasal or percutaneous routes. Lung function, barrier integrity, immune response and microbiota composition were analyzed. RESULTS: Severe impairment of respiratory function was observed in the mice sensitized by the nasal and cutaneous paths. It was associated with epithelial dysfunction characterized by an increased permeability secondary to junction protein disruption. Such sensitization paths induced a mixed eosinophilic and neutrophilic inflammatory response with high interleukin (IL)-17 airway secretion. In contrast, orally sensitized mice showed a mild impairment of respiratory function. Epithelial dysfunction was mild with increased mucus production, but preserved epithelial junctions. Regarding lung microbiota, sensitization provoked a significant loss of diversity. At the genus level, Cutibacterium, Acinetobacter, Streptococcus and Lactobacillus were found to be modulated according to the sensitization pathway. An increase in theanti-inflammatory microbiota metabolites was observed in the oral-sensitization group. CONCLUSIONS: Our study highlights the strong impact of the sensitization route on the pathophysiology and the critical phenotypic diversity of allergic asthma in a mouse model.

Bronchial smooth muscle cell in asthma: where does it fit?

Asthma is a frequent respiratory condition whose pathophysiology relies on altered interactions between bronchial epithelium, smooth muscle cells (SMC) and immune responses. Those leads to classical hallmarks of asthma: airway hyper-responsiveness, bronchial remodelling and chronic inflammation. Airway smooth muscle biology and pathophysiological implication in asthma are now better understood. Precise deciphering of intracellular signalling pathways regulating smooth muscle contraction highlighted the critical roles played by small GTPases of Rho superfamily. Beyond contractile considerations, active involvement of airway smooth muscle in bronchial remodelling mechanisms is now established. Not only cytokines and growth factors, such as fibroblats growth factor or transforming growth factor-ß, but also extracellular matrix composition have been demonstrated as potent phenotype modifiers for airway SMC. Although basic science knowledge has grown significantly, little of it has translated into improvement in asthma clinical practice. Evaluation of airway smooth muscle function is still limited to its contractile activity. Moreover, it relies on tools, such as spirometry, that give only an overall assessment and not a specific one. Interesting technics such as forced oscillometry or specific imagery (CT and MRI) give new perspectives to evaluate other aspects of airway muscle such as bronchial remodelling. Finally, except for the refinement of conventional bronchodilators, no new drug therapy directly targeting airway smooth muscle proved its efficacy. Bronchial thermoplasty is an innovative and efficient therapeutic strategy but is only restricted to a small proportion of severe asthmatic patients. New diagnostic and therapeutic strategies specifically oriented toward airway smooth muscle are needed to improve global asthma care.

Rac1 as a therapeutic anticancer target: Promises and limitations.

Small molecule inhibitors of GTPases are increasingly considered for the treatment of multiple human pathologies. The GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) plays major roles in vital cellular processes, notably in the control cell motility and dynamic, the regulation of oxidative stress, and in inflammatory and immune surveillance. As such, Rac1 is viewed as a potential target to combat cancers but also diverse inflammatory, metabolic, neurodegenerative, respiratory, cardiovascular, viral, and parasitic diseases. Potent and selective Rac1 inhibitors have been identified and designed, such as compounds GYS32661 and MBQ-167 both in preclinical development for the treatment of advanced solid tumors. The pleiotropic roles and ubiquitous expression of the protein can be viewed as limitations for anticancer approaches. However, the frequent overexpression and/or hyperactivation of the Rac1 in difficult-to-treat chemoresistant cancers, make Rac1 an attractive target in oncology. The key roles of Rac1 in multiple cellular pathways, together with its major implications in carcinogenesis, tumor proliferation and metastasis, support the development of small molecule inhibitors. The challenge is high and the difficulty shall not be underestimated, but the target is innovative and promising in combination with chemo- and/or immuno-therapy. Opportunities and challenges associated with the targeting of Rac1 are discussed.

Rac1 as a Target to Treat Dysfunctions and Cancer of the Bladder.

Bladder pathologies, very common in the aged population, have a considerable negative impact on quality of life. Novel targets are needed to design drugs and combinations to treat diseases such as overactive bladder and bladder cancers. A promising new target is the ubiquitous Rho GTPase Rac1, frequently dysregulated and overexpressed in bladder pathologies. We have analyzed the roles of Rac1 in different bladder pathologies, including bacterial infections, diabetes-induced bladder dysfunctions and bladder cancers. The contribution of the Rac1 protein to tumorigenesis, tumor progression, epithelial-mesenchymal transition of bladder cancer cells and their metastasis has been analyzed. Small molecules selectively targeting Rac1 have been discovered or designed, and two of them-NSC23766 and EHT 1864-have revealed activities against bladder cancer. Their mode of interaction with Rac1, at the GTP binding site or the guanine nucleotide exchange factors (GEF) interaction site, is discussed. Our analysis underlines the possibility of targeting Rac1 with small molecules with the objective to combat bladder dysfunctions and to reduce lower urinary tract symptoms. Finally, the interest of a Rac1 inhibitor to treat advanced chemoresistance prostate cancer, while reducing the risk of associated bladder dysfunction, is discussed. There is hope for a better management of bladder pathologies via Rac1-targeted approaches.

The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model.

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human ß 3-adrenoreceptor (Tgß 3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgß 3 rats. The aortic expression of • NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate • NO and O2 •- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgß 3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgß 3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgß 3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgß 3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgß 3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgß 3: 0.13 ± 0.05; p < 0.05). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgß 3 rats (+52% vs. WT). Aortic O2 •- production was increased in Tgß 3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.

Overexpressed or hyperactivated Rac1 as a target to treat hepatocellular carcinoma.

Despite novel targeted and immunotherapies, the prognosis remains bleak for patients with hepatocellular carcinoma (HCC), especially for advanced and/or metastatic forms. The rapid emergence of drug resistance is a major obstacle in the success of chemo-, targeted-, immuno-therapies of HCC. Novel targets are needed. The prominent roles of the small GTPase Rac1 in the development and progression of HCC are discussed here, together with its multiple protein partners, and the targeting of Rac1 with RNA-based regulators and small molecules. We discuss the oncogenic functions of Rac1 in HCC, including the contribution of Rac1 mutants and isoform Rac1b. Rac1 is a ubiquitous target, but the protein is frequently overexpressed and hyperactivated in HCC. It contributes to the aggressivity of the disease, with key roles in cancer cell proliferation, tumor metastasis and resistance to treatment. Small molecule targeting Rac1, indirectly or directly, have shown anticancer effects in HCC experimental models. Rac1-binding agents such as EHT 1864 and analogues offer novel opportunities to combat HCC. We discuss the different modalities to repress Rac1 overactivation in HCC with small molecules and the combination with reference drugs to promote cancer cell death and to repress cell invasion. We highlight the necessity to combine Rac1-targeted approach with appropriate biomarkers to select Rac1 activated tumors. Our analysis underlines the prominent oncogenic functions of Rac1 in HCC and discuss the modalities to target this small GTPase. Rac1 shall be considered as a valid target to limit the acquired and intrinsic resistance of HCC tumors and their metastatic potential.

Smooth muscle Rac1 contributes to pulmonary hypertension.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a multifactorial chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signalling in PA plays a major role in PH. We recently described the involvement of the Rho protein Rac1 in the control of systemic blood pressure through its involvement in NO-mediated relaxation of arterial smooth muscle cell (SMC). The aim of this study was to analyse the role of SMC Rac1 in PH. EXPERIMENTAL APPROACH: PH is induced by exposure of control and SMC Rac1-deficient (SM-Rac1-KO) mice to chronic hypoxia (10% O2 , 4 weeks). PH is assessed by the measurement of RV systolic pressure and hypertrophy. PA reactivity is analysed by isometric tension measurements. PA remodelling is quantified by immunofluorescence in lung sections and ROS are detected using the dihydroethidium probe and electronic paramagnetic resonance analysis. Rac1 activity is determined by immunofluorescence. KEY RESULTS: Rac1 activation in PA of hypoxic mice and patients with idiopathic PH. Hypoxia-induced rise in RV systolic pressure, RV hypertrophy and loss of endothelium-dependent relaxation were significantly decreased in SM-Rac1-KO mice compared to control mice. SMC Rac1 deletion also limited hypoxia-induced PA remodelling and ROS production in pulmonary artery smooth muscle cells (PASMCs). CONCLUSION AND IMPLICATIONS: Our results provide evidence for a protective effect of SM Rac1 deletion against hypoxic PH. Rac1 activity in PASMCs plays a causal role in PH by favouring ROS-dependent PA remodelling and endothelial dysfunction induced by chronic hypoxia.

New Functions of Vav Family Proteins in Cardiovascular Biology, Skeletal Muscle, and the Nervous System.

Vav proteins act as tyrosine phosphorylation-regulated guanosine nucleotide exchange factors for Rho GTPases and as molecular scaffolds. In mammals, this family of signaling proteins is composed of three members (Vav1, Vav2, Vav3) that work downstream of protein tyrosine kinases in a wide variety of cellular processes. Recent work with genetically modified mouse models has revealed that these proteins play key signaling roles in vascular smooth and skeletal muscle cells, specific neuronal subtypes, and glia cells. These functions, in turn, ensure the proper regulation of blood pressure levels, skeletal muscle mass, axonal wiring, and fiber myelination events as well as systemic metabolic balance. The study of these mice has also led to the discovery of new physiological interconnection among tissues that contribute to the ontogeny and progression of different pathologies such as, for example, hypertension, cardiovascular disease, and metabolic syndrome. Here, we provide an integrated view of all these new Vav family-dependent signaling and physiological functions.

Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling.

BACKGROUND: Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. METHODS AND RESULTS: Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. CONCLUSION: This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

Der p 2.1 Peptide Abrogates House Dust Mites-Induced Asthma Features in Mice and Humanized Mice by Inhibiting DC-Mediated T Cell Polarization.

Asthma is a chronic airway disease often due to sensitization to aeroallergens, especially house dust mite allergens (HDMs). The Dermatophagoides pteronyssinus group 2 (Der p 2), is one of the most representative HDM allergens and is recognized by more than 90% of HDM-allergic patients. In mouse models, all asthma-related features can be prevented by prophylactic administration of Dermatophagoides pteronyssinus 2-derived peptide (Der p 2.1). However, it is unknown whether it is able to treat well-established asthma in mice and humans. We aimed here to evaluate the efficacy of Der p 2.1 immunotherapy in a mouse, humanized mouse, and asthmatic patients. Asthma related-features were analyzed through airway hyperresponsiveness (AHR), allergen-specific IgE, and lung histology in mice and humanized mice. Immune profile was analyzed using lung and blood from mice and severe asthmatic patients respectively. T cell and dendritic cell (DC) polarization was evaluated using co-culture of bone marrow derived cells (BMDCs) and naïve T cell from naïve mice. Mice and humanized mice both have a reduced AHR, lung tissue alteration, and HDM-specific IgE under Der p 2.1 treatment. Concerning the immune profile, T helper 2 cells (Th2) and T helper 17 cells (Th17) were significantly reduced in both mice and humanized mice lung and in peripheral blood mononuclear cells (PBMCs) from severe asthmatic patients after Der p 2.1 incubation. The downregulation of T cell polarization seems to be linked to an increase of IL-10-secreting DC under Der p 2.1 treatment in both mice and severe asthmatic patients. This study shows that allergen-derived peptide immunotherapy abrogates asthma-related features in mice and humanized mice by reducing Th2 and Th17 cells polarization via IL-10-secreting DC. These results suggest that Der p 2.1 peptide immunotherapy could be a promising approach to treat both Th2 and Th17 immunity in asthma.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome.

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

[New protagonists in asthma pathophysiology]. / Nouveaux acteurs dans la physiopathologie de l'asthme.

Asthma is often associated with a Th2-type immune response with well-known cellular and molecular actors such as eosinophils, Th2 lymphocytes and associated cytokines such as interleukin-5 or IL-4. Nevertheless, some of the asthmatic patients show clinical manifestations and characteristics that do not correspond to the current pattern of the pathophysiology of asthma. Thus, recently new cellular and molecular actors in the development of asthma have been demonstrated in animal models and in humans. Among these are components of the innate immune system such as type 2 innate lymphoid cells or adaptive immune system such as Th9 lymphocytes. At the cellular level, the role of small G proteins in asthma is also highlighted as well as the role of major cytokines like IL-17 or those derived from the epithelium. A better knowledge of the physiopathology of asthma and the taking into account of these new actors allows the identification of new therapeutic targets for different endotypes of patients.

Synthesis and biological evaluation of 3-amino-, 3-alkoxy- and 3-aryloxy-6-(hetero)arylpyridazines as potent antitumor agents.

Various 3-amino-, 3-aryloxy- and alkoxy-6-arylpyridazines have been synthesized by an electrochemical reductive cross-coupling between 3-amino-, 3-aryloxy- or 3-alkoxy-6-chloropyridazines and aryl or heteroaryl halides. In vitro antiproliferative activity of these products was evaluated against a representative panel of cancer cell lines (HuH7, CaCo-2, MDA-MB-231, HCT116, PC3, NCI-H727, HaCaT) and oncogenicity prevention of the more efficient derivatives was highlighted on human breast cancer cell line MDA-MB 468-Luc prior establishing their interaction with p44/42 and Akt-dependent signaling pathways.

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 ß2 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.

In Vitro and In Vivo Approaches to Assess Rho Kinase Activity.

Increased arterial tone and the resulting rise in peripheral vascular resistance are major determinants of the elevated arterial pressure in hypertension. The RhoA/Rho kinase signaling pathways are now recognized as a major regulator of vascular smooth muscle contraction and arterial tone. Here we describe methods to directly and indirectly assess Rho kinase activity in vitro and in cells and tissues.

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.

Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.

Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26(+) lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26(+) macrophages expressed M2 phenotype, while the innate PKH26(-) macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26(+) MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836-1845.

Smooth muscle specific Rac1 deficiency induces hypertension by preventing p116RIP3-dependent RhoA inhibition.

BACKGROUND: Increasing evidence implicates overactivation of RhoA as a critical component of the pathogenesis of hypertension. Although a substantial body of work has established that Rac1 functions antagonize RhoA in a broad range of physiological processes, the role of Rac1 in the regulation of vascular tone and blood pressure is not fully elucidated. METHODS AND RESULTS: To define the role of Rac1 in vivo in vascular smooth muscle cells (vSMC), we generated smooth muscle (SM)-specific Rac1 knockout mice (SM-Rac1-KO) and performed radiotelemetric blood pressure recordings, contraction measurements in arterial rings, vSMC cultures and biochemical analyses. SM-Rac1-KO mice develop high systolic blood pressure sensitive to Rho kinase inhibition by fasudil. Arteries from SM-Rac1-KO mice are characterized by a defective NO-dependent vasodilation and an overactivation of RhoA/Rho kinase signaling. We provide evidence that Rac1 deletion-induced hypertension is due to an alteration of cGMP signaling resulting from the loss of Rac1-mediated control of type 5 PDE activity. Consequently, cGMP-dependent phosphorylation and binding of RhoA with its inhibitory partner, the phosphatase-RhoA interacting protein (p116(RIP3)), are decreased. CONCLUSIONS: Our data reveal that the depletion of Rac1 in SMC decreases cGMP-dependent p116(RIP3)/RhoA interaction and the subsequent inhibition of RhoA signaling. Thus, we unveil an in vivo role of Rac1 in arterial blood pressure regulation and a new pathway involving p116(RIP3) that contributes to the antagonistic relationship between Rac1 and RhoA in vascular smooth muscle cells and their opposite roles in arterial tone and blood pressure.