PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification.

Atherosclerosis is the primary cause of cardiovascular disease. The development of plaque complications, such as calcification and neo-angiogenesis, strongly impacts plaque stability and is a good predictor of mortality in patients with atherosclerosis. Despite well-known risk factors of plaque complications, such as diabetes mellitus and chronic kidney disease, the mechanisms involved are not fully understood. We and others have identified that the concentration of circulating leucine-rich α-2 glycoprotein 1 (LRG1) was increased in diabetic and chronic kidney disease patients. Using apolipoprotein E knockout mice (ApoE-/-) (fed with Western diet) that developed advanced atherosclerosis and using human carotid endarterectomy, we showed that LRG1 accumulated into an atherosclerotic plaque, preferentially in calcified areas. We then investigated the possible origin of LRG1 and its functions on vascular cells and found that LRG1 expression was specifically enhanced in endothelial cells via inflammatory mediators and not in vascular smooth muscle cells (VSMC). Moreover, we identified that LRG1 was able to induce calcification and SMAD1/5-signaling pathways in VSMC. In conclusion, our results identified for the first time that LRG1 is a direct contributor to vascular calcification and suggest a role of this molecule in the development of plaque complications in patients with atherosclerosis.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

A non-catalytic function of PI3Kγ drives smooth muscle cell proliferation after arterial damage.

Arterial remodeling in hypertension and intimal hyperplasia involves inflammation and disrupted flow, both of which contribute to smooth muscle cell dedifferentiation and proliferation. In this context, our previous results identified phosphoinositide 3-kinase γ (PI3Kγ) as an essential factor in inflammatory processes of the arterial wall. Here, we identify for the first time a kinase-independent role of nonhematopoietic PI3Kγ in the vascular wall during intimal hyperplasia using PI3Kγ-deleted mice and mice expressing a kinase-dead version of the enzyme. Moreover, we found that the absence of PI3Kγ in vascular smooth muscle cells (VSMCs) leads to modulation of cell proliferation, associated with an increase in intracellular cAMP levels. Real-time analysis of cAMP dynamics revealed that PI3Kγ modulates the degradation of cAMP in primary VSMCs independently of its kinase activity through regulation of the enzyme phosphodiesterase 4. Importantly, the use of an N-terminal competing peptide of PI3Kγ blocked primary VSMC proliferation. These data provide evidence for a kinase-independent role of PI3Kγ in arterial remodeling and reveal novel strategies targeting the docking function of PI3Kγ for the treatment of cardiovascular diseases.

Protein phosphatase 1 activity controls a balance between collective and single cell modes of migration.

Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.

PI3KC2α-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress.

Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and this specific autophagic response depends on primary cilium (PC) signaling and leads to cell size regulation. Here we report that PI3KC2α, required for ciliogenesis and PC functions, promotes the synthesis of a local pool of PI3P upon shear stress. We show that PI3KC2α depletion in cells subjected to shear stress abolishes ciliogenesis as well as the autophagy and related cell size regulation. We finally show that PI3KC2α and VPS34, the two main enzymes responsible for PI3P synthesis, have different roles during autophagy, depending on the type of cellular stress: while VPS34 is clearly required for starvation-induced autophagy, PI3KC2α participates only in shear stress-dependent autophagy.

Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγ-dependent T cells response.

AIMS: Defects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects. METHODS AND RESULTS: Using in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine. CONCLUSION: Altogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing.

Small GTPases orchestrate cell-cell communication during collective cell movement.

Collective cell migration is a critical mechanism involved in cell movement during various physiological and pathological processes such as angiogenesis and metastasis formation. During collective movement, cells remain functionally connected and can coordinate individual cell behaviors to ensure efficient migration. A cell-cell communication process ensures this complex coordination. Although the mechanisms regulating cell-cell communication remain unclear, recent findings indicate that it is based on acto-myosin cytoskeleton tension transmission from cell to cell through adherens junctions. As for single cell migration, small GTPases of the Rho and Rab families have been shown to be critical regulators of collective motion. Here, we discuss our current understanding on how these small GTPases are themselves regulated and how they control cell-cell communication during collective migration. Moreover, we also shed light on the key role of cell-cell communication and RhoGTPases in the physiological context of endothelial cell migration during angiogenesis.

Immune and Smooth Muscle Cells Interactions in Atherosclerosis: How to Target a Breaking Bad Dialogue?

Inflammation is a well-known pathophysiological factor of atherosclerosis but its therapeutic targeting has long been ignored. However, recent advances in the understanding of the immune mechanisms implicated in atherosclerosis have unveiled several therapeutic targets currently undergoing clinical trials. These studies have also shed light on a dialogue between the immune compartment and vascular smooth muscle cells (VSMCs) that plays a critical role in atherosclerotic disease initiation, progression, and stabilization. Our review focuses on the link between cellular and soluble immune effectors and VSMC behavior at different phases of the pathology. Furthermore, we discuss the potential targeting of these interactions to efficiently prevent cardiovascular diseases.

Non-autonomous role of Cdc42 in cell-cell communication during collective migration.

Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, we show here that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, we demonstrate that Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, we found that the endocytic small GTPase Rab11, controls Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. In addition, we showed that Cdc42 acts upstream of Moesin, a cytoskeletal regulator known to function downstream of rab11. Thus, our study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11.

Myosin II governs collective cell migration behaviour downstream of guidance receptor signalling.

Border cell migration during Drosophila oogenesis is a potent model to study collective cell migration, a process involved in development and metastasis. Border cell clusters adopt two main types of behaviour during migration: linear and rotational. However, the molecular mechanism controlling the switch from one to the other is unknown. Here, we demonstrate that non-muscle Myosin II (NMII, also known as Spaghetti squash) activity controls the linear-to-rotational switch. Furthermore, we show that the regulation of NMII takes place downstream of guidance receptor signalling and is critical to ensure efficient collective migration. This study thus provides new insight into the molecular mechanism coordinating the different cell behaviours in a migrating cluster.

Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1.

PtdIns5P is a lipid messenger acting as a stress-response mediator in the nucleus, and known to maintain cell activation through traffic alterations upon bacterial infection. Here, we show that PtdIns5P regulates actin dynamics and invasion via recruitment and activation of the exchange factor Tiam1 and Rac1. Restricted Rac1 activation results from the binding of Tiam1 DH-PH domains to PtdIns5P. Using an assay that mimics Rac1 membrane anchoring by using Rac1-His and liposomes containing Ni(2+)-NTA modified lipids, we demonstrate that intrinsic Tiam1 DH-PH activity increases when Rac1 is anchored in a PtdIns5P-enriched environment. This pathway appears to be general since it is valid in different pathophysiological models: receptor tyrosine kinase activation, bacterial phosphatase IpgD expression and the invasive NPM-ALK(+) lymphomas. The discovery that PtdIns5P could be a keystone of GTPases and cytoskeleton spatiotemporal regulation opens important research avenues towards unravelling new strategies counteracting cell invasion.

Cell coordination of collective migration by Rab11 and Moesin.

Cell migration is an important process involved in developmental events and in pathologies such as cancer. Cell migration can be classified into two types: individual and collective cell movements. Compared with individual migration, collective cell migration is less understood and has drawn increasing attention lately because of its emerging role in cancer spreading. We have recently established that Rab11 is absolutely required for spatial control of Rac1 activity through the control of cell-cell communication during collective movements (Ramel, et al. 2013). Moreover, we demonstrated that Rab11 acts through the control of Moesin activity. Here, we discuss how Rab11 and Moesin could cooperate to transfer forces from cell to cell in order to insure coordinated collective cell migration.

GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation.

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.

Rab11 regulates cell-cell communication during collective cell movements.

Collective cell movements contribute to development and metastasis. The small GTPase Rac is a key regulator of actin dynamics and cell migration but the mechanisms that restrict Rac activation and localization in a group of collectively migrating cells are unknown. Here, we demonstrate that the small GTPases Rab5 and Rab11 regulate Rac activity and polarization during collective cell migration. We use photoactivatable forms of Rac to demonstrate that Rab11 acts on the entire group to ensure that Rac activity is properly restricted to the leading cell through regulation of cell-cell communication. In addition, we show that Rab11 binds to the actin cytoskeleton regulator Moesin and regulates its activation in vivo during migration. Accordingly, reducing the level of Moesin activity also affects cell-cell communication, whereas expressing active Moesin rescues loss of Rab11 function. Our model suggests that Rab11 controls the sensing of the relative levels of Rac activity in a group of cells, leading to the organization of individual cells in a coherent multicellular motile structure.

Evi5 promotes collective cell migration through its Rab-GAP activity.

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.

Shigella flexneri infection generates the lipid PI5P to alter endocytosis and prevent termination of EGFR signaling.

The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase-Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.

The nucleophosmin-anaplastic lymphoma kinase oncogene interacts, activates, and uses the kinase PIKfyve to increase invasiveness.

NPM-ALK is a chimeric tyrosine kinase detected in most anaplastic large cell lymphomas that results from the reciprocal translocation t(2,5)(p23;q35) that fuses the N-terminal domain of nucleophosmin (NPM) to the catalytic domain of the anaplastic lymphoma kinase (ALK) receptor. The constitutive activity of the kinase is responsible for its oncogenicity through the stimulation of several downstream signaling pathways, leading to cell proliferation, migration, and survival. We demonstrated previously that the high level of phosphatidylinositol 5-phosphate measured in NPM-ALK-expressing cells is controlled by the phosphoinositide kinase PIKfyve, a lipid kinase known for its role in vesicular trafficking. Here, we show that PIKfyve associates with NPM-ALK and that the interaction involves the 181-300 region of the oncogene. Moreover, we demonstrate that the tyrosine kinase activity of the oncogene controls PIKfyve lipid kinase activity but is dispensable for the formation of the complex. Silencing or inhibition of PIKfyve using siRNA or the PIKfyve inhibitor YM201636 have no effect on NPM-ALK-mediated proliferation and migration but strongly reduce invasive capacities of NPM-ALK-expressing cells and their capacity to degrade the extracellular matrix. Accordingly, immunofluorescence studies confirm a perturbation of matrix metalloproteinase 9 localization at the cell surface and defect in maturation. Altogether, these results suggest a role for PIKfyve in NPM-ALK-mediated invasion.

Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration.

Border cell migration is a stereotyped migration occurring during the development of the Drosophila egg chamber. During this process, a cluster composed of six to eight follicle cells migrates between nurse cells toward the oocyte. Receptor tyrosine kinases (RTKs) are enriched at the leading edge of the follicle cells and establish the directionality of their migration. Endocytosis has been shown to play a role in the maintenance of this polarization; however, the mechanisms involved are largely unknown. In this study, we show that border cell migration requires the function of the small GTPases Rab5 and Rab11 that regulate trafficking through the early and the recycling endosome, respectively. Expression of a dominant negative form of rab11 induces a loss of the polarization of RTK activity, which correlates with a severe migration phenotype. In addition, we demonstrate that the exocyst component Sec15 is distributed in structures that are polarized during the migration process in a Rab11-dependent manner and that the down-regulation of different subunits of the exocyst also affects migration. Together, our data demonstrate a fundamental role for a plasma membrane-endosome trafficking cycle in the maintenance of active RTK at the leading edge of border cells during their migration.

Matrix metalloproteinase-9 is upregulated in nucleophosmin-anaplastic lymphoma kinase-positive anaplastic lymphomas and activated at the cell surface by the chaperone heat shock protein 90 to promote cell invasion.

Many anaplastic large cell lymphomas (ALCL) express the chimeric oncogene NPM-ALK, which drives malignant transformation and invasion. In this study, we show that NPM-ALK expression increases matrix metalloproteinase-9 (MMP-9) expression. Accordingly, we found that 100% of a large panel of ALK(+) ALCL biopsies examined were also MMP-9(+), in contrast to only 36.3% of ALK(-) tumors. Mechanistic studies revealed that Rac1 drove MMP-9 secretion. The MMP inhibitor GM6001 and MMP-9 blocking antibodies abolished the invasiveness of NPM-ALK(+) cells. Interestingly, the hyaluronan receptor CD44 acted as a docking surface for MMP-9 and the chaperone heat shock protein 90 on the cell surface, where MMP-9 was cleaved and activated. Membrane-associated MMP-9 was localized to invadopodia, which display a strong gelatinase activity. Taken together, our observations strengthen the concept that chaperones have a major extracellular role in the regulation of protein activation status, and reveal new factors that are crucial for spreading and invasion of ALK(+) ALCL. They also point out new factors crucial for ALK(+) ALCL.