175 research outputs found
Signaling pathways in the specification of arteries and veins
No full textThe establishment of arterial and venous identity of endothelial cells is critical for the proper anatomic configuration and function of the vascular tree. Arterial and venous specification of endothelial cells is determined by genetic factors, although surrounding cells and hemodynamic forces may also contribute to vascular remodeling. This review provides an overview of the signaling pathways and related transcription factors implicated in differentiation of endothelial cells. We will discuss, in particular, the role of upstream and downstream effectors of Wnt, Sox, and Notch pathways. The understanding of the molecular mechanisms that orchestrate endothelial differentiation may have therapeutic relevance for diseases such as atherosclerosis, arteriovenous malformations, aneurysms, and others
The molecular basis of the blood brain barrier differentiation and maintenance. Is it still a mystery?
No full textEndothelial cells (ECs) differ in morphology and functional responses in the different regions of the vascular tree. During embryo development they acquire organ specific characteristics to respond to the needs of the perfused organs. The brain microvasculature is a striking example of this process. This particular vasculature develops unique properties to assure a tight control of permeability between blood and the underlying nervous system. To this end, these cells present well developed cell to cell junctions, tight basement membrane and express a series of transporters which support the passage of nutrients and toxic agents inside or outside the brain, respectively. This highly differentiated EC phenotype is induced and maintained by the cross-talk with the surrounding cells such as pericytes and astrocytes. Recent evidence highlights the molecular basis of this cross-talk (constituting the neurovascular unit) and opens new perspectives in the development of drugs which modulate blood-brain-barrier (BBB) permeability properties. In this review we describe the specific features of the BBB and we discuss recent data on the role of Wnt as a mediator of brain angiogenesis and BBB differentiatio
Wnt/beta-catenin signaling controls development of the blood–brain barrier
No full textThe blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown
Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro
No full textReproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation
Timing and Strategy for Weaning From Venoarterial ECMO are Complex Issues
No full textOBJECTIVE:Weaning from venoarterial extracorporeal membrane oxygenation (VA ECMO) usually is performed without clear guidelines; yet, patients still die after removal of extracorporeal circulation because of inadequate heart or end-organ recovery. The aim of the study was to address the weaning procedure, analyzing the hemodynamic and echocardiographic picture of patients weaned and to identify predictors of poor outcome among this population.DESIGN:Observational study.SETTING:University hospital.PARTICIPANTS:One hundred twenty-nine VA ECMO cases.INTERVENTIONS:None.MEASUREMENTS AND MAIN RESULTS:Forty-nine patients (38%) were weaned, 7 (5.4%) were bridged to a ventricular assist device, and 6 (5.2%) were listed for heart transplantation. Weaned patients showed a significant increase of pulse pressure (35 [0-50] mmHg before ECMO, 59 [53-67] mmHg at weaning, 61 [51-76] mmHg after ECMO (p<0.001]) and reduction of dose of inotropes (inotropic score [as defined in the text] 20 [14-40] before ECMO, 10 [3-15] at weaning, and 10 [5-15] after ECMO, p<0.001). Left ventricular ejection fraction (LVEF) increased from 19 (0-22.5)% before ECMO to 35 (22-55)% after ECMO (p<0.001). A significant improvement of right ventricular (RV) function was observed in weaned patients (RV dysfunction from 52% to 21%, p<0.001). Among weaned patients, 15 (31%) died. Patients who died after weaning had longer ECMO duration compared to discharged patients (8 [5-11] v 4 [2-6] days, p = 0.01) and more transfusions (22 [10-37] v 7 [0.5-15] units, p = 0.02); survival was lower in patients with central ECMO (postcardiotomy) compared to peripheral ECMO (p = 0.045). Mortality was higher in those with persistence of RV failure, continuous venovenous hemofiltration, higher inotropic score, lower systolic pressure, or higher leucocyte count at weaning.CONCLUSIONS:Successful weaning from ECMO is a multifaceted process, which encompasses consistent recovery of myocardial and end-organ function; LVEF, though improved, is still low at weaning. Hospital survival is correlated significantly to the duration of ECMO support and to bleeding complications
Overlapping and divergent signaling pathways of N-cadherin and VE-cadherin in endothelial cells
No full textEndothelial cells (ECs) express 2 members of the cadherin family, VE and N-cadherin. Although VE-cadherin induces EC homotypic adhesion, N-cadherin function in ECs remains largely unknown. EC-specific inactivation of either VE or N-cadherin leads to early fetal lethality suggesting that these cadherins play a nonredundant role in vascular development. We report here that VE-cadherin negatively controls junctional localization and expression of N-cadherin by limiting p120-catenin availability and reducing β-catenin transcriptional activity. Using EC lines expressing either VE or N-cadherin we found that both cadherins inhibit cell proliferation and apoptosis. Both trigger the phosphatidylinositol-3- OH-kinase (PI3K)-AKT-Forkhead-box protein-O1 (FoxO1) pathway and reduce β-catenin transcriptional activity. The extent of signaling correlates with the total level of cadherins regardless of the type of cadherin expressed. In contrast, basal and fibroblast growth factor (FGF)-induced cell motility is promoted by N-cadherin and strongly inhibited by VE-cadherin. This opposite effect is partly because of the ability of VE-cadherin to associate with FGF receptor and the density- enhanced phosphatase-1 (Dep-1) which, in turn, inhibits receptor signaling. We conclude that VE and N-cadherin have both additive and divergent effects on ECs. Differences in signaling are due, in part, to cadherin association with growth factor receptors and modulation of their downstream signaling
JAM-A promotes neutrophil chemotaxis by controlling integrin internalization and recycling
No full textThe membrane-associated adhesion molecule JAM-A is required for neutrophil infiltration in inflammatory or ischemic tissues. JAM-A expressed in both endothelial cells and neutrophils has such a role, but the mechanism of action remains elusive. Here we show that JAM-A has a cell-autonomous role in neutrophil chemotaxis both in vivo and in vitro, which is independent of the interaction of neutrophils with endothelial cells. On activated neutrophils, JAM-A concentrates in a polarized fashion at the leading edge and uropod. Surprisingly, a significant amount of this protein is internalized in intracellular endosomal-like vesicles where it codistributes with integrin β1. Clustering of β1 integrin leads to JAM-A co-clustering, whereas clustering of JAM-A does not induce integrin association. Neutrophils derived from JAM-A-null mice are unable to correctly internalize β1 integrins upon chemotactic stimuli and this causes impaired uropod retraction and cell motility. Consistently, inhibition of integrin internalization upon treatment with BAPTA-AM induces a comparable phenotype. These data indicate that JAM-A is required for the correct internalization and recycling of integrins during cell migration and might explain why, in its absence, the directional migration of neutrophils towards an inflammatory stimulus is markedly impaired
The Wnt/β-Catenin Pathway Modulates Vascular Remodeling and Specification by Upregulating Dll4/Notch Signaling
No full textSummaryThe Wnt/β-catenin pathway is evolutionary conserved signaling system that regulates cell differentiation and organogenesis. We show that endothelial specific stabilization of Wnt/β-catenin signaling alters early vascular development in the embryo. The phenotype resembles that induced by upregulation of Notch signaling, including lack of vascular remodeling, altered elongation of the intersomitic vessels, defects in branching, and loss of venous identity. Both in vivo and in vitro data show that β-catenin upregulates Dll4 transcription and strongly increases Notch signaling in the endothelium, leading to functional and morphological alterations. The functional consequences of β-catenin signaling depend on the stage of vascular development and are lost when a gain-of-function mutation is induced at a late stage of development or postnatally. Our findings establish a link between Wnt and Notch signaling in vascular development. We propose that early and sustained β-catenin signaling prevents correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification
Importance of junctional adhesion molecule-A for neointimal lesion formation and infiltration in atherosclerosis-prone mice
No full textObjective - Although junctional adhesion molecule-A (JAM-A) has recently been implicated in leukocyte recruitment on early atherosclerotic endothelium and after reperfusion injury, its role in neointima formation after arterial injury remains to be elucidated. Methods and Results - Here we show that the genetic deletion of JAM-A in apolipoprotein E - deficient (apoE(-/-)) mice significantly reduced neointimal hyperplasia after wire injury of carotid arteries without altering medial area. This was associated with a significant decrease in neointimal macrophage content, whereas the relative content of smooth muscle cells and endothelial recovery was unaltered in JAM-A(-/-) apoE(-/-) compared with JAM-A(-/-) apoE(-/-) lesions. In carotid arteries perfused ex vivo, deficiency in JAM-A significantly impaired the recruitment of monocytes 1 week, but not 1 day, after injury. These effects were paralleled by an attenuation of monocyte arrest and transmigration on activated JAM-A(-/-) apoE(-/-) versus JAM-A(-/-) apoE(-/-) endothelial cells under flow conditions in vitro. A mechanism underlying reduced recruitment was implied by findings that the luminal expression of the arrest chemokine RANTES in injured arteries and its endothelial deposition by activated platelets in vitro were diminished by JAM-A deficiency. Conclusions - Our data provide the first evidence to our knowledge for a crucial role of JAM-A in accelerated lesion formation and monocyte infiltration in atherosclerosis-prone mice
VE-cadherin is a critical endothelial regulator of TGF-beta signalling
No full textVE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-β (TGF-β) signalling in endothelial cells (ECs). TGF-β antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-β/ALK1- and TGF-β/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-β receptor complex, TβRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TβRII and may promote TGF-β signalling by enhancing TβRII/TβRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-β signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-β signalling is impaired
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