Endothelial Adherens Junctions Research Articles

Long-term dioxin exposure promotes systemic endothelial damage and diastolic cardiac dysfunction in mice

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic and persistent organic pollutant that bioaccumulates in the body through the food chain. The toxicity of TCDD is mediated by the activation of aryl hydrocarbon receptor (AHR), a transcription factor regulating detoxification enzyme expression. Although human exposure to TCDD has been shown to be associated with increased risk of cardiovascular disease and deaths, the impact of TCDD exposure on the heart at cellular and molecular levels remains poorly understood. To assess the cardiac and vascular stress response to long-term TCDD exposure in mice. Height-week-old C57BL/6N male mice were exposed for 12 months to low-dose TCDD (4 μg/kg, once per week) or vehicle by oral gavage. One-year TCDD exposure did not affect body weight or arterial pressure in mice, but cardiac mass index was increased. Echocardiographic analysis showed that long-term TCDD exposure led to diastolic LV dysfunction with preserved ejection fraction. Diastolic LV dysfunction was confirmed by PV loop analysis showing an increased diastolic stiffness with preserved systolic contractility in TCDD-exposed mice. Using permeabilized cardiac myofibers, we showed that chronic TCDD administration alters cardiac mitochondrial energetics and increases ROS production. Confocal imaging and q-PCR analysis revealed that AHR is expressed in the mouse heart, especially in endothelial cells that respond better than cardiomyocytes to TCDD exposure. Cardiac MRI showed decreased LV perfusion in TCDD-exposed mice. This was associated with a loss of endothelium-mediated relaxation responses to acetylcholine and to flow in mesenteric arteries of TCDD-exposed mice. In parallel, en face microscopy revealed profound disruptions of endothelial adherens junctions in the aorta of mice after TCDD exposure. These data demonstrate severe cardiac and systemic endothelial dysfunction upon TCDD exposure. This endothelial dysfunction was associated with a metabolic adaptation of endothelial cells, as assessed by the enhanced mitochondrial oxidative phosphorylation in TCDD-exposed primary muscle endothelial cells. Long-term exposure to TCDD promotes endothelial dysfunction and diastolic LV impairment, possibly through an increased oxidative stress and disrupted mitochondrial bioenergetics in both endothelial cells and cardiomyocytes.

C-peptide attenuates hyperglycemia-induced pulmonary fibrosis by inhibiting transglutaminase 2.

Proinsulin C-peptide has a protective effect against diabetic complications; however, its role in hyperglycemia-induced pulmonary fibrosis is unknown. In this study, we investigated the inhibitory effect of C-peptide on hyperglycemia-induced pulmonary fibrosis and the molecular mechanism of C-peptide action in the lungs of diabetic mice and in human pulmonary microvascular endothelial cells (HPMVECs). We found that, in the lungs of diabetic mice, C-peptide supplementation using osmotic pumps attenuated hyperglycemia-induced pulmonary fibrosis and expression of fibrosis-related proteins. In HPMVECs, C-peptide inhibited vascular endothelial growth factor-induced adherens junction disruption and endothelial cell permeability by inhibiting reactive oxygen species generation and transglutaminase (TGase) activation. In the lungs, C-peptide supplementation suppressed hyperglycemia-induced reactive oxygen species generation, TGase activation, and microvascular leakage. C-peptide inhibited hyperglycemia-induced inflammation and apoptosis, which are involved in the pathological process of pulmonary fibrosis. We also demonstrated the role of TGase2 in hyperglycemia-induced vascular leakage, inflammation, apoptosis, and pulmonary fibrosis in the lungs of diabetic TGase2-null (Tgm2-/-) mice. Furthermore, we demonstrated a long-term inhibitory effect of systemic delivery of C-peptide using K9-C-peptide hydrogels on hyperglycemia-induced fibrosis in diabetic lungs. Overall, our findings suggest that C-peptide alleviates hyperglycemia-induced pulmonary fibrosis by inhibiting TGase2-mediated microvascular leakage, inflammation, and apoptosis in diabetes.

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts invivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.

Angiopoietin-like 4-Induced 3D Capillary Morphogenesis Correlates to Stabilization of Endothelial Adherens Junctions and Restriction of VEGF-Induced Sprouting

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell–cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.

Vascular Endothelial (VE)-cadherin-mediated adherens junctions involvement in cardiovascular progenitor cell specification.

Vascular Endothelial cadherin, a type II classical cadherin, is the major cadherin molecule participating in homotypic cell-cell adhesion structures between endothelial cells. It associates with cytoplasmic and membrane cytoskeletal elements to form endothelial adherens junctions (AJs), pivotal in regulating endothelial barrier function in the adult. VE-cadherin-mediated AJs are also involved in signaling via direct or indirect associations with receptors. The generation of mutant animals, especially mice and zebrafish, revealed many details concerning the role of VE-cadherin-mediated AJs in cardiovascular development. In general, VE-cadherin knockout (KO) in mice is embryonic lethal due to severe cardiovascular defects, and major signaling pathways as well as vascular formation cues were discovered in developing endothelium. However, there is little information regarding AJs formation and their components in cardiovascular progenitors. We have characterized in detail the activation pattern of mouse VE-cadherin promoter (Pvec) in a mouse embryonic stem cells (ESCs) differentiation system in vitro. Surprisingly, we found that it is activated transiently in cardiac progenitors that belong to the second heart field. Based on Pvec activation, we isolated this population in vitro and found that it can self-renew by induction of the Wnt/β-catenin pathway. Next, we successfully established cell culture conditions that allowed self-renewal of this population that consists of endothelial and cardiac progenitors. Transplantation in rat hearts showed that they can survive and differentiate to cardiomyocytes and endothelial cells. Although further characterization is needed, these cells can be used in cell-based therapies as well as in drug screening.

Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors.

VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts.Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.

Generating Monocyte-Derived Endothelial-like Cells for Vascular Regeneration.

A major limitation in engineering vascular grafts is the lack of proper endothelium to prevent thrombosis and subsequent graft failure. Obtaining endothelial cells from patients' vasculature is intrusive and requires extensive culture time. Here we present an alternative strategy wherein abundant and easily accessible monocytes from peripheral blood are cultured and differentiated towards an endothelial-like state capable of preventing thrombosis through production of nitric oxide and formation of endothelial adherens junctions. Considering the plethora of monocytes present within peripheral blood, this method provides a robust alternative to generating endothelial cells required for vascular graft production.

Interleukin-1RA Mitigates SARS-CoV-2-Induced Inflammatory Lung Vascular Leakage and Mortality in Humanized K18-hACE-2 Mice.

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Strategies to Attenuate Myocardial Infarction and No-Reflow Through Preservation of Vascular Integrity by Pigment Epithelium-Derived Factor.

The phenomenon of no-reflow seriously limits the therapeutic value of coronary recanalization and leads to poor prognosis. Recent studies have demonstrated the potential role of pigment epithelium-derived factor (PEDF) in stabilizing endothelial cell junction, reducing vascular permeability and maintaining a quiescent vasculature. In this study, intramyocardial gene delivery was performed 5 days before the acute myocardial infarction/recanalization experiment in male rats. Positron emission tomography perfusion imaging with 13N-NH3 indicated PEDF to promote microvascular reperfusion significantly 4 h postcoronary occlusion. PEDF was observed to maintain the stability of endothelial adherens junctions (AJs), thus preventing the occurrence of no-reflow. PEDF reduced the hypoxia-induced vascular endothelial (VE)-cadherin endocytosis through PEDF/LR/Src/VE-cadherin S665 axis in vitro, which was remarkably observed to maintain endothelial AJs. Generally, PEDF might function as a relevant target for therapeutic vasculoprotection by way of regulating the phosphorylation level of VE-cadherin according to our data, thus being crucial for preventing no-reflow.

SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

KRIT1 stabilizes endothelial adherens junctions independent of Rap1 via regulation of β1‐integrin

The junctional scaffold KRIT1 interacts with adherens junctions (AJ) proteins to stabilize endothelial cell-cell contacts. Loss of KRIT1 function leads to Cerebral Cavernous Malformations (CCM), a disease characterized by leaky vascular lesions as the result of perturbed endothelial proliferation and inflammatory signaling. As a true Rap1 effector, KRIT1 localizes to and associates with AJ after binding active Rap1. Inhibition of this interaction by mutation of the Rap1-binding site (KRIT1-R452E) prevents KRIT1 from localizing to AJ and stabilizing barrier function. However, it remains unclear whether KRIT1 can stabilize endothelial junctions independently of Rap1. In order to study the mechanisms of KRIT1-mediated junction stabilization, we targeted mCherry-tagged KRIT1-R452E to the plasma membrane using a C-terminal lipid modification (MT-R452E). Immunofluorescence studies indicated that this construct localized to cell-cell contacts similar to WT-KRIT1 in endothelial cells depleted of endogenous KRIT1 (KRIT1 shRNA). While MT-R452E partially rescued AJ staining in these cells, it was unable to rescue barrier function, suggesting that both KRIT1 and Rap1 may be necessary at AJ to stabilize the endothelial barrier. As Rap1 binding may also regulate a putative intramolecular interaction between KRIT1's first NPxY motif and its C-terminal PTB domain, we sought to evaluate whether stabilization of endothelial junctions was dependent on KRIT1 conformation. The NPxY motif of MT-R452E was mutated (N192A-Y195A) to yield a constitutively open, membrane-targeted construct (MT-R452E-ΔNPxY). Immunofluorescence confirmed that this construct localized to cell-cell contacts, and appeared to rescue AJ staining after loss of endogenous KRIT1. Interestingly, open conformation R452E lacking any targeting sequence (R452E-ΔNPxY) also rescued AJ staining despite remaining primarily cytosolic. Further, both open conformation constructs rescued barrier function independent of Rap1 binding, indicating that KRIT1's junction stabilizing effects may be exerted without binding of Rap1 or localization to cell-cell contacts. As this NPxY (192-195) motif mediates binding to ICAP1a, which has been shown to regulate β1-integrin activity, we next examined whether junctional stability was associated with changes in β1-integrin activation. Using an activation-specific antibody against β1-integrin, we observed an increase in large fibrillar adhesions in cells depleted of KRIT1 (KRIT1 shRNA), or rescued with either R452E or MT-R452E, constructs which notably failed to rescue loss of barrier function. Conversely, expression of WT, MT-R452E-ΔNPXY, and R452E-ΔNPXY constructs, which do rescue barrier function, was associated with smaller, peripheral staining of focal contacts. These results suggest that KRIT1 may stabilize endothelial cell junctions independent of Rap1 by regulating β1-integrin-containing adhesive complexes. We expect that these novel findings will further our understanding of junctional physiology while facilitating the development of novel CCM therapeutics.

SARS‐CoV‐2 spike protein induces degradation of junctional proteins that maintain endothelial barrier integrity

BackgroundSARS‐CoV‐2 enters cells through the Angiotensin‐converting enzyme 2 (ACE‐2) receptor present on the cell surface. ACE‐2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. Reports suggest that COVID‐19 patients also showed symptoms related to endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. We investigated this in vitro using endothelial cell culture and recombinant SARS‐CoV‐2 spike protein S1 Receptor‐Binding Domain (S1RBD).Materials and MethodsMouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. The expression of VE‐Cadherin, PECAM‐1, JAM‐A and Connexin 43, was probed by Western blot in normal and diabetic cells, before and after treatment with S1RBD.ResultsThe expression of VE‐Cadherin, an endothelial adherens junction protein, was not affected by diabetes or by S1RBD treatment. PECAM‐1 expression showed a small but significant (~15%) decrease after S1RD treatment in control cells. Junctional Adhesion Molecule (JAM‐A), a tight junction protein, and the gap junction protein, Connexin‐43, decreased by ~50 and 80% respectively in the presence of S1RBD when compared to untreated controls. Expression of PECAM‐1, JAM‐A and connexin‐43 were significantly lower in untreated diabetic endothelial cells, compared to untreated control cells. This expression level further decreased significantly in the presence of S1RBD, with maximal effects observed with JAM‐A and Connexin‐43 expression (~80 and 90% decrease, respectively). These results indicate that healthy and diabetic endothelial cells respond differently when challenged with SARS‐CoV‐2 S1RBD.ConclusionS1RBD‐induces degradation of endothelial junctional proteins that likely affects endothelial barrier function and causes vascular damage.

ENOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin.

Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics may aggravate hypoxia; therefore, therapy development is needed. Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) in pathological neovascularization and vessel permeability. Suppression of NO formation was achieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine (S1176A) mutant mice. Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remaining vascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions, manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage. We conclude that NO destabilizes adheren junctions, resulting in vascular hyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway. This study was supported by the Swedish Cancer foundation (19 0119 Pj ), the Swedish Research Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and a Fondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD 03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supported by Grants R35 HL139945, P01 HL1070205, AHA MERIT Award. DV was supported by grants from the Deutsche Forschungsgemeinschaft, SFB1450, B03, and CRU342, P2.

Mechanosensitive Piezo1 channel activation promotes ventilator-induced lung injury via disruption of endothelial junctions in ARDS rats

ObjectiveThis study aimed to investigate the role of endothelial Piezo1 in mediating ventilator-induced lung injury secondary to acute respiratory distress syndrome (ARDS). MethodsRats and lung endothelial cells (ECs) were transfected with Piezo1 shRNA (shPiezo1) and Piezo1 siRNA, respectively, to knock down Piezo1. Intratracheal instillation or incubation with lipopolysaccharide (LPS) was used to establish an ARDS model, and high tidal volume (HVT) ventilation or 20% cyclic stretch (CS) was administered to simulate a two-hit injury. Lung injury, alterations in lung endothelial barrier, disruption of adherens junctions (AJs), and Ca2+ influx were assessed. ResultsLung vascular hyperpermeability was further increased in ARDS rats following HVT ventilation, which was abrogated in shPiezo1-treated rats. 20% CS led to severer rupture of AJs following LPS stimulation as indicated by immunofluorescence staining. The internalization and degradation of VE-cadherin were blocked by knockdown of Piezo1. Additionally, 20% CS induced Piezo1 activation, manifesting as elevated intracellular Ca2+ concentration in LPS-treated ECs, and subsequently increased calcium-dependent calpain activity. Pharmacological inhibition of calpain or Piezo1 knockdown prevented the loss of VE-cadherin, p120-catenin, and β-catenin in ARDS rats undergoing HVT ventilation and LPS-treated ECs exposed to 20% CS. ConclusionExcessive mechanical stretch during ARDS induces the activation of Piezo1 channel and its downstream target, calpain, via Ca2+ influx. This results in the disassembly of endothelial AJs and further facilitates lung endothelial barrier breakdown and vascular hyperpermeability.

Molecular Genetic Features of Cerebral Cavernous Malformations (CCM) Patients: An Overall View from Genes to Endothelial Cells.

Cerebral cavernous malformations (CCMs) are vascular lesions that affect predominantly microvasculature in the brain and spinal cord. CCM can occur either in sporadic or familial form, characterized by autosomal dominant inheritance and development of multiple lesions throughout the patient’s life. Three genes associated with CCM are known: CCM1/KRIT1 (krev interaction trapped 1), CCM2/MGC4607 (encoding a protein named malcavernin), and CCM3/PDCD10 (programmed cell death 10). All the mutations identified in these genes cause a loss of function and compromise the protein functions needed for maintaining the vascular barrier integrity. Loss of function of CCM proteins causes molecular disorganization and dysfunction of endothelial adherens junctions. In this review, we provide an overall vision of the CCM pathology, starting with the genetic bases of the disease, describing the role of the proteins, until we reach the cellular level. Thus, we summarize the genetics of CCM, providing a description of CCM genes and mutation features, provided an updated knowledge of the CCM protein structure and function, and discuss the molecular mechanisms through which CCM proteins may act within endothelial cells, particularly in endothelial barrier maintenance/regulation and in cellular signaling.

PCB and TCDD derived embryonic cardiac defects result from a novel AhR pathway

Polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contaminants known to impact cardiac development, a key step in the embryonic development of most animals. To date, little is understood of the molecular mechanism driving the observed cardiac defects in exposed fishes. The literature shows PCB & TCDD derived cardiac defects are concurrent with, but not caused by, expression of cyp1A, due to activation of the aryl hydrocarbon receptor (AhR) gene activation pathway. However, in this study, detailed visualization of fish hearts exposed to PCBs and TCDD show that, in addition to a failure of cardiac looping in early heart development, the inner endocardial lining of the heart fails to maintain proper cell adhesion and tissue integrity. The resulting gap between the endocardium and myocardium in both zebrafish and Atlantic sturgeon suggested functional faults in endothelial adherens junction formation. Thus, we explored the molecular mechanism triggering cardiac defects using immunohistochemistry to identify the location and phosphorylation state of key regulatory and adhesion molecules. We hypothesized that PCB and TCDD activates AhR, phosphorylating Src, which then phosphorylates the endothelial adherens junction protein, VEcadherin. When phosphorylated, VEcadherin dimers, found in the endocardium and vasculature, separate, reducing tissue integrity. In zebrafish, treatment with PCB and TCDD contaminants leads to higher phosphorylation of VEcadherin in cardiac tissue suggesting that these cells have reduced connectivity. Small molecule inhibition of Src phosphorylation prevents contaminant stimulated phosphorylation of VEcadherin and rescues both cardiac function and gross morphology. Atlantic sturgeon hearts show parallels to contaminant exposed zebrafish cardiac phenotype at the tissue level. These data suggest that the mechanism for PCB and TCDD action in the heart is, in part, distinct from the canonical mechanism described in the literature and that cardiac defects are impacted by this nongenomic mechanism.

Dissecting the Cellular Mechanism of Prostacyclin Analog Iloprost in Reversing Vascular Dysfunction in Scleroderma.

Intravenous iloprost improves Raynaud's phenomenon (RP) and promotes healing of digital ulcers in systemic sclerosis (SSc; scleroderma). Despite a short half-life, its clinical efficacy lasts weeks. Endothelial adherens junctions, which are formed by VE-cadherin clustering between endothelial cells (ECs), regulate endothelial properties including barrier function, endothelial-to-mesenchymal transition (EndoMT), and angiogenesis. We undertook this study to investigate the hypothesis that junctional disruption contributes to vascular dysfunction in SSc, and that the protective effect of iloprost is mediated by strengthening of those junctions. Dermal ECs from SSc patients and healthy controls were isolated. The effect of iloprost on ECs was examined using immunofluorescence, permeability assays, Matrigel tube formation, and quantitative polymerase chain reaction. Adherens junctions in SSc were disrupted compared to normal ECs, as indicated by reduced levels of VE-cadherin and increased permeability in SSc ECs (P < 0.05). Iloprost increased VE-cadherin clustering at junctions and restored junctional levels of VE-cadherin in SSc ECs (mean ± SD 37.3 ± 4.3 fluorescence units) compared to normal ECs (mean ± SD 29.7 ± 3.4 fluorescence units; P < 0.05), after 2 hours of iloprost incubation. In addition, iloprost reduced permeability of monolayers, increased tubulogenesis, and blocked EndoMT in both normal and SSc ECs (n ≥ 3; P < 0.05). The effects in normal ECs were inhibited by a function-blocking antibody that prevents junctional clustering of VE-cadherin. Our data suggest that the long-lasting effects of iloprost reflect its ability to stabilize adherens junctions, resulting in increased tubulogenesis and barrier function and reduced EndoMT. These findings provide a mechanistic basis for the use of iloprost in treating SSc patients with RP and digital ulcers.

Sirt3 Maintains Microvascular Endothelial Adherens Junction Integrity to Alleviate Sepsis-Induced Lung Inflammation by Modulating the Interaction of VE-Cadherin and β-Catenin.

Inflammatory injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). However, the mechanisms underlying inflammatory injury remain obscure. Here, we developed the novel strategy to suppress lung inflammation through maintaining microvascular endothelial barrier integrity. VE-cadherin is the main adherens junction protein that interacts with β-catenin and forms a complex. We found that lung inflammation was accompanied by decreased VE-cadherin expression and increased β-catenin activity in animal models and human pulmonary microvascular endothelial cells (HPMECs), illuminating the relationship among VE-cadherin/β-catenin complex, microvascular endothelial barrier integrity, and inflammation. Furthermore, we showed that the VE-cadherin/β-catenin complex dissociated upon lung inflammation, while Sirt3 promoted the stability of such a complex. Sirt3 was decreased during lung inflammation in vivo and in vitro. Sirt3 deficiency not only led to the downregulation of VE-cadherin but also enhanced the transcriptional activity of β-catenin that further increased β-catenin target gene MMP-7 expression, thereby promoting inflammatory factor COX-2 expression. Sirt3 overexpression promoted VE-cadherin expression, inhibited β-catenin transcriptional activity, strengthened the stability of the VE-cadherin/β-catenin complex, and suppressed inflammation in HPMECs. Notably, Sirt3 deficiency significantly damaged microvascular endothelial barrier integrity and intensified lung inflammation in animal model. These results demonstrated the role of Sirt3 in modulating microvascular endothelial barrier integrity to inhibit inflammation. Therefore, strategies that aim at enhancing the stability of endothelial VE-cadherin/β-catenin complex are potentially beneficial for preventing sepsis-induced lung inflammation.