Benoît Melchior

PECAM-1 is a critical mediator of atherosclerosis

SUMMARY Atherosclerosis is a chronic inflammatory disease of large arteries in which lesion development preferentially occurs at vessel sites exposed to rapid changes in flow. We have previously shown that platelet endothelial cell adhesion molecule (PECAM-1), a surface receptor of the immunoglobulin superfamily, is involved in mechanosensing of rapid changes in flow. We wondered whether apolipoprotein E deficient (ApoE−/−) mice, predisposed to development of atheromas, would be protected from atherosclerosis by deficiency in PECAM-1. Using double knockout (DKO) mice for both PECAM-1 and ApoE (ApoE−/−/PECAM-1−/−) we found a significant reduction of sudanophilic lesions in their aortae compared to single knockout (SKO) (ApoE−/−/PECAM-1+/+) mice maintained on a high-fat Western diet. Immunostaining of aortic sinus cross sections demonstrated significantly lower ICAM-1 expression in DKO lesions compared with SKO lesions, and en face preparations of vessel regions subjected to disturbed and laminar flow showed less disruption of junctional connexin 43 in DKO than in SKO mice. Thus, PECAM-1 deficiency reduced the extent of lesions at the aortic arch and the aortic sinus, and lowered atherogenic indices. These results suggest that PECAM-1 is an important factor in the atherogenic changes seen in the ApoE-deficient mouse model and thus should be considered as a potential target for protection against atherosclerosis.

Shear-induced endothelial cell-cell junction inclination

Atheroprone regions of the arterial circulation are characterized by time-varying, reversing, and oscillatory wall shear stress. Several in vivo and in vitro studies have demonstrated that flow reversal (retrograde flow) is atherogenic and proinflammatory. The molecular and structural basis for the sensitivity of the endothelium to flow direction, however, has yet to be determined. It has been hypothesized that the ability to sense flow direction is dependent on the direction of inclination of the interendothelial junction. Immunostaining of the mouse aorta revealed an inclination of the cell-cell junction by 13 degrees in direction of flow in the descending aorta where flow is unidirectional. In contrast, polygonal cells of the inner curvature where flow is disturbed did not have any preferential inclination. Using a membrane specific dye, the angle of inclination of the junction was dynamically monitored using live cell confocal microscopy in confluent human endothelial cell monolayers. Upon application of shear the junctions began inclining within minutes to a final angle of 10 degrees in direction of flow. Retrograde flow led to a reversal of junctional inclination. Flow-induced junctional inclination was shown to be independent of the cytoskeleton or glycocalyx. Additionally, within seconds, retrograde flow led to significantly higher intracellular calcium responses than orthograde flow. Together, these results show for the first time that the endothelial intercellular junction inclination is dynamically responsive to flow direction and confers the ability to endothelial cells to rapidly sense and adapt to flow direction.

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule‐1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule‐1 (PECAM‐1) expression is pro‐atherogenic. PECAM‐1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane‐bound G proteins Gαq/11 also mediate flow‐induced responses. Here, we investigated whether PECAM‐1 and Gαq/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co‐localization of Gαq/11 and PECAM‐1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gαq/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM‐1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gαq/11–PECAM‐1 complex within 30 s and a partial relocalization of the Gαq/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow‐induced Gαq/11–PECAM‐1 dissociation. These results allow us to conclude that Gαq/11–PECAM‐1 forms a mechanosensitive complex and its localization suggests the Gαq/11–PECAM‐1 complex is a critical mediator of vascular diseases.

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.

Heparan Sulfates Mediate the Interaction between Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) and the Gαq/11 Subunits of Heterotrimeric G Proteins

Background: The mechanisms by which the PECAM-1·Gαq/11 mechanosensitive complex mediates endothelial flow responses remain unclear. Results: The PECAM-1·Gαq/11 complex contains heparan sulfate proteoglycans (HSPGs) and is disrupted by inhibition of HS. Conclusion: The interaction between PECAM-1 and Gαq/11 may be mediated by the HS of the HSPG syndecan-1. Significance: Targeting specific HSPGs may be an effective strategy for the therapeutic treatment of vascular diseases. The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits Gαq and 11 (Gαq/11) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1·Gαq/11 interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1·Gαq/11 binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1·Gαq/11 interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1·Gαq/11 mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.

Transdermal glyceryl trinitrate as an effective adjunctive treatment with artemether for late-stage experimental cerebral malaria.

ABSTRACT Cerebral malaria (CM) is associated with low nitric oxide (NO) bioavailability, cerebrovascular constriction, occlusion, and hypoperfusion. Administration of exogenous NO partially prevents the neurological syndrome and associated vascular pathology in an experimental CM (ECM) mouse model. In this study, we evaluated the effects of transdermal glyceryl trinitrate in preventing ECM and, in combination with artemether, rescuing late-stage ECM mice from mortality. The glyceryl trinitrate and/or artemether effect on survival and clinical recovery was evaluated in C57BL/6 mice infected with P. berghei ANKA. NO synthase (NOS) expression in mouse brain was determined by Western blots. Mean arterial pressure (MAP) and pial arteriolar diameter were monitored using a tail-cuff blood pressure system and a cranial window preparation, respectively. Preventative administration of glyceryl trinitrate at 0.025 mg/h decreased ECM mortality from 67 to 11% and downregulated inducible NOS expression in the brain. When administered as adjunctive rescue therapy with artemether, glyceryl trinitrate increased survival from 47 to 79%. The adjunctive therapy caused a sustained reversal of pial arteriolar vasoconstriction in ECM mice, an effect not observed with artemether alone. Glyceryl trinitrate induced a 13% decrease in MAP in uninfected mice but did not further affect MAP in hypotensive ECM mice. Glyceryl trinitrate, when combined with artemether, was an effective adjunctive rescue treatment for ECM. This treatment ameliorated pial arteriolar vasospasm and did not significantly affect MAP. These results indicate that transdermal glyceryl trinitrate has potential to be considered as a candidate for adjunctive therapy for CM.

Early VEGFR2 activation in response to flow is VEGF-dependent and mediated by MMP activity.

Although several potential mechanosensors/mechanotransducers have been proposed, the precise mechanisms by which ECs sense and respond to mechanical forces and translate them into biochemical signals remains unclear. Here, we report that two major ligand-dependent tyrosine autophosphorylation sites of VEGFR2, Y1175 and Y1214, are rapidly activated by shear stress in human coronary artery endothelial cells (HCAECs). Neutralizing antibody against VEGFR2 not only abrogates flow-induced phosphorylation of these tyrosine residues, but also has a marked inhibitory effect on downstream eNOS activation. In situ proximity ligation assay revealed that VEGF and VEGFR2 are closely associated in HCAECs, and more importantly, this association is increased with flow. Finally, we show that flow-induced VEGFR2 activation is attenuated in the presence of the broad spectrum matrix metalloproteinase (MMP) inhibitor, GM6001. Taken together, our results suggest that a ligand-dependent mechanism involving the activity of MMPs plays a key role in the early, shear stress-induced activation of VEGFR2.

Distinctive subcellular Akt-1 responses to shear stress in endothelial cells.

Endothelial cells undergo a rapid cell–cell junction inclination following exposure to atheroprotective unidirectional flow. In contrast, atherosclerotic lesions correlate with a heterogeneous distribution of the junctional wall inclination in cells exposed to time‐varying, reversing, and oscillatory flow as well as to low mean shear stress. However, the underlying biochemical events by which endothelial cells distinctively respond to unidirectional versus flow reversal remain unclear. Here, we show that the subcellular distribution of flow‐induced Akt‐1 phosphorylation in endothelial cells lining the mouse aorta varies depending on local hemodynamics. Activated Akt‐1 accumulated in perinuclear areas of cells in regions predisposed to disturbed flow but were localized at the cell–cell junction in regions of high unidirectional laminar shear stress. In flow‐adapted human endothelial cells, reversal in flow direction was associated within minutes with a subcellular concentration of phosphorylated Akt‐1 at the upstream edge of cells. Interestingly, oscillatory flow (with a zero mean shear stress) failed to activate Akt‐1, whereas a unidirectional pulsatile flow of similar amplitude induced an increase in Akt‐1 phosphorylation. Finally, silencing of the G protein αq/11 subunit abrogated both flow‐induced Akt‐1 and GSK‐3β activation. Together, these results characterize the existence of a Gαq/11‐mediated Akt‐1 signaling pathway that is dynamically responsive to flow direction, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to flow reversal. J. Cell. Biochem. 115: 121–129, 2014.