Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology.

The renin-angiotensin-aldosterone system plays crucial roles in cardiovascular physiology and pathophysiology. However, many of the signaling mechanisms have been unclear. The angiotensin II (ANG II) type 1 receptor (AT1R) is believed to mediate most functions of ANG II in the system. AT1R utilizes various signal transduction cascades causing hypertension, cardiovascular remodeling, and end organ damage. Moreover, functional cross-talk between AT1R signaling pathways and other signaling pathways have been recognized. Accumulating evidence reveals the complexity of ANG II signal transduction in pathophysiology of the vasculature, heart, kidney, and brain, as well as several pathophysiological features, including inflammation, metabolic dysfunction, and aging. In this review, we provide a comprehensive update of the ANG II receptor signaling events and their functional significances for potential translation into therapeutic strategies. AT1R remains central to the system in mediating physiological and pathophysiological functions of ANG II, and participation of specific signaling pathways becomes much clearer. There are still certain limitations and many controversies, and several noteworthy new concepts require further support. However, it is expected that rigorous translational research of the ANG II signaling pathways including those in large animals and humans will contribute to establishing effective new therapies against various diseases.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

Glucose consumption of vascular cell types in culture: toward optimization of experimental conditions.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Transduction Efficiency of Adenovirus Vectors in Endothelial Cells and Vascular Smooth Muscle Cells.

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

78 kDa Glucose-Regulated Protein Attenuates Protein Aggregation and Monocyte Adhesion Induced by Angiotensin II in Vascular Cells.

Investigations of vascular smooth muscle cell (VSMC) phenotypic modulation due to angiotensin II (AngII) stimulation are important for understanding molecular mechanisms contributing to hypertension and associated vascular pathology. AngII induces endoplasmic reticulum (ER) stress in VSMCs, which has been implicated in hypertensive vascular remodeling. Under ER stress, 78 kDa glucose-regulated protein (GRP78) acts as an endogenous chaperone, as well as a master controller of unfolded protein response (UPR) to maintain protein quality control. However, the potential downstream consequences of ER stress induced by AngII on protein quality control and pro-inflammatory phenotype in VSMCs remain elusive. This study aims to identify protein aggregation as evidence of the disruption of protein quality control in VSMCs, and to test the hypothesis that preservation of proteostasis by overexpression of GRP78 can attenuate the AngII-induced pro-inflammatory phenotype in VSMCs. Increases in protein aggregation and enhanced UPR were observed in VSMCs exposed to AngII, which were mitigated by overexpression of GRP78. Moreover, GRP78 overexpression attenuated enhanced monocyte adhesion to VSMCs induced by AngII. Our results thus indicate that the prevention of protein aggregation can potentially mitigate an inflammatory phenotype in VSMCs, which may suggest an alternative therapy for the treatment of AngII-associated vascular disorders.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated ß galactosidase activity. The number of ß galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

MicroRNA-155 Deficiency Leads to Decreased Atherosclerosis, Increased White Adipose Tissue Obesity, and Non-alcoholic Fatty Liver Disease: A NOVEL MOUSE MODEL OF OBESITY PARADOX.

Obesity paradox (OP) describes a widely observed clinical finding of improved cardiovascular fitness and survival in some overweight or obese patients. The molecular mechanisms underlying OP remain enigmatic partly due to a lack of animal models mirroring OP in patients. Using apolipoprotein E knock-out (apoE-/-) mice on a high fat (HF) diet as an atherosclerotic obesity model, we demonstrated 1) microRNA-155 (miRNA-155, miR-155) is significantly up-regulated in the aortas of apoE-/- mice, and miR-155 deficiency in apoE-/- mice inhibits atherosclerosis; 2) apoE-/-/miR-155-/- (double knock-out (DKO)) mice show HF diet-induced obesity, adipocyte hypertrophy, and present with non-alcoholic fatty liver disease; 3) DKO mice demonstrate HF diet-induced elevations of plasma leptin, resistin, fed-state and fasting insulin and increased expression of adipogenic transcription factors but lack glucose intolerance and insulin resistance. Our results are the first to present an OP model using DKO mice with features of decreased atherosclerosis, increased obesity, and non-alcoholic fatty liver disease. Our findings suggest the mechanistic role of reduced miR-155 expression in OP and present a new OP working model based on a single miRNA deficiency in diet-induced obese atherogenic mice. Furthermore, our results serve as a breakthrough in understanding the potential mechanism underlying OP and provide a new biomarker and novel therapeutic target for OP-related metabolic diseases.

HIF1α in aortic aneurysms and beyond.

Abdominal aortic aneurysm (AAA) is a permanent expansion of the vessel wall with a high prevalence in those 65 years of age and older. Aneurysms are prone to dissection and rupture that carry a mortality rate of over 85%. Currently, surgical repair is the only option to treat this disease. The need to intervene prior to these events has set off a flurry of basic studies in an effort to understand the cellular and molecular mechanisms that govern AAA formation, progression and rupture. In the present study, the role of myeloid cells in contributing to AAA development has been confirmed. More specifically, the transcription factor, hypoxia-inducible factor-1α (HIF1α), was demonstrated to be a necessary component for regulating the expression of extracellular matrix modifying enzymes and their endogenous inhibitors in these cells. This new discovery may lead to therapeutic targets to prohibit the degradation and weakening of the vessel wall with the hope of limiting AAA formation and/or growth.

AT1 receptor signaling pathways in the cardiovascular system.

The importance of the renin angiotensin aldosterone system in cardiovascular physiology and pathophysiology has been well described whereas the detailed molecular mechanisms remain elusive. The angiotensin II type 1 receptor (AT1 receptor) is one of the key players in the renin angiotensin aldosterone system. The AT1 receptor promotes various intracellular signaling pathways resulting in hypertension, endothelial dysfunction, vascular remodeling and end organ damage. Accumulating evidence shows the complex picture of AT1 receptor-mediated signaling; AT1 receptor-mediated heterotrimeric G protein-dependent signaling, transactivation of growth factor receptors, NADPH oxidase and ROS signaling, G protein-independent signaling, including the ß-arrestin signals and interaction with several AT1 receptor interacting proteins. In addition, there is functional cross-talk between the AT1 receptor signaling pathway and other signaling pathways. In this review, we will summarize an up to date overview of essential AT1 receptor signaling events and their functional significances in the cardiovascular system.

Epidermal growth factor receptor inhibitor protects against abdominal aortic aneurysm in a mouse model.

Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and ß-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress.

Caveolin 1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase.

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and ß-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

A caveolae-targeted L-type Ca²+ channel antagonist inhibits hypertrophic signaling without reducing cardiac contractility.

RATIONALE: The source of Ca(2+) to activate pathological cardiac hypertrophy is not clearly defined. Ca(2+) influx through the L-type Ca(2+) channels (LTCCs) determines "contractile" Ca(2+), which is not thought to be the source of "hypertrophic" Ca(2+). However, some LTCCs are housed in caveolin-3 (Cav-3)-enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. OBJECTIVE: To test the idea that LTCCs in Cav-3-containing signaling domains are a source of Ca(2+) to activate the calcineurin-nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. METHODS AND RESULTS: We developed reagents that targeted Ca(2+) channel-blocking Rem proteins to Cav-3-containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca(2+) influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. CONCLUSIONS: We provide proof of concept that Ca(2+) influx through LTCCs within caveolae signaling domains can activate "hypertrophic" signaling, and this Ca(2+) influx can be selectively blocked without reducing cardiac contractility.

Mitochondrial fission inhibition protects against hypertension induced by angiotensin II.

Mitochondrial dysfunction has been implicated in various types of cardiovascular disease including hypertension. Mitochondrial fission fusion balance is critical to mitochondrial quality control, whereas enhanced fission has been reported in several models of cardiovascular disease. However, limited information is available regarding the contribution of mitochondrial fission in hypertension. Here, we have tested the hypothesis that inhibition of mitochondrial fission attenuates the development of hypertension and associated vascular remodeling. In C57BL6 mice infused with angiotensin II for 2 weeks, co-treatment of mitochondrial fission inhibitor, mdivi1, significantly inhibited angiotensin II-induced development of hypertension assessed by radiotelemetry. Histological assessment of hearts and aortas showed that mdivi1 inhibited vessel fibrosis and hypertrophy induced by angiotensin II. This was associated with attenuation of angiotensin II-induced decline in mitochondrial aspect ratio seen in both the endothelial and medial layers of aortas. Mdivi1 also mitigated angiotensin II-induced cardiac hypertrophy assessed by heart weight-to-body weight ratio as well as by echocardiography. In ex vivo experiments, mdivi1 inhibited vasoconstriction and abolished the enhanced vascular reactivity by angiotensin II in small mesenteric arteries. Proteomic analysis on endothelial cell culture media with angiotensin II and/or mdivi1 treatment revealed that mdivi1 inhibited endothelial cell hypersecretory phenotype induced by angiotensin II. In addition, mdivi1 attenuated angiotensin II-induced protein induction of periostin, a myofibroblast marker in cultured vascular fibroblasts. In conclusion, these data suggest that mdivi1 prevented angiotensin II-induced hypertension and cardiovascular remodeling via multicellular mechanisms in the vasculature.

Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice.

BACKGROUND: Phosphatidylserine (PS) is a procoagulant phospholipid enriched on surfaces of activated vascular cells including platelets, endothelium, monocytes, and microvesicles. As a molecular driver of thrombosis accessible to drug blockade, PS is an attractive pharmacological target for modulating thrombogenesis, with potentially reduced bleeding risk compared to anticoagulant and antiplatelet therapies. OBJECTIVES: Test antithrombotic capabilities of a liposomal formulation, Zn-dipicolylamine cyanine-3[22,22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPC(molar ratio 3:97), designated as DPAL, which we previously described binds selectively to PS-enriched cell surfaces, compared with effects on bleeding, in mouse models. METHODS: PS-dependent DPAL binding to human and murine platelets was tested in vitro. Thrombosis and bleeding after DPAL intravenous administration were tested in C57Bl/6J mice following FeCl3 carotid arterial injury and tail tip amputation, respectively. Incorporation in hemostatic clots was investigated in the cremaster muscle laser injury model. Toxicity was tested by direct exposure to human endothelial cell cultures. RESULTS: DPAL bound agonist-stimulated, PS-positive human and murine platelets, blocked by Annexin V or Ano6 deletion which ablate PS exposure. DPAL prolonged prothrombin time, but did not prevent thrombin-induced fibrinogen receptor activation or aggregation, nor alter blood cell counts including platelets. Following arteriolar laser injury, DPAL bound wound surfaces and edges without destabilizing plugs. DPAL dose-dependently blocked FeCl3-induced arterial thrombosis, but did not substantially increase bleeding, or induce endothelial cell death. CONCLUSIONS: DPAL reduces thrombogenesis with minimal effects on bleeding in mouse models via selective binding to PS. DPAL may support novel approaches to modulate pathogenic thrombin generation with improved safety profiles in multiple contexts.

Volume overload induces differential spatiotemporal regulation of myocardial soluble guanylyl cyclase in eccentric hypertrophy and heart failure.

Nitric oxide activation of soluble guanylyl cyclase (sGC) blunts the cardiac stress response, including cardiomyocyte hypertrophy. In the concentric hypertrophied heart, oxidation and re-localization of myocardial sGC diminish cyclase activity, thus aggravating depressed nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling in the pressure-overloaded failing heart. Here, we hypothesized that volume-overload differentially disrupts myocardial sGC activity during early compensated and late decompensated stages of eccentric hypertrophy. To this end, we studied the expression, redox state, subcellular localization, and activity of sGC in the left ventricle of dogs subjected to chordal rupture-induced mitral regurgitation (MR). Unoperated dogs were used as Controls. Animals were studied at 4weeks and 12months post chordal rupture, corresponding with early (4wkMR) and late stages (12moMR) of eccentric hypertrophy. We found that the sGC heterodimer subunits relocalized away from caveolae-enriched lipid raft microdomains at different stages; sGCß1 at 4wkMR, followed by sGCα1 at 12moMR. Moreover, expression of both sGC subunits fell at 12moMR. Using the heme-dependent NO donor DEA/NO and NO-/heme-independent sGC activator BAY 60-2770, we determined the redox state and inducible activity of sGC in the myocardium, within caveolae and non-lipid raft microdomains. sGC was oxidized in non-lipid raft microdomains at 4wkMR and 12moMR. While overall DEA/NO-responsiveness remained intact in MR hearts, DEA/NO responsiveness of sGC in non-lipid raft microdomains was depressed at 12moMR. Caveolae-localization protected sGC against oxidation. Further studies revealed that these modifications of sGC were also reflected in caveolae-localized cGMP-dependent protein kinase (PKG) and MAPK signaling. In MR hearts, PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) disappeared from caveolae whereas caveolae-localization of phosphorylated ERK5 increased. These findings show that differential oxidation, re-localization, and expression of sGC subunits distinguish eccentric from concentric hypertrophy as well as compensated from decompensated heart failure.

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells.

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium.

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

Fluid shear stress induces the clustering of heparan sulfate via mobility of glypican-1 in lipid rafts.

The endothelial glycocalyx plays important roles in mechanotransduction. We recently investigated the distribution and interaction of glycocalyx components on statically cultured endothelial cells. In the present study, we further explored the unknown organization of the glycocalyx during early exposure (first 30 min) to shear stress and tested the hypothesis that proteoglycans with glycosaminoglycans, which are localized in different lipid microdomains, respond distinctly to shear stress. During the initial 30 min of exposure to shear stress, the very early responses of the glycocalyx and membrane rafts were detected using confocal microscopy. We observed that heparan sulfate (HS) and glypican-1 clustered in the cell junctions. In contrast, chondroitin sulfate (CS), bound albumin, and syndecan-1 did not move. The caveolae marker caveolin-1 did not move, indicating that caveolae are anchored sufficiently to resist shear stress during the 30 min of exposure. Shear stress induced significant changes in the distribution of ganglioside GM1 (a marker for membrane rafts labeled with cholera toxin B subunit). These data suggest that fluid shear stress induced the cell junctional clustering of lipid rafts with their anchored glypican-1 and associated HS. In contrast, the mobility of CS, transmembrane bound syndecan-1, and caveolae were constrained during exposure to shear stress. This study illuminates the role of changes in glycocalyx organization that underlie mechanisms of mechanotransduction.