Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage.

RATIONALE: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. OBJECTIVES: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. METHODS: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. MEASUREMENTS AND MAIN RESULTS: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. CONCLUSIONS: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Palmitic acid increases pro-oxidant adaptor protein p66Shc expression and affects vascularization factors in angiogenic mononuclear cells: Action of resveratrol.

A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control.

The shear stress-induced transcription factor KLF2 affects dynamics and angiopoietin-2 content of Weibel-Palade bodies.

BACKGROUND: The shear-stress induced transcription factor KLF2 has been shown to induce an atheroprotective phenotype in endothelial cells (EC) that are exposed to prolonged laminar shear. In this study we characterized the effect of the shear stress-induced transcription factor KLF2 on regulation and composition of Weibel-Palade bodies (WPBs) using peripheral blood derived ECs. METHODOLOGY AND PRINCIPAL FINDINGS: Lentiviral expression of KLF2 resulted in a 4.5 fold increase in the number of WPBs per cell when compared to mock-transduced endothelial cells. Unexpectedly, the average length of WPBs was significantly reduced: in mock-transduced endothelial cells WPBs had an average length of 1.7 µm versus 1.3 µm in KLF2 expressing cells. Expression of KLF2 abolished the perinuclear clustering of WPBs observed following stimulation with cAMP-raising agonists such as epinephrine. Immunocytochemistry revealed that WPBs of KLF2 expressing ECs were positive for IL-6 and IL-8 (after their upregulation with IL-1ß) but lacked angiopoietin-2 (Ang2), a regular component of WPBs. Stimulus-induced secretion of Ang2 in KLF2 expressing ECs was greatly reduced and IL-8 secretion was significantly lower. CONCLUSIONS AND SIGNIFICANCE: These data suggest that KLF2 expression leads to a change in size and composition of the regulated secretory compartment of endothelial cells and alters its response to physiological stimuli.

Expression of a retinoic acid signature in circulating CD34 cells from coronary artery disease patients.

BACKGROUND: Circulating CD34+ progenitor cells have the potential to differentiate into a variety of cells, including endothelial cells. Knowledge is still scarce about the transcriptional programs used by CD34+ cells from peripheral blood, and how these are affected in coronary artery disease (CAD) patients. RESULTS: We performed a whole genome transcriptome analysis of CD34+ cells, CD4+ T cells, CD14+ monocytes, and macrophages from 12 patients with CAD and 11 matched controls. CD34+ cells, compared to other mononuclear cells from the same individuals, showed high levels of KRAB box transcription factors, known to be involved in gene silencing. This correlated with high expression levels in CD34+ cells for the progenitor markers HOXA5 and HOXA9, which are known to control expression of KRAB factor genes. The comparison of expression profiles of CD34+ cells from CAD patients and controls revealed a less naïve phenotype in patients' CD34+ cells, with increased expression of genes from the Mitogen Activated Kinase network and a lowered expression of a panel of histone genes, reaching levels comparable to that in more differentiated circulating cells. Furthermore, we observed a reduced expression of several genes involved in CXCR4-signaling and migration to SDF1/CXCL12. CONCLUSIONS: The altered gene expression profile of CD34+ cells in CAD patients was related to activation/differentiation by a retinoic acid-induced differentiation program. These results suggest that circulating CD34+ cells in CAD patients are programmed by retinoic acid, leading to a reduced capacity to migrate to ischemic tissues.

The NR1 subunit of NMDA receptor regulates monocyte transmigration through the brain endothelial cell barrier.

Normal neuronal functioning is dependent on the blood-brain barrier. This barrier is confined to specialized brain endothelial cells lining the inner vessel wall, and tightly controlling transport of nutrients, efflux of potentially harmful molecules and entry of immune cells into the brain. Loss of blood-brain barrier function is an early and significant event which contributes to inflammation in the brain and subsequent progression of neuronal deficits in a number of brain disorders and has been well-documented for the auto-immune disease multiple sclerosis. Extravasation of cells happens by paracellular transport across the endothelial junctions, transcellularly across the endothelial cells, or both, and requires the active participation of endothelial cells. We and others have shown that this process requires the activity of proteases, including tissue-type plasminogen activator. We here describe a novel role for NMDA receptor, a potential cellular target of tissue-type plasminogen activator, in human brain endothelial cells. Our results show that the NMDA receptor subunit 1 (NR1) is expressed in brain endothelial cells, regulates tissue-type plasminogen activator-induced signal transduction and controls the passage of monocytes through the brain endothelial cell barrier. Together, our results hold significant promise for the treatment of chronic inflammation in the brain.

KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium.

The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

Tissue-type plasminogen activator is a regulator of monocyte diapedesis through the brain endothelial barrier.

Inflammatory cell trafficking into the brain complicates several neurological disorders including multiple sclerosis. Normally, reliable brain functioning is maintained and controlled by the blood-brain barrier (BBB), which is essential to restrict the entry of potentially harmful molecules and cells from the blood into the brain. The BBB is a selective barrier formed by dedicated brain endothelial cells and dependent on the presence of intracellular tight junctions. In multiple sclerosis, a severe dysfunction of the BBB is observed, which is key to monocyte infiltration and inflammation in the brain. Proteolytic activity has been associated with these inflammatory processes in the brain. Our studies in plasma of rats indicated that the extracellular protease tissue-type plasminogen activator (tPA) correlates with the clinical signs of experimental allergic encephalomyelitis, a rat model of multiple sclerosis. In this study, we studied the function of the tPA during diapedesis of monocytes through a rat and human brain endothelial barrier. Monocyte-brain endothelial cell coculture experiments showed that monocytes induce the release of tPA by brain endothelial cells, which subsequently activates the signal transduction protein extracellular signal related kinase (ERK1/2), both involved in monocyte diapedesis. Importantly, live imaging and immunoblot analyses of rat brain endothelial cells revealed that tPA and ERK1/2 control the breakdown of the tight junction protein occludin. These studies identify tPA as a novel and relevant pathological mediator of neuroinflammation and provide a potential mechanism for this.