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Abstract Background The incidence of ventilator-associated pneumonia (VAP) within the first 48 hours of intensive care unit (ICU) stay has been poorly investigated. The objective was to estimate early-onset VAP occurrence in ICUs within 48 hours after admission. Methods We analyzed data from prospective surveillance between 01/01/2001 and 31/12/2009 in 11 ICUs of Lyon hospitals (France). The inclusion criteria were: first ICU admission, not hospitalized before admission, invasive mechanical ventilation during first ICU day, free of antibiotics at admission, and ICU stay ≥ 48 hours. VAP was defined according to a national protocol. Its incidence was the number of events per 1,000 invasive mechanical ventilation-days. The Poisson regression model was fitted from day 2 (D2) to D8 to incident VAP to estimate the expected VAP incidence from D0 to D1 of ICU stay. Results Totally, 367 (10.8%) of 3,387 patients in 45,760 patient-days developed VAP within the first 9 days. The predicted cumulative VAP incidence at D0 and D1 was 5.3 (2.6-9.8) and 8.3 (6.1-11.1), respectively. The predicted cumulative VAP incidence was 23.0 (20.8-25.3) at D8. The proportion of missed VAP within 48 hours from admission was 11% (9%-17%). Conclusions Our study indicates underestimation of early-onset VAP incidence in ICUs, if only VAP occurring ≥ 48 hours are considered to be hospital-acquired. Clinicians should be encouraged to develop a strategy for early detection after ICU admission.

Background: Endothelial colony forming cells (ECFC) represent a subpopulation of endothelial progenitor cells involved in endothelial repair. The activation of procoagulant mechanisms associated with the vascular wall's inflammatory responses to injury plays a crucial role in the induction and progression of atherosclerosis. However, little is known about ECFC proinflammatory potential., Aims: To explore the role of the thrombin receptor PAR-1 proinflammatory effects on ECFC chemotaxis/recruitment capacity., Methods and Results: The expression of 30 genes known to be associated with inflammation and chemotaxis was quantified in ECFC by real-time qPCR. PAR-1 activation with the SFLLRN peptide (PAR-1-ap) resulted in a significant increase in nine chemotaxis-associated genes expression, including CCL2 and CCL3 whose receptors are present on ECFC. Furthermore, COX-2 expression was found to be dramatically up-regulated consequently to PAR-1 activation. COX-2 silencing with the specific COX-2-siRNA also triggered down-regulation of the nine target genes. Conditioned media (c.m.) from control-siRNA- and COX-2-siRNA-transfected ECFC, stimulated or not with PAR-1-ap, were produced and tested on ECFC capacity to recruit leukocytes in vitro as well in the muscle of ischemic hindlimb in a preclinical model. The capacity of the c.m. from ECFC stimulated with PAR-1-ap to recruit leukocytes was abrogated when COX-2 gene expression was silenced in vitro (in terms of U937 cells migration and adhesion to endothelial cells) as well as in vivo. Finally, the postnatal vasculogenic stem cell derived from infantile hemangioma tumor (HemSC) incubated with PAR-1-ap increased leukocyte recruitment in Matrigel(®) implant., Conclusions: PAR-1 activation in ECFC increases chemotactic gene expression and leukocyte recruitment at ischemic sites through a COX-2-dependent mechanism.

Published clinical trials in patients with ischemic diseases show limited benefit of adult stem cell-based therapy, likely due to their restricted plasticity and commitment toward vascular cell lineage. We aim to uncover the potent regenerative ability of MesP1/stage-specific embryonic antigen 1 (SSEA-1)-expressing cardiovascular progenitors enriched from human embryonic stem cells (hESCs). Injection of only 10(4) hESC-derived SSEA-1(+) /MesP1(+) cells, or their progeny obtained after treatment with VEGF-A or PDGF-BB, was effective enough to enhance postischemic revascularization in immunodeficient mice with critical limb ischemia (CLI). However, the rate of incorporation of hESC-derived SSEA-1(+) /MesP1(+) cells and their derivatives in ischemic tissues was modest. Alternatively, these cells possessed a unique miR-21 signature that inhibited phosphotase and tensin homolog (PTEN) thereby activating HIF-1α and the systemic release of VEGF-A. Targeting miR-21 limited cell survival and inhibited their proangiogenic capacities both in the Matrigel model and in mice with CLI. We next assessed the impact of mR-21 in adult angiogenesis-promoting cells. We observed an impaired postischemic angiogenesis in miR-21-deficient mice. Notably, miR-21 was highly expressed in circulating and infiltrated monocytes where it targeted PTEN/HIF-1α/VEGF-A signaling and cell survival. As a result, miR-21-deficient mice displayed an impaired number of infiltrated monocytes and a defective angiogenic phenotype that could be partially restored by retransplantation of bone marrow-derived cells from wild-type littermates. hESC-derived SSEA-1(+) /MesP1(+) cells progenitor cells are powerful key integrators of therapeutic angiogenesis in ischemic milieu and miR-21 is instrumental in this process as well as in the orchestration of the biological activity of adult angiogenesis-promoting cells., (© 2014 AlphaMed Press.)

Objectives: We studied whether plasma levels of angiogenic factors VEGF and placental growth factor (PlGF) in coronary artery disease patients or undergoing cardiac surgery are modified, and whether those factors modulate endothelial progenitor's angiogenic potential., Methods and Results: A total of 143 patients' plasmas from two different studies were analyzed (30 coronary artery disease patients, 30 patients with stable angina, coupled with 30 age and sex-matched controls; 53 patients underwent cardiac surgery). Among factors screened, only PlGF was found significantly increased in these pathological populations. PlGF-1 and PlGF-2 were then tested on human endothelial-colony-forming cells (ECFCs). We found that PlGF-1 and PlGF-2 induce VEGFR1 phosphorylation and potentiate ECFCs tubulogenesis in vitro. ECFCs VEGFR1 was further inhibited using a specific small interfering RNA (siRNA) and the chemical compound 4321. We then observed that the VEGFR1-siRNA and the compound 4321 decrease ECFCs tubulogenesis potential in vitro. Finally, we tested the compound 4321 in the preclinical Matrigel(®)-plug model with C57Bl/6J mice as well as in the murine hindlimb ischemia model. We found that 4321 inhibited the plug vascularization, attested by the hemoglobin content and the VE-Cadherin expression level and that 4321 inhibited the post-ischemic revascularization., Conclusion: PlGF plasma levels were found increased in cardiovascular patients. Disrupting PlGF/VEGFR1 pathway could modulate ECFC-induced tubulogenesis, the cell type responsible for newly formed vessels in vivo.

Rationale for Study: MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein expression at post-transcriptional level. We hypothesized that a specific pool of endothelial miRNAs could be selectively regulated by flow conditions and inflammatory signals, and as such be involved in the development of atherosclerosis., Objective: To identify miRNAs, called atheromiRs, which are selectively regulated by shear stress and oxidized low-density lipoproteins (oxLDL), and to determine their role in atherogenesis., Methods and Results: Large-scale miRNA profiling in HUVECs identified miR-92a as an atheromiR candidate, whose expression is preferentially upregulated by the combination of low shear stress (SS) and atherogenic oxLDL. Ex vivo analysis of atheroprone and atheroprotected areas of mouse arteries and human atherosclerotic plaques demonstrated the preferential expression of miR-92a in atheroprone low SS regions. In Ldlr(-/-) mice, miR-92a expression was markedly enhanced by hypercholesterolemia, in particular in atheroprone areas of the aorta. Assessment of endothelial inflammation in gain- and loss-of-function experiments targeting miR-92a expression revealed that miR-92a regulated endothelial cell activation by oxLDL, more specifically under low SS conditions, which was associated with modulation of Kruppel-like factor 2 (KLF2), Kruppel-like factor 4 (KLF4), and suppressor of cytokine signaling 5. miR-92a expression was regulated by signal transducer and activator of transcription 3 in SS- and oxLDL-dependent manner. Furthermore, specific in vivo blockade of miR-92a expression in Ldlr(-/-) mice reduced endothelial inflammation and altered the development of atherosclerosis, decreasing plaque size and promoting a more stable lesion phenotype., Conclusions: Upregulation of miR-92a by oxLDL in atheroprone areas promotes endothelial activation and the development of atherosclerotic lesions. Therefore, miR-92a antagomir seems as a new atheroprotective therapeutic strategy.

Upregulation of hypoxia-inducible transcription factor-1α (HIF-1α), through prolyl-hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF-1α were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow-derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF-1α and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF-1α or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF-1α. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1α and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies., (© AlphaMed Press.)

Purpose: To assess the feasibility of loading iron oxide nanoparticles in endothelial microparticles (EMPs), thereby enabling their noninvasive monitoring with magnetic resonance (MR) imaging in mice., Materials and Methods: Experiments were approved by the French Ministry of Agriculture. Endothelial cells, first labeled with anionic superparamagnetic nanoparticles, were stimulated to generate EMPs, carrying the nanoparticles in their inner compartment. C57BL/6 mice received an intravenous injection of nanoparticle-loaded EMPs, free nanoparticles, or the supernatant of nanoparticle-loaded EMPs. A 1-week follow-up was performed with a 4.7-T MR imaging device by using a gradient-echo sequence for imaging spleen, liver, and kidney and a radial very-short-echo time sequence for lung imaging. Comparisons were performed by using the Student t test., Results: The signal intensity loss induced by nanoparticle-loaded EMPs or free nanoparticles was readily detected within 5 minutes after injection in the liver and spleen, with a more pronounced effect in the spleen for the magnetic EMPs. The kinetics of signal intensity attenuation differed for nanoparticle-loaded EMPs and free nanoparticles. No signal intensity changes were observed in mice injected with the supernatant of nanoparticle-loaded EMPs, confirming that cells had not released free nanoparticles, but only in association with EMPs. The results were confirmed by using Perls staining and immunofluorescence analysis., Conclusion: The strategy to generate EMPs with magnetic properties allowed noninvasive MR imaging assessment and follow-up of EMPs and opens perspectives for imaging the implications of these cellular vectors in diseases., (© RSNA, 2012.)