Identification of Heparin-Induced Thrombocytopenia in Surgical Critically Ill Patients by Using the HIT Expert Probability Score: An Observational Pilot Study.

Background: Heparin-induced thrombocytopenia (HIT) remains a challenging diagnosis especially in surgical intensive care unit (SICU) patients. The aim of the study was to evaluate for the first time the diagnostic accuracy of the HIT Expert Probability (HEP) score in the early identification of HIT in SICU patients. Methods: The HEP and 4Ts scores were calculated in all patients with suspected HIT during their stay in our SICU. The diagnosis of HIT was finally confirmed (HIT+ group) or excluded (HIT− group) by an independent committee blinded to the HEP and 4Ts score values. The primary outcome was the sensitivity and specificity of a HEP score ≥ 5 for the diagnosis of HIT. The secondary outcome was the area under the ROC curve (AUC) of the HEP and 4Ts scores in the diagnosis of HIT. Results: Respectively 6 and 113 patients were included in the HIT+ and HIT− groups. A HEP score value ≥ 5 had a sensitivity (95% confidence interval (95% CI)) of 1.00 (0.55−1.00), and a specificity (95% CI) of 0.92 (0.86−0.96). The AUC (95% CI) was significantly higher for the HEP score versus for the 4Ts score (0.967 (0.922−1.000) versus 0.707 (0.449−0.965); p = 0.035). Conclusions: A HEP score value < 5 could be helpful to rule out HIT in SICU patients.

Egfl7 Represses the Vasculogenic Potential of Human Endothelial Progenitor Cells.

Egfl7 (VE-statin) is a secreted protein mostly specific to the endothelial lineage during development and in the adult and which expression is enhanced during angiogenesis. Egfl7 involvement in human postnatal vasculogenesis remains unresolved yet. Our aim was to assess Egfl7 expression in several angiogenic cell types originating from human bone marrow, peripheral blood, or cord blood. We found that only endothelial colony forming cells (ECFC), which are currently considered as the genuine endothelial precursor cells, expressed large amounts of Egfl7. In order to assess its potential roles in ECFC, Egfl7 was repressed in ECFC by RNA interference and ECFC angiogenic capacities were tested in vitro and in vivo. Cell proliferation, differentiation, and migration were significantly improved when Egfl7 was repressed in ECFC in vitro, whereas miR-126-3p levels remained unchanged. In vivo, repression of Egfl7 in ECFC significantly improved post-ischemic revascularization in a model of mouse hind-limb ischemia. In conclusion, ECFC are the sole postnatal angiogenic cells which express large amounts of Egfl7 and whose angiogenic properties are repressed by this factor. Thus, Egfl7 inhibition may be considered as a therapeutic option to improve ECFC-mediated postnatal vasculogenesis and to optimize in vitro ECFC expansion in order to develop an optimized cell therapy approach.

Thrombin receptor PAR-1 activation on endothelial progenitor cells enhances chemotaxis-associated genes expression and leukocyte recruitment by a COX-2-dependent mechanism.

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.

Targeting VEGFR1 on endothelial progenitors modulates their differentiation potential.

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.

HIF-prolyl hydroxylase 2 inhibition enhances the efficiency of mesenchymal stem cell-based therapies for the treatment of critical limb ischemia.

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.

Forearm ischemia decreases endothelial colony-forming cell angiogenic potential.

BACKGROUND AIMS: Circulating endothelial progenitor cells and especially endothelial colony-forming cells (ECFCs) are promising candidate cells for endothelial regenerative medicine of ischemic diseases, but the conditions for an optimal collection from adult blood must be improved. METHODS: On the basis of a recently reported vascular niche of ECFCs, we hypothesized that a local ischemia could trigger ECFC mobilization from the vascular wall into peripheral blood to optimize their collection for autologous implantation in critical leg ischemia. Because the target population with critical leg ischemia is composed of elderly patients in whom a vascular impairment has been documented, we also analyzed the impact of aging on ECFC mobilization and vascular integrity. RESULTS: After having defined optimized ECFC culture conditions, we studied the effect of forearm ischemia on ECFC numbers and functions in 26 healthy volunteers (13 volunteers ages 20-30-years old versus 13 volunteers ages 60-70 years old). The results show that forearm ischemia induced an efficient local ischemia and a normal endothelial response but did not mobilize ECFCs regardless of the age group. Moreover, we report an alteration of angiogenic properties of ECFCs obtained after forearm ischemia, in vitro as well as in vivo in a hindlimb ischemia murine model. This impaired ECFC angiogenic potential was not associated with a quantitative modification of the circulating endothelial compartment. CONCLUSIONS: The procedure of local ischemia, although reulting in a preserved endothelial reactivity, did not mobilize ECFCs but altered their angiogenic potential.

Imbalance of circulating endothelial cells and progenitors in idiopathic pulmonary fibrosis.

BACKGROUND: Fibrogenesis during idiopathic pulmonary fibrosis (IPF) is strongly associated with abnormal vascular remodeling. Respective abundance of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair and potentially serve as biomarkers of the disease. OBJECTIVES AND METHODS: We postulated that CEC and EPC subtypes might be differently modulated in IPF. Sixty-four consecutive patients with newly diagnosed IPF were prospectively enrolled and compared to thirteen healthy volunteers. CEC were counted with immunomagnetic CD146-coated beads; progenitors CD34+45(dim)/CD34+133+/CD34+KDR+were assessed through flow cytometry and EPC (colony-forming-units-Endothelial Cells, CFU-EC, and endothelial colonies forming cells, ECFC) were quantified by cell culture assays. RESULTS: IPF patients were characterized by a marked increase in CEC associated to an EPC defect: both CD34(+)KDR(+) cells and CFU-EC were decreased versus controls. Moreover, in IPF subjects with a low diffusing capacity of the lung for carbon monoxide (DL(CO)) < 40 %, CFU-EC and ECFC were higher compared to those with DL(CO) > 40 %. Finally, ECFC were negatively correlated with DL(CO). During an 18 month follow up, CEC levels increased in patients with exacerbation, including those who died during follow up. Finally, ECFC from patients with exacerbation proliferative potential was strongly increased. CONCLUSION: IPF is basically associated with both a vascular injury and a repair defect. This study highlights an adaptative process of EPC mobilization in the most severe forms of IPF, that could reflect enhanced homing to the pulmonary vasculature, which clinical consequences remain to be determined.

Treprostinil increases the number and angiogenic potential of endothelial progenitor cells in children with pulmonary hypertension.

BACKGROUND: Pulmonary vasodilators in general and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary arterial hypertension (PAH). As endothelial dysfunction is a key feature of PAH, and as endothelial progenitor cells (EPC) may contribute to vascular repair in PAH, we suspected that prostacyclin therapy might enhance EPC numbers and functions. In the present study, objectives were to determine whether EPC may contribute to vasodilator treatment efficacy in PAH. METHODS: We quantified CD34+ cells, CFU-Hill and ECFC (endothelial colony forming cells) in peripheral blood from children with idiopathic PAH (n = 27) or PAH secondary to congenital heart disease (n = 52). CD34+ were enumerated by flow cytometry, CFU-Hill and ECFC by a culture assay. ECFC grown ex vivo were tested for their angiogenic capacities before and after prostacyclin analog therapy (subcutaneous treprostinil). RESULTS: ECFC counts were significantly enhanced in the 8 children treated with treprostinil, while no change was observed in children receiving oral therapy with endothelin antagonists and/or PDE5 inhibitors. CD34+ cell and CFU-Hill counts were unaffected. ECFC from patients treated with treprostinil had a hyperproliferative phenotype and showed enhanced angiogenic potential in a nude mouse preclinical model of limb ischemia. CONCLUSIONS: ECFC may partly mediate the clinical benefits of prostanoids in pulmonary arterial hypertension.

Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties.

OBJECTIVE: We examined whether plasma levels of angiogenic factors are altered in plasma of patients with peripheral arterial disease (PAD) and whether these factors affect endothelial progenitor cell-induced angiogenesis. METHODS AND RESULTS: Plasma was collected from 184 patients with PAD and 330 age-matched healthy controls. Vascular endothelial growth factor and placental growth factor concentrations did not differ between the groups, whereas we found a linear correlation between PAD disease and thrombospondin (TSP)-1 plasma level. TSP-1 was expressed in newly formed vessels in PAD patients having received local injections of bone marrow mononuclear cells. To analyze the functional role of TSP-1 during neoangiogenesis, we used a Matrigel-plug assay and showed that vascularization of implanted Matrigel-plugs was increased in TSP-1(-/-) mice. Moreover, injections of TSP-1 in C57Bl6/J mice after hindlimb ischemia induced a significant decrease of blood flow recovery. To investigate the effects of TSP-1 on human endothelial colony-forming cell (ECFC) angiogenic potential, recombinant human TSP-1 and a small interfering RNA were used. In vitro, TSP-1 N-terminal part significantly enhanced ECFC adhesion, whereas recombinant human TSP-1 had a negative effect on ECFC angiogenic potential. This effect, mediated by CD47 binding, modulated stromal cell-derived factor 1/CXC chemokine receptor 4 pathway. CONCLUSIONS: TSP-1 is a potential biomarker of PAD and ECFC-induced angiogenesis, suggesting that TSP-1 modulation might improve local tissue ischemia in this setting. ( CLINICAL TRIAL REGISTRATION: NCT00377897.).

The Wnt antagonist Dickkopf-1 increases endothelial progenitor cell angiogenic potential.

OBJECTIVE: To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. METHODS AND RESULTS: Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. CONCLUSIONS: DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

Interleukin 8 is differently expressed and modulated by PAR-1 activation in early and late endothelial progenitor cells.

The proinflammatory chemokine interleukin 8 exerts potent angiogenic effects on endothelial cells by interacting with its receptors CXCR1 and CXCR2. As thrombin is also a potent inflammatory factor, and as endothelial progenitor cells (EPC) express functional PAR-1 thrombin receptor, we examined whether PAR-1 stimulation interferes with the IL-8 pathway in EPC. EPC were obtained from adult blood (AB) and cord blood (CB). The effect of PAR-1 stimulation by the peptide SFLLRN on IL-8, CXCR1 and CXCR2 expression was examined by RTQ-PCR and at the protein level in AB and CB late EPC and in AB early EPC. Specific siRNA was used to knock down PAR-1 expression. The IL-8 gene was expressed strongly in AB early EPC and moderately in late EPC. In contrast, CXCR1 and CXCR2 gene expression was restricted to AB early EPC. The IL-8 level in AB early EPC conditioned medium was high in basal conditions and did not change after PAR-1 activation. By contrast, IL-8 secretion by late EPC was low in basal conditions and strongly up-regulated upon PAR-1 activation. PAR-1 activation induced a number of genes involved in activating protein-1 (AP-1) and nuclear factor (NF)-kappaB pathways. Conditioned medium of PAR-1-activated late EPC enhanced the migratory potential of early EPC, and this effect was abrogated by blocking IL-8. Target-specific siRNA-induced PAR-1 knockdown, and fully inhibited PAR-1-induced IL-8 synthesis. In conclusion, PAR-1 activation induces IL-8 synthesis by late EPC. This could potentially enhance cooperation between late and early EPC during neovascularization, through a paracrine effect.