Discovery and Validation of Urinary Molecular Signature of Early Sepsis.

Identify alterations in gene expression unique to systemic and kidney-specific pathophysiologic processes using whole-genome analyses of RNA isolated from the urinary cells of sepsis patients. DESIGN: Prospective cohort study. SETTING: Quaternary care academic hospital. PATIENTS: A total of 266 sepsis and 82 control patients enrolled between January 2015 and February 2018. INTERVENTIONS: Whole-genome transcriptomic analysis of messenger RNA isolated from the urinary cells of sepsis patients within 12 hours of sepsis onset and from control subjects. MEASUREMENTS AND MAIN RESULTS: The differentially expressed probes that map to known genes were subjected to feature selection using multiple machine learning techniques to find the best subset of probes that differentiates sepsis from control subjects. Using differential expression augmented with machine learning ensembles, we identified a set of 239 genes in urine, which show excellent effectiveness in classifying septic patients from those with chronic systemic disease in both internal and independent external validation cohorts. Functional analysis indexes disrupted biological pathways in early sepsis and reveal key molecular networks driving its pathogenesis. CONCLUSIONS: We identified unique urinary gene expression profile in early sepsis. Future studies need to confirm whether this approach can complement blood transcriptomic approaches for sepsis diagnosis and prognostication.

Maternal endothelial function, circulating endothelial cells, and endothelial progenitor cells in pregnancies conceived with or without in vitro fertilization.

In women who conceived with or without assisted reproduction, we evaluated endothelial function by EndoPAT [reactive hyperemia index (RHI)], circulating numbers of endothelial cells (CEC) and endothelial progenitor cells (EPC), and their function before during and after pregnancy. In vitro fertilization (IVF) pregnancies were stratified by method of conception and corpus luteum (CL) number-controlled ovarian stimulation (>1 CL) or programmed (0 CL) cycles and spontaneous singleton pregnancies (1 CL). We observed 1) comparable gestational decline of RHI in the three participant groups secondary to gestational rise of baseline preocclusion pulse-wave amplitude (PWA) incorporated into the RHI calculation by EndoPAT software; 2) progressive rise in "normalized" RHI throughout pregnancy (calculated by substituting prepregnancy baseline preocclusion PWA into the RHI equation), greater in spontaneous conception vs. IVF cohorts; 3) similar gestational increase of maximum PWA and time to maximum PWA after the ischemia stimulus among the three participant groups; 4) modest gestational increase of ischemia response (reactive hyperemia) in the spontaneous conception group and no change or significant decline, respectively, in women who conceived using programmed or controlled ovarian stimulation cycles; 5) enhanced basal nitric oxide production by early (primitive) outgrowth EPC during pregnancy in women who conceived spontaneously, but not through IVF; and 6) gestational increase in CEC in all three participant cohorts, more pronounced in women who conceived by IVF using programmed cycles. On balance, the evidence supported enhanced endothelial function during pregnancy in spontaneous conceptions but less so in IVF pregnancies using either controlled ovarian stimulation or programmed cycles.

Activation of the ß-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR2 ) and the heterodimeric EPOR/ß-common receptor (ßCR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First-order, mesenteric arteries were isolated from 16-week-old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO-stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/ßCR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium-dependent (acetylcholine, bradykinin, A23187) and endothelium-independent (sodium nitroprusside) vasodilator responses. Only ACh-induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh-induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium-independent vasodilation by SNP. Additionally, a novel ßCR inhibitory peptide (ßIP), which was computationally developed, prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP, further implicating the EPOR/ßCR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/ßCR in endothelial cells leads to a selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries.

Computational design and experimental characterization of a novel ß-common receptor inhibitory peptide.

In short-term animal models of ischemia, erythropoietin (EPO) signaling through the heterodimeric EPO receptor (EPOR)/ß-common receptor (ßCR) is believed to elicit tissue protective effects. However, large, randomized, controlled trials demonstrate that targeting a higher hemoglobin level by administering higher doses of EPO, which are more likely to activate the heterodimeric EPOR/ßCR, is associated with an increase in adverse cardiovascular events. Thus, inhibition of long-term activation of the ßCR may have therapeutic implications. This study aimed to design and evaluate the efficacy of novel computationally designed ßCR inhibitory peptides (ßIP). These novel ßIPs were designed based on a truncated portion of Helix-A from EPO, specifically residues 11-26 (VLERYLLEAKEAEKIT). Seven novel peptides (P1 to P7) were designed. Peptide 7 (P7), VLERYLHEAKHAEKIT, demonstrated the most robust inhibitory activity. We also report here the ability of P7 to inhibit ßCR-induced nitric oxide (NO) production and angiogenesis in human umbilical vein endothelial cells (HUVECs). Specifically, we found that P7 ßIP completely abolished EPO-induced NO production. The inhibitory effect could be overcome with super physiological doses of EPO, suggesting a competitive inhibition. ßCR-induced angiogenesis in HUVEC's was also abolished with treatment of P7 ßIP, but P7 ßIP did not inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis. In addition, we demonstrate that the novel P7 ßIP does not inhibit EPO-induced erythropoiesis with use of peripheral blood mononuclear cells (PBMCs). These results, for the first time, describe a novel, potent ßCR peptide inhibitor that inhibit the actions of the ßCR without affecting erythropoiesis.

Effects of Long-Term Type I Interferon on the Arterial Wall and Smooth Muscle Progenitor Cells Differentiation.

OBJECTIVE: Patients with systemic lupus erythematosis are at risk for premature atherosclerosis and half of the patients with systemic lupus erythematosis have elevated type I interferon (IFN-I) levels. We hypothesized that IFN-I would induce premature atherosclerosis by increasing the number of smooth muscle progenitor cells (SMPC) in the bloodstream and promoting atherosclerotic lesions within the vasculature. APPROACH AND RESULTS: SMPC isolated from wild-type and IFN receptor knockout animals were cultured in medium±IFN-I. In vivo, we used electroporation to generate stable IFN-I expression for as long as 4 months. The number of SMPC was determined in mice that expressed IFN-I and in control mice and sections from the bifurcation of the abdominal aorta were analyzed 3 months after electroporation of an IFN-I expression plasmid or a control plasmid. Adding IFN-I to the media increased the number of cultured wild-type SMPC and increased mRNA for SM22, but had no effect on SMPC isolated from IFN receptor knockout mice. Our in vivo results demonstrated a positive relationship between the preatherosclerotic-like lesions and endothelial damage. Although, there were no significant differences in smooth muscle cell density or thickness of the medial layer between groups, the IFN-I-expressing mice had a significant increase in preatherosclerotic-like lesions and immature smooth muscle cells, cells that expressed CD34 and smooth muscle α-actin; but lacked smooth muscle myosin heavy chain. CONCLUSIONS: IFN-I seems to enhance SMPC number in vitro. In vivo IFN-I expression may maintain SMPC in an immature state. These immature smooth muscle cells could give rise to macrophages and eventually foam cells.

DNA Aptamer Assembly as a Vascular Endothelial Growth Factor Receptor Agonist.

Controlling receptor-mediated processes in cells is paramount in many research areas. The activity of small molecules and growth factors is difficult to control and can lead to off-target effects through the activation of nonspecific receptors as well as binding affinity to nonspecific cell types. In this study, we report the development of a molecular trigger in the form of a divalent nucleic acid aptamer assembly toward vascular endothelial growth factor receptor-2 (VEGFR2). The assembly binds to VEGFR2 and functions as a receptor agonist with targeted receptor binding, promoting receptor phosphorylation, activation of the downstream Akt pathway, upregulation of endothelial nitric oxide synthase, and endothelial cell capillary tube formation. The agonist action we report makes this aptamer construct a promising strategy to control VEGFR2-mediated cell signaling.

Number and function of bone-marrow derived angiogenic cells and coronary flow reserve in women without obstructive coronary artery disease: a substudy of the NHLBI-sponsored Women's Ischemia Syndrome Evaluation (WISE).

BACKGROUND: In women with ischemia and no obstructive coronary artery disease, the Women's Ischemic Syndrome Evaluation (WISE) observed that microvascular coronary dysfunction (MCD) is the best independent predictor of adverse cardiovascular events. Since coronary microvascular tone is regulated in part by endothelium, we hypothesized that circulating endothelial cells (CEC), which reflect endothelial injury, and the number and function of bone-marrow derived angiogenic cells (BMDAC), which could help repair damaged endothelium, may serve as biomarkers for decreased coronary flow reserve (CFR) and MCD. METHODS: We studied 32 women from the WISE cohort. CFR measurements in response to intracoronary adenosine were taken as an index of MCD. We enumerated BMDAC colonies and CEC in peripheral blood samples. BMDAC function was assessed by assay of migration of CD34+ cells toward SDF-1 and measurement of bioavailable nitric oxide (NO). These findings were compared with a healthy reference group and also entered into a multivariable model with CFR as the dependent variable. RESULTS: Compared with a healthy reference group, women with MCD had lower numbers of BMDAC colonies [16 (0, 81) vs. 24 (14, 88); P = 0.01] and NO [936 (156, 1875) vs. 1168 (668, 1823); P = 0.02]. Multivariable regression analysis showed strong correlation of CFR to the combination of BMDAC colony count and CD34+ cell function (migration and NO) (R(2) = 0.45; P<0.05). CONCLUSIONS: The BMDAC function and numbers of BMDAC colonies are decreased in symptomatic women with MCD and are independently associated with CFR. These circulating cells may provide mechanistic insights into MCD in women with ischemia.

Induction of nitric oxide by erythropoietin is mediated by the {beta} common receptor and requires interaction with VEGF receptor 2.

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have profound effects on the endothelium and endothelial progenitor cells (EPCs), which originate from the bone marrow and differentiate into endothelial cells. Both EPO and VEGF have demonstrated an ability to increase the number and performance properties of EPCs. EPC behavior is highly dependent on nitric oxide (NO), and both VEGF and EPO can stimulate intracellular NO. EPO can bind to the homodimeric EPO receptor (EPO-R) and the heterodimeric receptor, EPO-R and the common beta receptor (betaC-R). Although VEGF has several receptors, VEGF-R2 appears most critical to EPC function. We demonstrate that EPO induction of NO is dependent on the betaC-R and VEGF-R2, that VEGF induction of NO is dependent on the expression of the betaC-R, and that the betaC-R and VEGF-R2 interact. This is the first definitive functional and structural evidence of an interaction between the 2 receptors and has implications for the side effects of EPO.