Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1ß, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

Integrating Clinical and Air Quality Data to Improve Prediction of COPD Exacerbations.

Several studies have found associations between air pollution and respiratory disease outcomes. However, there is minimal prognostic research exploring whether integrating air quality into clinical prediction models can improve accuracy and utility. In this study, we built models using both logistic regression and random forests to determine the benefits of including air quality data with meteorological and clinical data in prediction of COPD exacerbations requiring medical care. Logistic models were not improved by inclusion of air quality. However, the net benefit curves of random forest models showed greater clinical utility with the addition of air quality data. These models demonstrate a practical and relatively low-cost way to include environmental information into clinical prediction tools to improve the clinical utility of COPD prediction. Findings could be used to provide population level health warnings as well as individual-patient risk assessments.

MicroRNA-335-5p and -3p synergize to inhibit estrogen receptor alpha expression and promote tamoxifen resistance.

microRNAs (miRNAs) are small noncoding RNA molecules involved in the regulation of gene expression and play critical roles in human malignancies. Next-generation sequencing analysis of the MCF-7 breast cancer cell line overexpressing miR-335-5p and miR-335-3p demonstrated that the miRNA duplex repressed genes involved in the ERα signaling pathway, and enhanced resistance of MCF-7 cells to the growth inhibitory effects of tamoxifen. These data suggest that despite its conventional role in tumor suppression, the miR-335 transcript can also play an oncogenic role in promoting agonistic estrogen signaling in a cancerous setting.

Argonaute 2 Expression Correlates with a Luminal B Breast Cancer Subtype and Induces Estrogen Receptor Alpha Isoform Variation.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.