The presence of angiographic collaterals in non-ST elevation myocardial infarction is a predictor of long-term clinical outcomes.

OBJECTIVES: To determine whether the presence of angiographic coronary collaterals is a predictor of long-term clinical outcomes in patients with non-ST elevation myocardial infarction (NSTEMI). BACKGROUND: The presence of coronary collaterals on angiography provides prognostic information in patients with STEMI, but it is unknown whether they provide prognostic information in patients with NSTEMI. METHODS: This was a prospective cohort study of 931 consecutive patients undergoing coronary angiography of which 269 (29%) had a NSTEMI. Baseline characteristics, angiographic details, and long-term clinical outcomes including death, recurrent MI, coronary artery bypass graft surgery (CABG), percutaneous coronary intervention (PCI), stroke, and congestive heart failure (CHF) were collected. Each clinical outcome as well as the combined endpoint of death, recurrent MI, CABG, PCI stroke and CHF was compared in subjects with and without collaterals. RESULTS: At one year, individuals with collaterals had significantly increased rates of the combined endpoint compared with those without (25% vs. 16%, P = 0.0001). On multivariate analysis, the presence of collaterals was a strong predictor of the combined endpoint of death, recurrent MI, CABG, PCI, stroke and CHF (HR 1.95, CI 95% 1.08-3.52; P = 0.027). Similarly, in the subset of 115 patients (43%) in whom the culprit artery was identified, the presence of collaterals was a strong negative predictor (HR 3.71, CI 1.31-10.57, P = 0.014), driven by a 13-fold increase in subsequent CABG. CONCLUSIONS: In patients with NSTEMI the presence of angiographic coronary collaterals is a predictor of long-term clinical outcomes primarily driven by increased rates of surgical revascularization.

Circulating hematopoietic progenitor cells are decreased in COPD.

RATIONALE: Bone marrow derived progenitor cells participate in the repair of injured vessels. The lungs of individuals with emphysema have reduced alveolar capillary density and increased endothelial apoptosis. We hypothesized that circulating levels of endothelial and hematopoietic progenitor cells would be reduced in this group of patients. OBJECTIVES: The goal of this study was to measure circulating levels of endothelial progenitor cells (EPCs) and hematopoietic progenitor cells (HPCs) in subjects with COPD and to determine if progenitor levels correlated with disease severity and the presence of emphysema. METHODS: Peripheral blood mononuclear cells were isolated from 61 patients with COPD and 32 control subjects. Levels of EPCs (CD45(dim) CD34+) and HPCs (CD45(+) CD34(+) VEGF-R2(+)) were quantified using multi-parameter flow cytometry. Progenitor cell function was assessed using cell culture assays. All subjects were evaluated with spirometry and CT scanning. MEASUREMENTS AND MAIN RESULTS: HPC levels were reduced in subjects with COPD compared to controls, whereas circulating EPC levels were similar between the two groups. HPC levels correlated with severity of obstruction and were lowest in subjects with severe emphysema. These associations remained after correction for factors known to affect progenitor cell levels including age, smoking status, the use of statin medications and the presence of coronary artery disease. The ability of mononuclear cells to form endothelial cell colony forming units (EC-CFU) was also reduced in subjects with COPD. CONCLUSIONS: HPC levels are reduced in subjects with COPD and correlate with emphysema phenotype and severity of obstruction. Reduction of HPCs may disrupt maintenance of the capillary endothelium, thereby contributing to the pathogenesis of COPD.

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Requirements for the destruction of human Aurora-A.

The mitotic kinase Aurora A (Aur-A) is overexpressed in a high proportion of human tumors, often in the absence of gene amplification. In somatic cells, Aur-A protein levels fall following mitosis or upon overexpression of Cdh1, an activator of the ubiquitin ligase APC/C. Thus, mutations that reduce or block the rate of Aur-A destruction might also be expected to contribute to its oncogenic potential. Previous work had defined two short sequences of Xenopus Aur-A that are required for its Cdh1-inducible destruction in extracts of Xenopus eggs, an N-terminal A box and a C-terminal D box, and a serine residue within the A box whose phosphorylation might inhibit destruction. Here, we show that these same sequences are required for the destruction of human Aur-A during mitotic exit and G1 in the somatic cell cycle. Expression of a dominant negative Cdh1 protein leads to accumulation of Aur-A, further indicating that the Cdh1-activated form of the APC/C is responsible for destruction of Aur-A during the somatic cell cycle in vivo. During the course of this work, we found some previously unsuspected problems in commonly used in vitro destruction assays, which can result in misleading results. Potentially confounding factors include: (i) the presence of D-box- and A-box-dependent destruction-promoting activities in the reticulocyte in vitro translation mix that is used to produce radiolabeled substrates for destruction assays; and (ii) the ability of green-fluorescent-protein tags to reduce the destruction rate of Aur-A substantially. These findings have direct relevance for studies of Aur-A destruction itself, and for broader approaches that use in vitro translation products in screens for additional APC/C targets.