Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development.

BACKGROUND: A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing. OBJECTIVE: To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development. PATIENTS AND METHODS: We examined 16 thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses. RESULTS: We classified thrombus regions as unorganized, organizing and organized according to their morphological characteristics. We then evaluated them, focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi. CONCLUSIONS: NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances.

Variation in adenovirus receptor expression and adenovirus vector-mediated transgene expression at defined stages of the cell cycle.

Detailed investigations have addressed the infection pathway of recombinant adenovirus (Ad) gene transfer vectors, but little attention has been paid to the influence of cell physiology on the outcome of Ad infection. Based on observations that Ad infection of clonal cell populations show cell-to-cell variability in the extent of capsid binding, we hypothesized that the cell cycle may influence the outcome of Ad infection. To address this hypothesis, we evaluated Ad association with cells in both unsynchronized and pharmacologically synchronized cell populations. In unsynchronized cell populations, elevated Ad association with cells correlated with expression of cyclin B1, a marker of entry into the M phase of mitosis. The same analysis conducted on cell populations that were synchronized at M phase (using paclitaxel or nocodazole) or at S phase (using aphidicolin) confirmed that M phase cells bound three- to sixfold more capsid compared with unsynchronized cells, which are primarily in the G(1) and G(2) phases. The elevated association of vectors with cells translated into 2.5- to 4-fold greater transgene expression 24 hours after infection. Assessment of cell surface expression of Ad receptors demonstrated that both the high-affinity coxsackie-adenovirus receptor for Ad fiber protein and the low-affinity alpha(v) integrin receptor for Ad penton base protein showed increased cell surface expression at M phase (1.5-fold and 2- to 3-fold increases, respectively). These data demonstrate that Ad infection of a homogenous population of cells can vary depending on the cell cycle stage, with enhanced Ad binding and expression correlating with the enhanced expression of Ad receptors during M phase. These observations have relevance to understanding the mechanisms of gene transfer by Ad vectors and should help in the design of in vivo gene transfer strategies.