Shur-Jen Wang

Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices.

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB4). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB4 using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB4 gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB4, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB4 is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB4 is not reduced in opposing gradients as compared to that in a single LTB4 gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.

Epidermal growth factor promotes breast cancer cell chemotaxis in CXCL12 gradients.

The chemokine receptor CXCR4 and its ligand CXCL12 play an important role in breast cancer invasion and metastasis, and induce the chemotaxis of various types of cancer cells. Previous studies of CXCL12‐induced chemotaxis have, for the most part, relied on endpoint assays (e.g., transwell assays) that provide poor control over the cell microenvironment. Specifically, these assays lacked the ability to dissect the role that autocrine and paracrine growth factors play in chemokine‐induced cancer cell chemotaxis. Here, we employ a microfluidic chemotaxis chamber that allows the effects of specific exogenous factors on cell migration to be directly characterized, without the interference of autocrine/paracrine signaling. Using this approach, we investigated the migration of MDA‐MB‐231 breast cancer cells in well‐defined CXCL12 gradients. We found that CXCL12 alone failed to stimulate chemotaxis of these cells; however, when the CXCL12 gradient was supplemented with a uniform stimulus of either EGF or conditioned media, a directional response was induced. This dependence on growth factor signaling points to the importance of autocrine and paracrine factors in determining the migratory response of the cells, and may play an important role in cancer metastasis. Biotechnol. Bioeng. 2008;100: 1205–1213.

Ancrod and Fibrin Formation Perspectives on Mechanisms of Action

Background and Purpose— Ancrod, derived from Malayan pit viper venom, has been tested as ischemic stroke treatment in clinical trials with inconsistent results. We studied the actions of ancrod on fibrinolysis pathways in patient plasma samples and endothelial cell culture systems. Methods— We analyzed fibrinogen levels during the first 6 hours of ancrod infusion in patients entered in the Stroke Treatment with Ancrod Trial. For the in vitro study, human brain microvascular endothelial cells incubated with plasminogen or with human brain microvascular endothelial cell-conditioned medium were co-incubated with ancrod and fibrinogen under normal or oxygen-glucose deprivation conditions over 6 hours. Results— Fibrinogen levels decreased both in vivo and in vitro. Ancrod generated fibrinopeptide A, caused visible clot formation, and reduced levels of tissue-type plasminogen activator antigen in the human brain microvascular endothelial cell system and in a cell-free system with conditioned media. Conclusions— The in vitro results indicate that ancrod causes local fibrin formation and secondary depletion of tissue-type plasminogen activator by binding to fibrin clot. Ancrod-induced fibrin formation could result in cerebral microvascular occlusion and may explain the suboptimal clinical effects of ancrod in human stroke trials.

PDE4 Regulates Tissue Plasminogen Activator Expression of Human Brain Microvascular Endothelial Cells

INTRODUCTION Factors regulating brain tissue plasminogen activator (tPA) are pertinent for stroke. Recent observations have suggested a role for the phosphodiesterase-4 (PDE4) pathway in stroke pathogenesis, via an uncertain mechanism. We studied PDE4 regulation of tPA expression by human brain microvascular endothelial cells in a variety of conditions, including an in vitro model of ischemia. MATERIALS AND METHODS We analyzed tPA antigen and mRNA of human brain microvascular endothelial cells (HBECs) during normoxia and oxygen-glucose deprivation (OGD) following inhibition of PDE4 and PDE4D, using HBEC monocultures and co-cultures with astrocytes and pericytes, and analyzed relevant signal transduction pathways. RESULTS PDE4 inhibitor rolipram enhanced OGD effects on endothelial tPA release in endothelial monocultures and co-cultures with astrocytes; there was a 54±10% (p<0.001) reduction of tPA release in astrocyte-endothelial co-cultures under OGD. PDE4D siRNA reduced endothelial tPA mRNA to 40-55% of control (p<0.05). Use of Epac inducer mimicked, while use of Epac siRNA inhibited, these effects. CONCLUSIONS Inhibition of PDE4 and PDE4D reduced expression of tPA by HBEC via Epac pathway.

Tissue Plasminogen Activator Expression and Barrier Properties of Human Brain Microvascular Endothelial Cells

Background: Tissue plasminogen activator (tPA) regulates fibrinolysis and is routinely used as ischemic stroke pharmacotherapy. We hypothesized that brain microvascular tPA expression and barrier properties of endothelial cells are substantially related. Methods: Human brain microvascular endothelial cells were incubated with two agents known to modify cAMP pathways: forskolin and rolipram. We analyzed development of endothelial barrier properties, i.e., trans-endothelial electrical resistance (TEER), permeability of endothelial cell monolayer, expression of influx transporter glut-1 and endothelial tight junction molecules occludin and claudin-5, tPA antigen release, and levels of endothelial tPA mRNA. Results: Forskolin plus rolipram-treated endothelial cells showed increased TEER compared to controls (174±20% of control at day six, p<0.01), while permeability to albumin and 70kDa dextran was reduced (21±6.8% of control and 3.8±0.3% of control, respectively, p<0.001). In addition, occludin and claudin-5 protein were up-regulated, occludin mRNA was increased to 206±60% of control (p<0.05), glut-1 mRNA was increased to 196±68% of control (p<0.05), levels of tPA protein were reduced to 35±7.0% of control (p<0.001) after six days, and tPA mRNA was reduced to 32±7.7% of control (p<0.01). TPA and occludin mRNA levels were inversely associated (r=-0.68, p<0.05). Conclusions: In this in vitro model, barrier properties were strongly linked (by inverse association) with tPA expression of brain microvascular endothelial cells.