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Madison, WI, United States

Belair D.G.,University of Wisconsin - Madison | Whisler J.A.,Massachusetts Institute of Technology | Valdez J.,Massachusetts Institute of Technology | Velazquez J.,Massachusetts Institute of Technology | And 14 more authors.
Stem Cell Reviews and Reports | Year: 2015

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. © 2014, Springer Science+Business Media New York.

Soto B.L.,University of Wisconsin - Madison | Hank J.A.,University of Wisconsin - Madison | Van De Voort T.J.,University of Wisconsin - Madison | Subramanian L.,University of Wisconsin - Madison | And 9 more authors.
Cancer Immunology, Immunotherapy | Year: 2011

We investigated the anti-tumor effect of peritumoral resveratrol in combination with immunotherapy in vivo in neuroblastoma-bearing mice. Subcutaneous NXS2 tumors were induced in A/J mice. On day 10, some mice received 15 mcg of intravenous immunocytokine for 5 days, mice received 20 mg of peritumoral resveratrol twice a week (starting on day 12) for a total of 5 injections, and a separate group received a combination of both regimens. Tumor progression and survival were assessed every 3-4 days. Blood and primary tumor tissue samples were collected on day 20 for Complete Blood Count and CD45 immunohistochemistry and histology, respectively. The primary tumor regressed in all mice receiving peritumoral resveratrol. Most of these mice receiving peritumoral resveratrol alone developed metastatic tumors and recurrence of the primary tumor after cessation of therapy. When resveratrol and immunocytokine regimens were combined, 61% of the mice receiving this combination therapy resolved their primary tumors and survived without developing metastatic tumors, compared to 15 and 13% receiving resveratrol or immunocytokine alone, respectively. None of the therapeutic regimes prevented lymphocyte infiltration or affected the complete blood count. Greater necrosis was observed microscopically in tumors from mice receiving the combination therapy. These results demonstrate that the combination therapy of peritumoral resveratrol plus intravenous immunocytokine provides better anti-tumor effects in this model than either therapy alone. © 2011 Springer-Verlag.

Wang B.,Wisconsin Institute for Medical Research | Zhang M.,Wisconsin Institute for Medical Research | Takayama T.,Wisconsin Institute for Medical Research | Takayama T.,University of Wisconsin - Madison | And 5 more authors.
EBioMedicine | Year: 2015

Background: Intimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic "readers" including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known. Methods: We investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model. Results: While BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(-), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(-), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced. Conclusions: Blocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases. © 2015 The Authors.

Sackmann E.K.,Wisconsin Institute for Medical Research | Berthier E.,Wisconsin Institute for Medical Research | Young E.W.K.,Wisconsin Institute for Medical Research | Shelef M.A.,University of Wisconsin - Madison | And 3 more authors.
Blood | Year: 2012

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays. © 2012 by The American Society of Hematology.

Sackmann E.K.,Wisconsin Institute for Medical Research | Sackmann E.K.,University of Wisconsin - Madison | Berthier E.,Wisconsin Institute for Medical Research | Berthier E.,University of Wisconsin - Madison | And 14 more authors.
Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 | Year: 2012

Improvements in neutrophil cliemotaxis assays have enabled significant advances in understanding the mechanisms of neutrophil recruitment, however current methods are still limiting due to high sample volume requirements, low experimental throughput, or complex assay protocols. We report a microfluidic technology that performs neutrophil sorting and cliemotaxis on-chip within minutes using nanoliters of whole blood and only requires a micropipette to operate. The platform was adapted to incorporate an endothelial cell monolayer: perform cliemotaxis on mouse neutrophils; and human neutrophil cliemotaxis in 3D. Finally, the platform was employed in a clinical setting to diagnose asthma in human patients.

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