Endothelial Progenitor Cells Promote Directional Three-Dimensional Endothelial Network Formation by Secreting Vascular Endothelial Growth Factor

Endothelial progenitor cell (EPC) transplantation induces the formation of new blood-vessel networks to supply nutrients and oxygen, and is feasible for the treatment of ischemia and cardiovascular diseases. However, the role of EPCs as a source of proangiogenic cytokines and consequent generators of an extracellular growth factor microenvironment in three-dimensional (3D) microvessel formation is not fully understood. We focused on the contribution of EPCs as a source of proangiogenic cytokines on 3D microvessel formation using an in vitro 3D network model. To create a 3D network model, EPCs isolated from rat bone marrow were sandwiched with double layers of collagen gel. Endothelial cells (ECs) were then cultured on top of the upper collagen gel layer. Quantitative analyses of EC network formation revealed that the length, number, and depth of the EC networks were significantly enhanced in a 3D model with ECs and EPCs compared to an EC monoculture. In addition, conditioned medium (CM) from the 3D model with ECs and EPCs promoted network formation compared to CM from an EC monoculture. We also confirmed that EPCs secreted vascular endothelial growth factor (VEGF). However, networks cultured with the CM were shallow and did not penetrate the collagen gel in great depth. Therefore, we conclude that EPCs contribute to 3D network formation at least through indirect incorporation by generating a local VEGF gradient. These results suggest that the location of EPCs is important for controlling directional 3D network formation in the field of tissue engineering

A three-dimensional microfluidic tumor cell migration assay to screen the effect of anti-migratory drugs and interstitial flow

Most anti-cancer drug screening assays are currently performed in two dimensions, on flat, rigid surfaces. However, there are increasing indications that three-dimensional (3D) platforms provide a more realistic setting to investigate accurate morphology, growth, and sensitivity of tumor cells to chemical factors. Moreover, interstitial flow plays a pivotal role in tumor growth. Here, we present a microfluidic 3D platform to investigate behaviors of tumor cells in flow conditions with anti-migratory compounds. Our results show that interstitial flow and its direction have significant impact on migration and growth of hepatocellular carcinoma cell lines such as HepG2 and HLE. In particular, HepG2/HLE cells tend to migrate against interstitial flow, and their growth increases in interstitial flow conditions regardless of the flow direction. Furthermore, this migratory activity of HepG2 cells is enhanced when they are co-cultured with human umbilical vein endothelial cells. We also found that migration activity of HepG2 cells attenuates under hypoxic conditions. In addition, the effect of Artemisinin, an anti-migratory compound, on HepG2 cells was quantitatively analyzed. The microfluidic 3D platform described here is useful to investigate more accurately the effect of anti-migratory drugs on tumor cells and the critical influence of interstitial flow than 2D culture models.Japan Society for the Promotion of Science (22680037)Japan Society for the Promotion of Science (G2212)National Cancer Institute (U.S.) (R21CA140096)Japan. Ministry of Education, Culture, Sports, Science and Technology (2009-00631)Japan. Ministry of Education, Culture, Sports, Science and Technology (2012-0009565)Korea (South). Ministry of Education & Human Resources Development (MOEHRD)Korea (South). Ministry of Education & Human Resources Development (MOEHRD) (20124010203250

Neurovascular coupling in primary auditory cortex investigated with voltage-sensitive dye imaging and laser-Doppler flowmetry

The spatiotemporal dynamics of the neurovascular response to brief acoustic stimuli were investigated in guinea pig primary auditory cortex. Neural activity and cortical tonotopic organization were measured with a voltage-sensitive dye (VSD) technique, whereas cerebral blood flow (CBF) response to neural stimulation was measured with laser-Doppler flowmetry (LDF). The acoustic stimulus was given as a wide band sound (click), which induced global activation or as one of two pure tones (1 kHz and 12 kHz), which induced distinct localizations in the auditory cortex. The VSD imaging showed that the sound-induced activation area varied dynamically, and that the spatial extent had peaks at 37+/-3 ms and 38+/-8 ms after the onset of stimulation during 1-kHz and 12-kHz tones, respectively. We observed that the average CBF response had a similar peak intensity irrespective of the type of stimuli: 16+/-9%, 18+/-11%, and 16+/-8% for click, 1-kHz, and 12-kHz tones, respectively. No significant differences in the CBF time course, time-to-onset ( approximately 0.6 s), or time-to-peak ( approximately 3.3 s) were found across the recording sites and stimulus types. These results showed that the CBF response measured with LDF produced a less specific spatial pattern relative to the neural map determined with VSD. The findings can be explained by the methodological limitations of LDF and/or neurovascular regulatory systems in the auditory cortex

pO2 profiles in tumor after carbon beams.

HCPBM and ENLIGHT-200