Abstract
Angiogenesis plays a critical role in many diseases, including macular degeneration. At present, the pathological mechanisms remain unclear while appropriate models dissecting regulation of angiogenic processes are lacking. We propose an in vitro angiogenesis process and test it by examining the co-culture of human retinal pigmental epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC) inside a microfluidic device. From characterisation of the APRE-19 monoculture, the tight junction protein (ZO-1) was found on the cells cultured in the microfluidic device but changes in the medium conditions did not affect the integrity of monolayers found in the permeability tests. Vascular endothelial growth factor (VEGF) secretion was elevated under low glucose and hypoxia conditions compared to the control. After confirming the angiogenic ability of HUVEC, the cell-cell interactions were analyzed under lowered glucose medium and chemical hypoxia by exposing ARPE-19 cells to cobalt (II) chloride (CoCl2). Heterotypic interactions between ARPE-19 and HUVEC were observed, but proliferation of HUVEC was hindered once the monolayer of ARPE-19 started breaking down. The above characterisations showed that alterations in glucose concentration and/or oxygen level as induced by chemical hypoxia causes elevations in VEGF produced in ARPE-19 which in turn affected directional growth of HUVEC.
Highlights
Angiogenesis, the growth of new capillary blood vessels from pre-existing vascular structures, occurs naturally in the body during reproduction and wound healing
We have examined responses of cells in a logical way, starting from characterising ARPE-19 and human umbilical vein endothelial cells (HUVEC) individually before examining the co-culture under different conditions
The device is designed in such a way that ARPE-19 cells and HUVEC are separated by a porous membrane, similar to the in vivo anatomy where retinal cells are separated from the choroids by Bruch’s membrane (BM) (Fig. 1)
Summary
Angiogenesis, the growth of new capillary blood vessels from pre-existing vascular structures, occurs naturally in the body during reproduction and wound healing. In the case of the more advanced type of age-related macular degeneration (wet AMD), abnormal blood vessels develop under the macula and compromise Bruch’s membrane, leading to leakage of fluid (exudate) or blood. Choroidal neovascularization of wet AMD occurs in response to the abnormal secretion of growth factors, of which vascular endothelial growth factor (VEGF) being the most important mediators of angiogenesis. Choroidal neovascularization is promoted and exacerbated when there are changes in the extracellular microenvironment in vivo, especially hypoxia, inflammation or oncogene products which lead to the upregulation of growth factors, integrins and proteinases, resulting in the formation of new vessels[10, 11]. We build a simplified microfluidic co-culture model of the ocular fundus tissue in an attempt to elucidate AMD pathology. The microdevice can be fabricated in a short amount of time; with the same fabrication methods and slight alteration of the design, the microfluidic system can be tailored to other applications, demonstrating a great potential in medical diagnosis and pharmacokinetics
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