Vascular wall biology and interactions with bloodstream components are areas of continued interest due to the high prevalence of cardiovascular disease and stroke. Cell types comprising the vascular wall, as well as those found in the bloodstream, are frequently isolated and cultured in vitro to determine their biological roles, including their involvement in disease progression. Recently, microfluidic systems have emerged as desirable platforms for mimicking biological systems, offering precise control of cellular environments. Here, work is presented that advances microfluidic technology and improves the utility of microfluidic systems for cell-based assays and cell culture. The endothelium is an important component of the vascular wall due to its role as a protective barrier, as well as its involvement in several important vascular functions. In vivo, the endothelium plays a vital role in the regulation of vascular tone by releasing several factors that cause vessel dilation, of which nitric oxide (NO) is one of the most potent. The endothelial cells comprising the endothelium are influenced by many environmental factors, such as shear stress generated by flowing blood and the degree of confluence of the endothelial monolayer. Presented here is the development of novel microfluidic system capable of mimicking the vasculature by incorporating microfluidic channels that enable blood flow past cultured endothelial monolayers. Importantly, this microfluidic vascular mimic is successfully integrated with a transendothelial electrical resistance (TEER) measurement system that allows for the monitoring of endothelial monolayer confluence and barrier integrity. This microfluidic system is used to highlight the impact of monolayer confluence on cellular behavior by showing that, in response to flowing red blood cells (RBCs), confluent endothelial monolayers produce significantly more NO than less confluent monolayers.C-peptide, a potential drug for type 1 diabetes, is known to improve blood flow by a previously unknown mechanism. This dissertation hypothesizes that C-peptide is capable of indirectly inducing vessel dilation by stimulating the release of adenosine triphosphate (ATP) from flowing RBCs, which can diffuse to the endothelium to bind purinergic receptors on the endothelial cell surface, ultimately resulting in endothelial NO production. To investigate this hypothesis, the microfluidic vascular mimic is utilized to monitor cell-cell communication, revealing C-peptide is capable of stimulating endothelial NO production by a mechanism mediated by the RBC which requires P2Y purinoreceptor activation by ATP. Importantly, it is shown that in order to observe this NO production, C-peptide must be prepared with Zn2+. This proposed mechanism describes how Zn2+/C-peptide may improve blood flow indirectly as a result of activity within the bloodstream. However, since C-peptide has never been observed exogenous of the bloodstream in vivo, it is unclear whether all C-peptide bioactivity results similarly, or if C-peptide can also escape the bloodstream to directly stimulate cells in surrounding tissue. To assess the ability of C-peptide to penetrate the vascular wall, the microfluidic TEER system is employed to determine the ability of C-peptide to permeate the endothelium, revealing that C-peptide can permeate confluent endothelial monolayers in vitro, suggesting that C-peptide may escape the bloodstream in vivo. Collectively, this work shows that C-peptide may have a more comprehensive biological role than is currently assumed.
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