Cardiovascular: Atherosclerosis, Drug Eluting Stents

Overview

Atherosclerosis is a chronic inflammatory disease that constitutes the primary cause of heart disease and stroke. It is a progressive condition characterized by the gradual accumulation of lipid material in the artery wall. The recruitment of leukocytes, their adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. These experimental conditions can easily be studied using Cellix's microfluidic pumps and biochips.

Stents are designed to line the inner walls of the blood vessel, holding the artery open and maintaining normal blood flow. New devices called drug-eluting stents have made a major contribution to reducing the incidence of restenosis. These devices consist of a metallic stent covered with a polymer coating, slowly releasing a medication that blocks inflammatory and proliferative processes in the vessel wall, thereby limiting the overgrowth of vascular tissue during the healing process. Cellix have optimized a microfluidic set-up to study the interaction of whole blood incubated with fractions eluted over time from different co-polymer combinations. The dimensions of the biochips can mimic those of human capillaries thereby enabling researchers to simulate in vivo microenvironments. Cellix's 8-channel Mirus Evo Nanopump enables researchers to conduct up to 8 assays simultaneously in microfluidic biochips while single assays may be conducted using the ExiGo pump.

 

Assay Examples:

 

Monocyte adhesion - chemokines – Oxidised LDL

Aim: To investigate the role of different stimuli recognized to play an important part in atherosclerosis development (chemokines, cytokines, oxidized lipoproteins) on leukocyte adhesion to endothe-lial cells or purified adhesion molecules, under physiological flow conditions using Cellix’s VenaFlux™ platform and biochips.

Vena 8™ biochip microchannels were coated with rhVCAM-1, overnight in humid conditions at 4°C. ThP-1 cells were incubated with anti-integrin αa mAB for 15 minutes. PBMC’s were incubated with Fractakine or MCP-1 for 30 minutes.

HUVECs were grown in static conditions for 48h prior to flow experiments on VenaEC biochips.

Perfusion was executed via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays at a shear stress of 0.5 dyne/cm2.

Results: Adhesion to VCAM-1, a major adhesion molecule expressed on inflamed endothelium, was completely inhibited when blocking the α4 integrin on THP1 cells.  Interestingly, adhesion was not significantly reduced when looking at adhesion to endothelial cells, even with a concentration of anti-α4 mAb 100 times higher.

In relation to oxidative stress, a central factor in atherosclerosis development, we studied the effects of oxidized LDL on adhesion molecule expression on endothelial cells and subsequent leukocyte adhesion.Three different levels of oxidation were considered, and the result of oxLDL incubation with regard to adhesion molecule upregulation onHUVECs was assessed via flow cytometric analysis. Only the highly oxidized LDL (Hox) was shown to cause a significant increase in VCAM-1, ICAM-1 and E-selectin expression. When endothelial cells were incubated with TNFα on top of oxLDL this result was not seen.

 

 

Discussion: These in vitro studies, closely mimicking the human in vivo vascular microenvironment, represent a useful model to dissect different mechanisms involved in atherogenesis. This allows for a better design of more focused experiments involving animal models, and for a more realistic prediction of the responses in humans, considering the difficulty in engaging into clinical trials, due to the dramaticend-points of the disease (such as myocardial infarction, stroke or death), and the lack of specific markers.

 

Drug Eluting Stents – Platelet Adhesion

Aim: To screen a range of novel thermoresponsive polymers designed to be used as dual drug-eluting systems in coating stents. Specifically, to assess the ability of xemilofiban released in conjunction with fluvastatin, to prevent thrombus formation / platelet adhesion to fibrinogen using Cellix’s VenaFlux™ platform.

Vena8™ biochips were coated with fibrinogen (20 μg/ml) and placed at 4°C, overnight. Prior to experimentation, the coated channels were further coated with BSA (10 μg/ml) for 30 mins at room temperature.

Blood was collected from healthy volunteers who were not taking any medication and were free from aspirin and other anti-platelet agents. Blood was drawn by venepuncture into light-blue Vacuette™ Tubes.  Blood was incubated with drug eluates for 5 minutes at room temperature and subjected to a constant shear stress of 32 dyne cm-2 for 3 min, followed by buffer for a further 3 min at similar shear stress via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays.

Platelet adhesion profiles were recorded using DucoCell software. Cell images were captured from three microscopic fields from each channel. Data were exported into Excel to allow further analysis.

Results:

 

 

Conclusion:  System A and System B illustrate the adhesion profile of platelets treated with drug eluates over the course of 14 days. Day 1 suggests that there is a significant release of drug resulting in inhibition of platelet adhesion to fibrinogen. Day 3 shows a similar pattern (as shown by the median). It is noted that the drug is released at a slower rate after Day 9 and Day 14 resulting in more platelets adhering to fibrinogen (as shown by the median). System A and System B result in a more rapid release of drug during the first 3-day period compared to System C. System C results in a steady release of drug from the microgel/matrix composition from Day 3. Under flow conditions 0.1 μM xemilofiban results in an almost complete inhibition of platelet adhesion, whereas static experiments show a decrease by approximately 50%.Interestingly, under flow conditions platelet adhesion tofibrinogen is inhibited at a lower concentration of xemilofiban. Data from flow experiments show that 0.01 μM xemilofiban results in a statistically significant inhibition of platelet adhesion compared to static experiments where the 0.01 μM concentration shows no significant inhibitory effects. Cellix’s VenaFlux™ platform allows researchers to mimic physiological shear stresses. In comparison to static experiments, the VenaFlux™ platform allows for a more dynamic and sensitive detection method for platelet adhesion