Human vascular permeability measurements in vitro
Microvascular function is key in the regulation of blood pressure and vascular tone, however, disruption to microvascular function, for example alteration to the integrity of endothelial tight junctions, is a factor in many diseases such as hypertension, diabetes heart failure and sepsis. Adverse drug effects may manifest through changes in microvascular function causing angioedema or vasodilatation. Angioedema is caused by an increase in vascular permeability, leading to swelling of the deep layers of subcutaneous or submucosal tissues. Drug-induced angioedema is a well-documented adverse effect of ACE inhibitors, SSRIs (selective serotonin reuptake inhibitors), COX-II inhibitors, angiotensin II antagonists, statins and proton pump inhibitors (Salih & Thomas, 2006). These adverse side effects can remain undetected until late stage human clinical trials due to the lack of a predictive in vitro model.
Typically, vascular permeability is measured using an in vivo animal model, such as the Evans Blue Dye (EBD) model. Evans Blue Dye, bound to albumin, is injected intravenously and extravasion of the dye into the vascular wall is used as a marker of vascular permeability (Radu & Chernoff, 2013). Such models suffer disadvantages such as potential species difference and an inability to track changes in real time.
Biopta’s PM-1 (perfusion myograph-1) is an automated perfusion myograph allowing sensitive and accurate measurements of vascular function. As well as measurements of vessel diameter, Evans Blue Dye (EBD) conjugated to bovine serum albumin can also be perfused through the lumen on the vessel, and using the PM-1’s novel imaging system, the flow of dye into the vascular wall under different conditions can be measured in real time.
Under normal physiological conditions, EBD conjugated with bovine serum albumin (BSA) at a ratio of 10:1, does not readily pass through the endothelial layer of blood vessels (Stopa et al., 2006). Changes in endothelial function, either under pathophysiologic conditions or due to direct drug effects, can alter this barrier function; allowing BSA to penetrate the tissue. Thus, perfusion of the BSA conjugated EBD in the presence and absence of test compounds provides real time images on the permeability of the vascular wall.
Figure 1 shows example pictures from the PM-1 taken during a single experiment, showing the accummulation of BSA-conjugated EBD in the vessel wall of a pressurised human subcutaneous artery in the presence and absence of 0.5 units/ml thrombin.
The transmittance blue light was determined from images taken throughout the BSA-conjugated EBD experiment and normalised to account for the total transmittance of light by the tissue.
Figure 2 demonstrates the percentage change of blue light in human subcutaneous arteries perfused with BSA-conjugated EBD over a 30 minute period in the presence and absence of thrombin (0.5 units/ml).
Infusion of thrombin into the lumen of the artery caused a significant time-dependent increase in transmission of blue light (p = 0.018; two-way ANOVA) indicating uptake of the BSA-conjugated dye into the vessel wall. Changes in light transmission were not observed in the absence of thrombin suggesting that it was not simply a time-dependent accumulation of dye into the vessel wall and that thrombin caused a measurable increase in vascular permeability.
Utilising Biopta’s PM-1 system allows real time investigations in vitro to be carried out in the target species and provides data on the safety and possible adverse effects of test compounds. This provides data allowing decisions to be made early on in the development process, thereby saving significant costs on poor drug candidates and allowing better prediction of the safest, most efficacious drugs in human tissue.