Abstract
Inflammation-induced impaired function of vascular endothelium may cause leakage of plasma proteins that can lead to edema. Proteins may leave the vascular lumen through two main paracellular and transcellular pathways. As the first involves endothelial cell (EC) junction proteins and the second caveolae formation, these two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during pathology that causes inflammation. Here we present a newly developed dual-tracer probing method that allows differentiation of transcellular from paracellular transport during pathology. This fluorescence-based method can be used in vitro to test changes in EC layer permeability and in vivo in various animal vascular preparations. The method is based on comparison of low molecular weight molecule (LMWM) transport to that of high molecular weight molecule (HMWM) transport through the EC layer or the vascular wall during physiological and pathological conditions. Since the LMWM will leak through mainly the paracellular and HMWM will move through paracellular (when gaps between the ECs are wide enough) and transcellular pathways, the difference in transport rate (during normal conditions and pathology) of these molecules will indicate the prevailing transport pathway involved in overall protein crossing of vascular wall. Thus, the novel approach of assessing the transport kinetics of different size tracers in vivo by intravital microscopy can clarify questions related to identification of target pathways for drug delivery during various pathologies associated with elevated microvascular permeability.
Highlights
Many inflammatory diseases such as hypertension (Johansson et al, 1978; Miller et al, 1986; Pedrinelli et al, 1999), diabetes (Bonnardel-Phu et al, 1999), and stroke (Hatashita and Hoff, 1990; Laursen et al, 1993; Wardlaw et al, 2003) are accompanied by impaired vascular wall integrity leading to enhanced microvascular leakage
This method is based on a comparison of leakage of a low molecular weight molecule (LMWM) and a high molecular weight molecule (HMWM; in our case albumin) through cultured mouse brain endothelial cells (ECs) (MBECs) and mouse pial venules during normal and pathological conditions
We showed that enhanced blood content of fibrinogen (Fg) compromises EC layer integrity resulting in albumin leakage through cultured EC layer (Tyagi et al, 2008; Muradashvili et al, 2011) and mouse pial venules (Muradashvili et al, 2012)
Summary
Many inflammatory diseases such as hypertension (Johansson et al, 1978; Miller et al, 1986; Pedrinelli et al, 1999), diabetes (Bonnardel-Phu et al, 1999), and stroke (Hatashita and Hoff, 1990; Laursen et al, 1993; Wardlaw et al, 2003) are accompanied by impaired vascular wall integrity leading to enhanced microvascular leakage. Paracellular transport occurs through gaps between endothelial cells (ECs) and involves alterations in tight, gap, and adherence junction proteins (Mehta and Malik, 2006). We developed a dual-tracer probing method to differentiate the relative involvement of transcellular and paracellular transport pathways in protein crossing the EC layer and vascular wall during pathology. This method is based on a comparison of leakage of a low molecular weight molecule (LMWM) and a high molecular weight molecule (HMWM; in our case albumin) through cultured mouse brain ECs (MBECs) and mouse pial venules during normal and pathological conditions. The prevailing role of one or the other transport mechanism was not defined
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