Novel Infrared Fluorescence Methodology Defines An Essential Role for Endothelial Protein C Receptor (EPCR) for Protection Against Vascular Leakage In Inflammation

Activated protein C (APC) reduces mortality in adult sepsis and murine sepsis models. In mice, this mortality reduction requires EPCR. Mice with low levels of EPCR (“EPCR low”) are more susceptible to death from lipopolysaccharide (LPS) than wild-type (wt) mice. Mice over-expressing EPCR are resistant to LPS. EPCR binds protein C (PC) and APC and this promotes both PC activation and APC's cytoprotective signaling. In vitro data show that EPCR is important for endothelial barrier integrity but in vivo data for this concept are lacking. To probe EPCR's role in vascular barrier stability in vivo, we challenged EPCR low and wt mice with LPS and assessed vascular permeability based on: 1) plasma extravasation of albumin-bound Evans Blue (EB) dye into organs quantified by Infrared Fluorescence (IRF); 2) intravascular volume contraction in relation to weight loss; and 3) organ pathology. First, we developed a highly novel method for vascular barrier leak in vivo in which albumin-bound EB in organs was quantified by IRF; this method was highly sensitive and allowed EB quantification in organs (lung, kidney, spleen, liver, heart) after i.v. injection of a wide range of EB doses (5 - 50 mg/kg EB). Even 5 mg/kg EB i.v. gave good IRF data showing detectable organ accumulation of EB in wt mice. This low dose of i.v. EB is one-tenth of the EB dose typically used for EB quantification by formamide extraction for 3 days with EB levels determined by absorbance spectrophotometry (“traditional method”). When EB plasma concentrations were determined by the traditional method and by our IRF method in 96 mice, EB values from the two methods correlated significantly (r²=0.9; n=192 plasma samples from 96 mice receiving LPS i.p. (n=48) or saline (n=48)). These comparisons validated our IRF method for vascular leakage. To define sensitivity in organs, the two methods were compared when 25 mg/kg EB i.v. was used in 59 mice receiving LPS (n=39) or saline (n=20). The results showed that EB levels were detected by IRF in all mice for all 7 organ systems studied (= 100%; kidney, lung, peritoneum, heart, spleen, liver, brain). In contrast, the traditional method detected useful EB levels in only 55% of instances and the detection was poorest in spleen (43%) and brain (14%). Nonetheless, in the 55% of instances where EB could be detected by the traditional method, correlations with IRF EB quantitation were significant (r²=0.85). Furthermore, our novel IRF method quantified EB in wet organs, thus permitting subsequent histology of the same organs. Histology is not possible with the traditional method since it requires drying of organs. Second, after we had developed IRF for reliable EB quantitation, we then used IRF to assess EB vascular leak in EPCR low compared to wt mice. Mice received LPS in doses leading to wt 7-day mortality rates of 25% (n=8), 40% (n=14) or 50% (n=8). At 18 hr after LPS or saline, EB was given and 30 min later, mice were sacrificed and EB was determined by IRF. In saline controls, EB levels in organs of EPCR low and wt mice were similar (p=0.3). However, EB levels in organs of EPCR low mice were higher than in wt mice for each LPS dose; e.g., at LD40 and LD50, EB in organs of EPCR low mice was double the EB levels seen for wt mice (p<0.0001) and was significantly increased over baseline (p=0.0006). Mean intravascular plasma volume of EPCR low mice but not of wt mice decreased significantly after LPS (p=0.0004). Since weight loss was identical (6.5%), the plasma volume loss in the EPCR low mice was caused by plasma extravasation. Histology analyses of wet organs previously used for IRF data showed that EPCR low mice had developed pronounced renal hemorrhage, extensive lung injury (H/E stains), and proteinuria (EB in urine). These histological findings were absent or significantly milder in wt mice. In summary, we have developed and applied a novel IRF method to quantify EB in murine organs for assessment of vascular permeability in organs of EPCR low mice and wt mice. EPCR low mice receiving LPS had greater vascular leakage, loss of intravascular plasma, renal hemorrhage, lung injury and proteinuria than wt mice, indicating severe vascular injury. These findings show that EPCR is required to maintain normal vascular integrity in vivo during LPS-induced inflammation, thus further demonstrating the importance of this key receptor that is central to the endogenous protein C pathway.

No relevant conflicts of interest to declare.