Vascular endothelial effects of collaborative binding to platelet/endothelial cell adhesion molecule-1 (PECAM-1)

Abstract

Targeting drugs to endothelial cells has shown the ability to improve outcomes in animal models of inflammatory, ischemic and thrombotic diseases. Previous studies have revealed that certain pairs of ligands (antibodies and antibody fragments) specific for adjacent, but distinct, epitopes on PECAM-1 enhance each other's binding, a phenomenon dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL. This discovery has been leveraged to enable simultaneous delivery of multiple therapeutics to the vascular endothelium. Given the known role of PECAM-1 in promoting endothelial quiescence and cell junction integrity, we sought here to determine if CEPAL might induce unintended vascular effects. Using a combination of in vitro and in vivo techniques and employing human and mouse endothelial cells under physiologic and pathologic conditions, we found only modest or non-significant effects in response to antibodies to PECAM-1, whether given solo or in pairs. In contrast, these methods detected significant elevation of endothelial permeability, pro-inflammatory vascular activation, and systemic cytokine release following antibody binding to the related endothelial junction protein, VE-Cadherin. These studies support the notion that PECAM-1-targeted CEPAL provides relatively well-tolerated endothelial drug delivery. Additionally, the analysis herein creates a template to evaluate potential toxicities of vascular-targeted nanoparticles and protein therapeutics.

Figure 1

Barrier function assay in mouse and human endothelial cells. Electric cell-substrate impedance sensing (ECIS) measurement of endothelial resistance. (A and C) Real time tracings (representative of 4 independent monolayers) in MECs and HUVECs starting at the time of addition of specific antibodies or isotype controls. Presented resistance is normalized to a pre-treatment time-point for comparison of mAb effect on barrier function. (B) Quantification of AUC normalized to no treatment control. Anti-PECAM-1 antibodies had no effects on the electrical resistance of MEC monolayers in comparison to no treatment or isotype control (^#p = 0.97, 0.99 for solo mAbs and 0.51 for a combination, respectively). Data shown as mean ± SD, dotted line illustrating a level of no treatment control. (D) In quiescent HUVEC cells, binding to PECAM-1, both solo and paired, results in significant drop in endothelial resistance (*p < 0.01, compared to no treatment). Effect mAbs to PECAM-1, when compared to IgG isotype control, was not significant (^#p = 0.36 for Ab62 vs. mouse IgG2A and p = 0.81, for Ab37 vs. mouse IgG1). In both systems, significant barrier disruption was seen after binding of mAb to VE-cadherin (**p = 0.0005; ***p < 0.0001, compared to IgG isotype control). Data shown as mean ± SD, dotted line illustrating a level of no treatment control.

Figure 2

Barrier function restoration in human endothelial cells. (A) ECIS measurement of HUVECs pre-incubated with mAbs and challenged with human α-thrombin. Data shown are mean ± SD for 4 independent monolayers. (B) Ratio of area under the curve (AUC) of antibody-treated vs. no treatment control. Anti-PECAM-1 antibodies (Ab62 and Ab37 solo or paired) do not result in additional drop in endothelial resistance (^#p = 0.99, 0.75, and 0.90, respectively), whereas anti-VE-cadherin antibody, clone BV9, exaggerates both the initial drop in permeability induced by thrombin (**p < 0.0001), and (C) barrier restoration, as reflected in the slope of recovery (**p < 0.0001). Anti-PECAM-1 antibodies slightly delayed barrier recovery but, unlike BV9, were not significantly different from isotype control (^#p = 0.93, 0.99, and 0.90, respectively).

Figure 3

Extravascular albumin accumulation of radiolabeled albumin in lungs of naïve (A) and endotoxin challenged (B) mice. Samples of mAbs were injected intravenously just after injection of [¹²⁵I]-BSA. (A) Anti-PECAM-1 mAbs 390 and Mec13.3 both cause significant elevation of BSA leakage in comparison to isotype control, IgG2A, as does the mixture of CEPAL antibodies (*p = 0.02, 0.04, and 0.04 for Mec13, 390, and CEPAL, respectively). Collaborative binding does not cause any additional effect, however, as compared to solo antibodies (^#p = 0.76 and p = 0.95 vs Mec13 and 390, respectively). Anti-VE-Cadherin mAb, in contrast, caused significant vascular leak (**p < 0.0001, compared to IgG2A control). Data shown as mean ± SD. (B) Neither anti-PECAM-1 antibodies nor CEPAL mixture exacerbate vascular leakage in animals treated with intratracheal LPS (^#p = 0.87, 0.23, and 0.74, respectively), whereas anti-VE-Cadherin results in further compromise of endothelial barrier function (*p = 0.02 compared to LPS + IgG2A control). Pulmonary extravascular albumin index (Equation 1) is calculated as described in Materials and Methods, with data shown as mean ± SD.

Figure 4

Expression of pulmonary activation markers in lung tissues. (A) Quantitative RT-qPCR revealed no significant increase in endothelial CAM expression in mice treated with paired anti-PECAM-1 antibodies, as compared to isotype control (^#p = 0.99, 0.99, and 0.12 for VCAM-1, ICAM-1, and eSelectin, respectively). Anti-VE-Cadherin mAb, BV13, a positive control for antibody mediated barrier disruption, caused significant elevation of mRNA expression of all three CAMs (*p < 0.001 vs. isotype control). Graph shows mean ± SD for n = 3 animals per group. HKG – housekeeping gene. (B) Western blot showing lung homogenates (10 μg total protein/lane) stained for mouse VCAM-1 and α-actin. Quantification (VCAM/actin) using ImageJ software demonstrates an increase in VCAM-1 protein expression induced by LPS (**p < 0.001) and BV13 (*p < 0.01), but for CEPAL antibodies (^#p = 0.28 vs. isotype control). Data presented as mean ± SD for n = 3 animals per group. Full-length western blot is presented in Supplemental Figure S3.

Figure 5

Systemic cytokine release in vivo. Cytokine concentrations from plasma samples of mice treated with mAbs were determined with the LEGENDplex Mouse Inflammation Panel (Biolegend). CEPAL mAbs cause significant increase in only two cytokines, GM-CSF and IFN-β (*p < 0.05, compared to isotype control). Results shown as mean ± SD. Complete dataset presented in Supplemental Table S1.