Immortalized Human Pulmonary Microvascular Endothelial Cells

Endothelial Inflammation as Assessed by ICAM-1, NLRP3, and P-Selectin in the In Vitro HPMVEC COVID-19 Model

Endothelial activation in COVID-19 induces inflammation, however, the mechanism is not well understood. Paul et al. investigated how COVID-19 leads to pulmonary endothelial activation and subsequent pro-inflammatory states. In order to recapitulate systemic inflammation in vitro, human pulmonary microvascular endothelial cells (HPMVEC) were treated with serum from severe COVID-19 patients.

Immune cell adhesion to the vascular wall is driven by inflammation in endothelial cells (EC). To determine the presence of a pro-inflammatory phenotype in HPMVEC, levels of ICAM-1, NLRP3, and P-selectin were quantified in an in vitro model of COVID-19. Cells treated with COVID-19 patient serum had a more robust inflammatory phenotype than those treated with normal human serum. HPMVECs treated with normal serum had low ICAM-1 and NLRP3 expression (green fluorescence) in comparison to COVID-19 serum treated cells (Fig. 1A and C). The fluorescence intensity was quantified using ImageJ, and the total integrated intensity was normalized to the cell area. Figure 1B and 1D present the quantified fluorescence data, with both ICAM-1 and NLRP3 significantly upregulated in the COVID-19 samples. P-selectin, which is upregulated and mediates neutrophil and monocyte binding, was also higher in the COVID-19 group, but not significantly (Fig 1E).

Fig. 1. Expression of inflammatory moieties in an in vitro HPMVEC COVID-19 model (Paul O, Alolia I K, et al., 2024).

Nicotine-free E-cigarette Aerosol Triggers Oxidative Stress and ICAM-1 Upregulation in Pulmonary Endothelium

Fine and ultrafine particles in e-cig vapor may induce oxidative stress and endothelial dysfunction, as has been shown for environmental pollutants. Chatterjee et al. determined the acute effects of e-cigarette aerosol inhalation (nicotine-free) on oxidative stress, inflammation, and endothelial activation in healthy nonsmokers by measuring serum biomarkers (CRP, sICAM-1, NOx) and endothelial cell responses (ROS, ICAM-1).

Serum-induced endothelial oxidative stress and inflammation were quantified by assessing ICAM-1 expression (Fig. 2) and ROS production (Fig. 3) in human pulmonary microvascular endothelial cells (HPMVEC) treated with pre- and post-e-cigarette exposure serum. Pre-exposure serum treatment resulted in minimal induction of ICAM-1 expression (barely detectable fluorescence; Fig. 2A), whereas serum collected 120 minutes after exposure caused a significant increase in ICAM-1 (Fig. 2A and B). Images taken at high magnification revealed perinuclear ICAM-1 staining (Fig. 2A, inset). Individual responses varied (subjects S7/S9 had a stronger response); however, ICAM-1 levels returned to baseline by 6 hours after exposure (Fig. 2B). The ICAM-1 response to 120-minute post-exposure serum was significantly elevated (P<0.001 vs baseline).

Fig. 2. Effect of serum on endothelial inflammation as monitored by intercellular adhesion molecule (Chatterjee S, Tao J Q, et al., 2019).

Fig. 3. Endothelial activation potential as monitored by reactive oxygen species (ROS) production by human pulmonary microvascular endothelial cells (HPMVEC) upon treatment with subjects' serum post e-cigarette (e-cig) vaping (Chatterjee S, Tao J Q, et al., 2019).