Proteomic Analysis of Control and Disrupted Endothelial Monolayers
Authors: Abdelmaboud Salma , Yuan Sarah Y. , Breslin Jerome W. 
Published: May 2022

The microvascular wall regulates normal blood‐tissue exchange, but under inflammatory conditions, elevated endothelial permeability leads to edema, poor tissue oxygenation, and disrupted tissue homeostasis. Much remains unknown about the mechanisms involved. Using an unbiased proteomic analysis of confluent human umbilical vein endothelial cell (HUVEC) monolayers that were stimulated with 0.1 U/mL thrombin, which produces a time‐dependent increase in permeability that peaks 30 min and recovers to baseline levels at 120 min, novel candidate proteins that may participate in this mechanism were identified. Cell lysates were collected, proteins digested, and the peptides were characterized using a Thermo Q‐exactive‐HF mass spectrometer coupled to a Thermo Easy nLC 1200 to get label‐free quantitation (LFQ) of relative protein expression. High‐resolution mass spectrometric data were analyzed on the MaxQuant processing suite, and the protein groups file subsequently analyzed with the Perseus processing suite. The analysis revealed identified 4627 unique proteins present in HUVEC. Ratios of the LFQ intensities of 30 min. thrombin vs. control and 120 min. thrombin vs. control were generated. Statistical significance (p < 0.05) for 30 min. thrombin vs. control or 120 min. thrombin vs. control was determined using Welch’s t‐test of log(2)‐transformed LFQ intensities. In addition, a z‐score cutoff calculated as [(t test difference of individual protein) − (median t test difference of dataset)] / (standard deviation of t test difference of dataset) was used. The criteria of |z‐score| > 1 and p < 0.05 with Welch’s t test were utilized to filter the dataset, identifying 214 proteins to have altered expression after 30 min thrombin treatment, and 172 proteins having altered expression after 120 min thrombin. When entering the filtered datasets into String‐db, tight junction signaling was identified as enriched with thrombin treatment. Employing a more stringent filtering approach to identify candidate proteins for further study (|z‐score| > 2), we found that the tight junction protein claudin‐11 (CLDN11) had significantly decreased expression after 30 min thrombin (73% decrease from control) but was not significant changed at 120 min. Expression of CLDN11 was confirmed to be decreased by thrombin at 30 min using SimpleWestern (WES) analysis. To determine whether experimental depletion of CLDN11 expression decreases endothelial barrier function, selective CLDN11 siRNA was used. However, evaluation of barrier function using electric cell‐substrate impedance sensing showed no significant changes for up to 48 h after transfection with the siRNA, when CLDN11 protein levels were significantly decreased. Immunofluorescence labeling of CLDN11 in confluent HUVEC revealed that while some CLDN11 could be found in junctions, most labeling was punctate in the cytoplasm, possibly residing in vesicles. Collectively, the results suggest that thrombin significantly alters endothelial tight junctions and reduces CLDN11 expression. However, selective reduction of CLDN11 levels alone does not appear to be sufficient to alter HUVEC monolayer barrier function, and this may be because most CLDN11 fails to localize in junctions in this cultured endothelial cell model. Future studies will be directed at evaluating CLDN11 localization in postcapillary venules and determining its potential role in microvascular barrier function using intact microvessels and in vivo.