ITGB4 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The Lentivirus ELISA kit was purchased from ZeptoMetrix (Buffalo, NY, United States). A protease and phosphatase inhibitor cocktail was purchased from Calbiochem (San Diego, CA, United States), and a bicinchoninic acid protein assay kit was purchased from Pierce (Rockford, IL, United States). ECM Gel from Engelbreth-Holm-Swarm murine sarcoma (Matrigel) was obtained from MilliporeSigma (Burlington, VT, United States). Unless otherwise stated, all other antibodies and reagents were purchased from Cell Signaling Technology (Beverly, MA, United States).

Human pulmonary artery ECs from Lonza (Walkersville, MD, United States) were cultured in an essential growth medium (EGM-2) containing 10% of fetal bovine serum (Lonza). Cells were placed in an incubator at 37°C, 5% of CO₂, and 95% of humidity to achieve contact-inhibited monolayers.

The ITGB4E variant DNA sequence was subcloned into a pLV-eGFP lentiviral mammalian expression vector (VectorBuilder, Chicago, IL, United States) (see supplementary material). Control vector (pLV-eGFP) or pLV carrying ITGB4E was then co-transfected into 293NT cells with packaging plasmid psPAX2, a gift from Didier Trono (Addgene plasmid #12260;¹ RRID:Addgene_12260) and envelope plasmid pMD2.g, a gift from Didier Trono (Addgene plasmid #12259;² RRID:Addgene_12259).

Briefly, 12 μg of psPAX2 plasmid, 5 μg of pMD2.G plasmid, and 15 μg of ITGB4E or vector plasmid DNA were added to 80 μl of Lipofectamine 2000 in 3.5 ml of Opti-MEM for 10 min at room temperature and then applied to a 15-cm plate of 293NT cells. The medium (Opti-MEM) was changed on the following day. On day 3, the medium was collected and centrifuged at 26,000 rpm for 90 min. The pellets were resuspended in phosphate-buffered saline (PBS), and the virus concentration was measured using an HIV-1 p24 antigen ELISA kit (ZeptoMetrix, NY, United States). The virus was then incubated with polybrene transfection reagent (TR-1003-G, MilliporeSigma, St. Louis, MO, United States) for 10 min at room temperature and then applied to human pulmonary artery ECs in media. The expression efficiency was determined by green fluorescent protein (GFP) detectable using fluorescent microscopy, and GFP-positive cells were then sorted using flow cytometry (Moflo Astrios Flow Cytometry Cell Sorter, Beckman Coulter, Indianapolis, IN, United States). Finally, purified cells were cultured in EGM in the presence of 1 μg/ml of puromycin for 2 days after which cells were used for experiments as described.

Total proteins were extracted using NP-40 lysis buffer (50 mM of Tris–HCl, pH 7.4, 150 mM of NaCl, 1% of NP-40, and 5 mM of ethylenediaminetetraacetic acid) supplemented with 40 mM of sodium fluoride, 0.1 M of sodium orthovanadate, 0.2 mM of phenylmethylsulfonyl fluoride, 10 mM of N′ ethylmalemide, and protease and phosphatase inhibitor cocktail (Calbiochem). Lung homogenates and cell lysates were briefly sonicated and were subjected to cycles of thawing and freezing on dry ice. The protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce). Western blotting was performed using standard protocols, and band densities were determined using ImageJ software (National Institutes of Health³).

Angiogenesis was assessed as we have described previously (Linz-Mcgillem et al., 2004). Briefly, 1 × 10⁵ ECs were seeded into 12-well plates containing Matrigel in EGM-2. On day 1 after seeding, representative images were obtained using fluorescence microscopy (Olympus BX 51/IX70, Olympus, Japan). Total segment lengths relative to the number of anchorage joints were quantified using ImageJ software as described by others (Carpentier et al., 2020).

To assess migration, 2 × 10⁵ ECs were seeded into 6-well plates in EGM-2 that were changed on day 2. Cells were then scratched using a 200 μl pipette tip, washed with PBS, and then placed in EGM-2. Images were obtained using fluorescence microscopy (Olympus BX 51/IX70, Olympus, Japan). For quantification of gap closure, the gap area after the scratch was measured at the time of scratch and again on day 1 using ImageJ software as described by others (Suarez-Arnedo et al., 2020).

For the LPS challenge, ECs were cultured in EGM-2 and challenged with 100 ng/ml of LPS for various time points as indicated. The resulting media were collected and briefly centrifuged using ELISA assay (Biolegend, San Diego, CA, United States) according to the manufacturer’s instructions. For CS experiments, ECs were plated onto six-well silicone elastomer Bioflex plates coated with type I collagen (FlexCell International, Hillsborough, NC, United States) and grown to 75–80% prior to transfection as described. Mechanical stretch was performed via the Flexcell Strain Unit (FX-3000; FlexCell International) placed in a 5% of CO₂ incubator at 37°C and 95% of humidity. The device uses a controlled vacuum to induce CS with 18% of elongation at a frequency of 30 cycles per minute (0.5 Hz) for 4 h. The media were then collected and briefly centrifuged prior to the ELISA assay.

Endothelial cells were plated into polycarbonate wells containing evaporated gold microelectrodes to measure TER that evaluates real-time changes in cell morphology, attachment, and locomotion using an electric cell-substrate impedance system (ECIS) (Applied Biophysics, Troy, NY, United States) as previously described (Garcia et al., 2001). Cells were grown to confluence in EGM-2. EC monolayers were then treated with thrombin (1 U/ml) to induce barrier disruption. TER values from each microelectrode were pooled at discrete time points and plotted vs. time as the mean ± SEM.

The Shapiro–Wilk’s test for normality was used. Student’s t-test was used to compare the means of data from two experimental groups while significant differences (p < 0.05) among multiple group comparisons were confirmed by a one-way ANOVA followed by Tukey’s range test. Results are expressed as means ± SE.

In the initial experiments, human pulmonary artery ECs were treated with simvastatin (5 μM, 24 h), and lysates were collected for Western blotting for ITGA6 and ITGB4 (Figure 1). These experiments confirmed a significant increase in expression in both proteins induced by simvastatin with two distinct bands observed after probing for ITGB4. The 202 kDa immunoreactive band is consistent with full-length ITGB4, and the second band at ∼150 kDa is consistent with ITGB4E. Furthermore, immunoprecipitation of ITGA6 followed by Western blotting for ITGB4 confirmed increased heterodimer formation of both ITGB4 and ITGB4E with ITGA6 after simvastatin treatment. These experiments were repeated to compare cell-specific responses using human pulmonary artery ECs and human colon adenocarcinoma cells (HT-29). ITGB4 and ITGB4E were both significantly increased in simvastatin-treated ECs relative to simvastatin-treated HT-29 cells and untreated ECs.

To study the effects of ITGB4E on EC expression and function, an ITBG4 construct was subcloned into pLV plasmid and then co-transfected with sPAX2 and pMD2.G into 293NT cells. The medium was collected, and the virus was then purified and transfected into human pulmonary artery ECs. GFP-positive cells were sorted using flow cytometry and cultured in EMG2. Images obtained on day 4 after transfection were notable for aggregation of cells overexpressing ITGB4E compared with cells transfected with vector alone (Figure 2). In addition, 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain to assess cell morphology. Notably, these studies did not demonstrate changes consistent with significant apoptosis associated with ITGB4E overexpression.

Human pulmonary artery ECs were used for two separate assays to assess the effects of ITGB4 overexpression on angiogenesis and migration. To assess effects on angiogenesis, cells transfected with ITGB4E or control plasmids were grown in Matrigel, and images were obtained on day 1 after seeding (Figure 3A). Compared with control cells, characteristic changes associated with angiogenesis including cell elongation, tube formation, and branching were significantly diminished in cells overexpressing ITGB4E. To study the effects on stimulated migration, cells transfected with ITGB4E or control plasmids were grown to confluence in media prior to being scratched with a pipette tip to create a gap in the monolayer. Images were then taken on day 0 and then day 1 to assess relative gap closure (Figure 3B). While the initial gap created on day 0 was similar to both cell types, gap closure was complete with control cells on day 1 while cells transfected with ITGB4E demonstrated cell aggregation and little evidence of migration to achieve gap closure.

Evidence of dysregulated EC angiogenesis and migration associated with overexpression of ITGB4E is consistent with a phenotypic change in these cells. To further characterize these changes, Western blots were performed using lysates from ITGB4E overexpressing and control cells probed for various EC marker proteins. These experiments confirmed nearly complete abrogation of the expression of VE-cadherin, PECAM-1, and VEGFR2 (Figure 4A). Additional experiments demonstrated significant increases in the expression of both E-cadherin and N-cadherin without altered expression of either vimentin or alpha-smooth muscle actin (α-SMA) (Figure 4B). As the ITGB4-mediated loss of EC markers in conjunction with increased N-cadherin is consistent with EMT, we further characterized potential EMT phenotypic changes. Western blots of ITGB4-overexpressing cells identified significant increases in the expression of Slug, Snail, Zeb-1, and Zeb-2 specific transcription factors known to mediate the spectrum of EMT (Figure 4C; Cano et al., 2000; Comijn et al., 2001; Bolos et al., 2003; Eger et al., 2005; Ma et al., 2021).

To investigate the effects of ITGB4E on EC signaling in response to inflammatory stimuli, human pulmonary artery ECs were transfected with ITGB4E or control vectors prior to treatment with LPS (100 ng/ml) for various time points (0–30 min). Lysates were then collected and subjected to Western blotting for specific MAPKs (Figure 5). These experiments confirmed a significant and consistent attenuation of LPS-induced Erk and JNK phosphorylation while no changes were appreciable with respect to total Erk and JNK. Of note, no changes were appreciable with respect to phosphorylated or total p38 MAPK. Collectively, these data are consistent with significant attenuation of EC inflammatory signaling by increased ITGB4E expression.

Next, in complementary experiments, human pulmonary artery ECs were transfected with ITGB4E or control vectors prior to being subject to distinct inflammatory stimuli, LPS (100 ng/ml, 4 h), or excessive mechanical stretch (18% CS, 4 h). Media were then collected and used for the measurement of inflammatory cytokines, IL-6 and IL-8, via ELISA (Figure 6). Increased ITGB4E expression was associated with a significant reduction of 75–80% in both IL-6 and IL-8 expressions in the media after either stimulus consistent with a decreased inflammatory response.

To determine the effect of ITGB4E overexpression on EC barrier function, human pulmonary artery ECs were transfected with ITBG4E or a control vector and then grown to confluence overlying gold-plated microelectrodes. Cells were then treated with thrombin (1 U/ml) to effect barrier disruption, and TER was measured (Figure 7). TER nadir and time to recovery after the treatment with thrombin (1 U/ml), a barrier-disrupting agent, were significantly decreased in cells overexpressing ITGB4E compared with controls. These changes are consistent with relative barrier protection associated with ITGB4E expression.