GHRH regulates the secretion of GH from the anterior pituitary gland, previously associated with cancer progression and inflammation. An emerging body of evidence suggests that GHRHAnt support endothelial barrier function, but the mechanisms mediating these events are not completely understood. In the present study, it is demonstrated that the GHRHAnt JV-1-36 counteracts barrier dysfunction due to LPS or LTA treatment in HUVECs, utilizing the Dextran–FITC assay. Moreover, it is shown in BPAECs that these bacterial toxins increase ROS generation, and that this effect is counteracted by JV-1-36, which reinstates the redox balance. The possible involvement of NEK2 in the beneficial activities of GHRHAnt in IFN-γ- and LPS-triggered hyperpermeability was also assessed, since that kinase is involved in inflammatory responses. NEK2 was increased in the inflamed cells, and JV-1-36 counteracted those endothelial events. Our data support the beneficial effects of GHRHAnt in toxin-induced endothelial injury.
Endothelial barrier dysfunction has been associated with a diverse variety of potentially lethal disorders, including direct and indirect lung injury, acute respiratory syndrome, and sepsis. Targeted efficient countermeasures for the aforementioned disorders are urgently needed, as indicated by the devastating outcomes of COVID-19. The delineation of the cellular pathways mediating endothelial homeostasis will most probably lead to the development of novel pharmacotherapies to support the inflamed endothelium and respiratory function.
In the present study, endothelial cells were treated with LPS, LTA, and IFN-γ. LPS exists in Gram-negative bacteria, whereas LTA is the endotoxin of Gram-positive bacteria and induces proinflammatory responses. ΙFN-γ is a downstream mediator of both bacterial toxins. Both human (HUVECs) and bovine endothelial cell lines (BPAECs) were cultured to study barrier function. Dextran–FITC was used to measure the paracellular permeability in the context of GHRHAnt in toxin-induced barrier dysfunction.
GHRHAnt are peptides designed to alleviate the inflammatory and tumor-promoting effects of the GH/GHRH axis in human and animal tissues and act—at least in part—by binding to the growth hormone-releasing hormone receptor and its splice variants. Those receptors are expressed in various tissues and endothelial cells, including BPAECs. GHRHAnt were initially synthesized to suppress tumors in experimental models of malignancies. Later on, it was recognized that they can exert beneficial effects in inflammatory lung disease, based on their ability to suppress ERK1/2 and JAk2/STAT3; and augment the wild-type P53. This tumor suppressor enhances barrier function and protects against inflammation. P53 deficient mice are more susceptible to LPS compared to animals expressing normal P53 levels, and super-P53 mice were resilient to LPS-induced lung injury. P53 interrelates with NEK2, since it serves as an NEK2 substrate.
NEK2 is a kinase involved in cytoskeletal remodeling, cell motility, and cell cycle regulation, participating in microtubule formation, stabilization, and centrosome regulation. Mice subjected to cecal and ligation-puncture-induced sepsis presented with elevated NEK2 expression levels in their lungs, and studies on experimental cancers suggest that this kinase is involved in cancer progression and drug resistance. Indeed, NEK2 inhibition was suggested to be a potential anticancer strategy.
In the present study, we investigated the possibility that GHRHAnt protect against toxin-induced endothelial barrier dysfunction in the context of paracellular permeability and ROS generation. Our observations suggest that GHRHAnt ameliorate endothelial leak and oxidative stress due to bacterial toxin treatment. Furthermore, NEK2 was induced in the inflamed cells, and GHRHAnt counteracted those effects. Since NEK2 was previously linked to inflammatory responses, we introduce the possibility that this kinase is involved in the GHRHAnt-related beneficial effects in the endothelium.
GHRHAnt JV-1-36 (031-23) is available from Phoenix Pharmaceuticals (Burlingame, CA, USA). Anti-mouse IgG HRP secondary antibody (95017-554), nitrocellulose membranes (10063-173) (BT142015-5G), IFN-γ (103014-494), and RIPA solution (AAJ63306-AP) were purchased from VWR (Radnor, PA, USA). Protease Inhibitor (AB287909) was purchased from Abcam (Cambridge, UK). NEK 2 (D-8) (sc-55601) antibody is available from Santa Cruz Biotechnology (Dallas, TX, USA). β-actin antibody (A6441), Corning trans-well cell culture inserts (CLS3470), LPS (L4130), and FITC–Dextran (46945) were acquired from Sigma-Aldrich (St. Louis, MO, USA).
Bovine pulmonary artery endothelial cells (BPAECs) are available from Genlantis (San Diego, CA, USA). The cells were maintained in DMEM (VWRL0101–0500), which included 10% fetal bovine serum, available from VWR (Radnor, PA, USA). Human umbilical vein endothelial cells (HUVECs) were subcultured in media specific to endothelial cells (PCS-100–030), and a special kit was purchased (PCS-100–040) to supplement it. The material is available from ATCC (Manassas, VA, USA). Moreover, to prevent infections/contaminations, we added a solution of 1 × penicillin/streptomycin to the cell media, available from VWR (Radnor, PA, USA). In all cases, the cells grew at 37 °C in a tightly adjusted humified environment of 5% CO 2–95% air.
First, 40 µg of protein was separated onto sodium dodecyl sulfate (SDS-PAGE) Tris-HCl gels. The separated proteins were transferred to nitrocellulose membranes, which were incubated for 60 min to a solution of 5% milk, to block non-specific binding sites. The membranes were exposed to the appropriate primary antibodies at a concentration of 1:1000 in a cold room for 16 h. Then, secondary antibodies (1:5000) were used to detect the corresponding primary antibodies, and a special chemiluminescent substrate was used (SuperSignal West Femto (PI34096)) to generate light, captured in ChemiDoc System from Bio-Rad (Hercules, CA, USA). VWR (Radnor, PA, USA) was the company which provided the material described in this section.
Paracellular permeability was estimated utilizing 24-well trans-well dishes. First, 200,000 endothelial cells were contained in each well, which were incubated for 20 min with FITC–Dextran (70 kDa, 1 mg/mL). Then, 100 mL of media was removed from each receiver well to assess fluorescence, measured with Synergy H1 Hybrid Multi-Mode Reader from Biotek (Winooski, Vermont). For this, 485 nm and 535 nm were the excitation and emission wavelengths, respectively.
DCFDA (25 μΜ) was used to measure ROS. The endothelial cells were incubated for 45 min with that compound, and the fluorescence intensity was captured by using the fluorescence plate reader described previously.
Densitometry was carried out by using Image J software (National Institute of Health), and the data are described as Means ± SEM (standard error of the Mean). Student’s t-test was utilized to evaluate statistically significant differences, whereas p< 0.05 was considered significant. To analyze data and draw figures, we used GraphPad Prism (version 5.01). In all cases, the letter n is used to indicate repeats.
Endothelial paracellular permeability was assessed to measure the barrier function. The HUVEC monolayers were treated with a vehicle (0.1% DMSO) or GHRHAnt (1 µM) for 8 h. Then, the cells were exposed to the vehicle (PBS) or LPS (10 μg/mL) and the vehicle (PBS) or LPS (10 μg/mL) for 4 h to assess their barrier function utilizing FITC–dextran (70 kDa, 1 mg/mL). GHRHAnt enhanced the endothelial barrier function and reduced the endothelial permeability.
BPAECs were pre-treated with GHRHAnt (1 μM) or a vehicle (0.1% DMSO) for 8 h and were subsequently exposed to LPS (10 μg/mL) or the vehicle (PBS) and LTA (10 μg/mL) or the vehicle (PBS) (4 h). The results reveal that both LPS and LTA increased the ROS levels in the BPAECs, and that this effect was counteracted by GHRHAnt. Furthermore, GHRHAnt reduced the basal endothelial ROS generation.
Endothelial cells were exposed to GHRHAnt (1 μM) (8 h) or a vehicle and were post-treated with LPS (10 μg/mL) (4 h) or IFN-γ (4 μg/mL) (24 h). NEK2 was induced due to the toxin (LPS, IFN-γ) treatments. On the other hand, GHRHAnt suppressed toxin-induced barrier dysfunction.
The endothelial cells form monolayers, which are tightly regulated. Increased ROS generation has been associated with numerous diseases, affecting the cardiovascular and neurological systems. Gram-negative bacteria can cause acute inflammatory responses by releasing inflammatory factors and ROS. LPS is the key component of Gram-negative bacteria, and it is recognized as a potent activator of monocytes/macrophages. Moreover, this toxin activates cells in the immune system (e.g., macrophages, neutrophils), which further induce the synthesis of proinflammatory cytokines (e.g., IL-1β, TNF-α). LTA is a toxin of Gram-positive bacteria.
