(M1030-04-27) Neflamapimod Inhibits Endothelial Cell Activation and Leukocyte Adhesion by Inhibiting p38 MAPKα and NF-κB Signaling

Purpose: Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), is undergoing clinical trials for the treatment of inflammatory and neurodegenerative diseases. Based on the reported anti-inflammatory actions, here we examined if Neflamapimod could inhibit lipopolysaccharide (LPS)-mediated activation of endothelial cells (ECs), induction of adhesion molecules and subsequent attachment of leukocytes to EC monolayer – events that lead to vascular inflammation and cardiovascular dysfunction.

Methods: To study the role of neflamapimod on endothelium activation, mouse mesenteric arterial endothelial cells (MECs) were pretreated with vehicle or increasing concentrations (1 nM to 1 μM) of neflamapimod followed by induction with 10 μg bacterial endotoxin lipopolysaccharide (LPS) for 16 hours. At the end of treatment, cells were homogenized in RIPA buffer, supernatants collected and analyzed using ELISA and Western blotting. Immunofluorescence was carried out on rat thoracic aortas by opening the luminal side and subjecting them to the same treatment protocol as the cultured endothelial cell monolayer. Leukocyte attachment assay to the endothelial cell monolayer was performed in accordance with the manufacturer’s instructions (Cell Biolabs). Student t-test was used for comparing paired and unpaired data from two populations and ANOVA with Student-Newman-Keuls post hoc test for multiple group comparisons. p< 0.05 is considered significant.

Results: ELISA data demonstrates that 10 μg LPS treatment causes maximum induction of adhesion molecule expression in MECs, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin by approximately 409% and 384% 210% respectively. Western blotting data also demonstrates that the concentration of 10 μg/ml of LPS robustly increases the abundance of ICAM-1, VCAM-1, and E-selectin proteins by ~292%, 318%, and 210%, respectively, compared to untreated control conditions. Our results reveal that while Neflamapimod, in the range of 10-100 nM, significantly reduces LPS-stimulated overexpression of adhesion molecules, the maximum suppression (~50%) of their expression is observed with 10 nM concentration. Western blotting data demonstrates that there is a low basal phosphorylation of p38 MAPKα in MECs, which is elevated to ~236% following overnight LPS treatment. Importantly, co-treatment of MECs with 1, 10, and 100 nM Neflamapimod significantly reduces the LPS-induced phosphorylation of p38 MAPKα to ~179%, 125%, and 144%. Western blotting data further shows that LPS treatment increases NF-κB p65 subunit protein expression to 222%, while Neflamapimod reduces the expression of p65, albeit, in a concentration-dependent manner. To assess the outcome of NF-κB activation post nuclear translocation, we also performed a NF-κB reporter assay and results indicate that LPS treatment produces strong activation of NF-κB, as evidenced by a 199% increase in relative luminescence (RLU), which corresponds to the presence of higher level of produced luciferase reporter. However, treatment with Neflamapimod reduces luciferase RLU in a dose-dependent manner. Leukocyte adhesion experiments show that LPS-treated wells have a robust increase in leukocyte attachment to 660%, as evidenced by the presence of green puncta. In contrast, the percentage of attached leukocytes in 1, 10, and 100 nM Neflamapimod-treated wells are 316%, 178%, and 173%, respectively. Likewise, our data demonstrates that the relative fluorescence for LPS-treated cells is 630% compared to the untreated control condition, while Neflamapimod in the range of 1-100 nM significantly reduced leukocyte fluorescence, suggesting that it effectively inhibits leukocyte-MEC adhesion.

Conclusion: Our study demonstrates that Neflamapimod is a potent inhibitor of p38 MAPKα and NF-κB pathways – two key regulators of inflammation. Overall, our data demonstrates that Neflamapimod can inhibit endothelium activation and leukocyte attachment, which could prevent vascular inflammation.

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Acknowledgements: Funding information: This work was supported by a start-up grant from the Mercer University College of Pharmacy and a SEED grant from the Mercer University Provost’s Office to Dr. Raquibul Hasan. Declaration of competing of interests: None

Fig. 1: LPS treatment stimulates the over expression of adhesion molecules in mouse mesenteric artery endothelial cells (MECs). (A) Western blot images illustrating induction of ICAM-1, VCAM-1 and E-selection proteins by LPS in MECs. (B) Mean data for ICAM-1, VCAM-1 and E-selection protein expression. n=4

Fig. 2: (A) Neflamapimod inhibits LPS-stimulated induction of ICAM-1 on MECs. Representative Western blot images showing ICAM-1 induction by LPS and inhibition of ICAM-1 expression by 1, 10 and 100 nM Neflamapimod treatment in MECs. Mean data for ICAM-1 protein expression. n=4. *p < 0.05 vs Control; #p < 0.05 vs LPS-treated group.
(B) Neflamapimod suppresses VCAM-1 overexpression by LPS. Representative Western blot images for VCAM-1 induction by LPS and its modulation by Neflamapimod treatment. Mean data for VCAM-1 expression. n=4. *p < 0.05 vs Control; #p < 0.05 vs LPS.
(C) Neflamapimod reduces LPS-mediated phosphorylation of p38 MAPKα in MECs. Western blot images showing the modulation of p38 MAPKα phosphorylation by LPS and the effect of Neflamapimod treatment. While Neflamapimod in the 1-100 nM range significantly reduces the phosphorylation of p38 MAPKα, 10 nM concentration appears to show the highest suppression. Mean data for phosphorylated p38 MAPKα relative to total p38 MAPKα. n=4. *p < 0.05 vs Control; #p < 0.05 vs LPS.
(D) Neflamapimod reduces LPS-evoked activation of NF-κB signaling in endothelial cells. (A) Western blot images showing the modulation of NF-κB subunit p65 expression by LPS and its inhibition by Neflamapimod. (B) Mean data for p65 expression relative to GAPDH. n=4. *p < 0.05 vs Control; #p < 0.05 vs LPS.

Fig. 3: Neflamapimod inhibits leukocyte (THP-1 cells) adhesion onto MECs. (A) Immunofluorescence images comparing attached THP-1 cells (green) onto MECs in LPS- and Neflamapimod -treated groups with untreated controls. (B) Mean data for the percentage attached THP-1 cells onto MEC monolayer, n=4. (C) Mean RFU of solubilized cells, n=4. * p < 0.05 vs Control, # p < 0.05 vs LPS. Scale bar in the images = 50 µm.