MHEC5-T

Neprilysin Inhibitors Exert Pro-Arteriogenic Effects Through the Bradykinin Receptor Signaling Pathway

Li's team explored the pleiotropic action of an angiotensin receptor-neprilysin inhibitor (ARNi) in collateral-dependent myocardial perfusion in the rat coronary arteriogenesis model and conducted further analyses to explore the underlying mechanism. In the in vitro study, murine heart endothelial cells (MHEC5-T) were exposed to a neprilysin inhibitor (NEPi) alone or in combination with bradykinin receptor antagonists to determine the effects on cell proliferation.

The results showed that 0.01 μM ACEi significantly increased cell proliferation compared with control (Fig. 1A). In addition, 0.01 μM and 0.1 μM NEPi also significantly increased cell proliferation (Fig. 1B). On the other hand, 10 μM and 20 μM ARB significantly inhibited cell proliferation (Fig. 1C). ARNi (NEPi + ARB) also significantly inhibited cell proliferation in a dose-dependent manner (Fig. 1D). To further examine if the pro-arteriogenic effect of ACEis and ARNis were mediated by the bradykinin signaling pathway, additional tests were performed. ACEi alone promoted cell proliferation, while ACEi combined with BDKRB1i or BDKRB2i inhibited cell proliferation. Nevertheless, ACEi combined with both BDKRB1i and BDKRB2i increased cell proliferation (Fig. 1E). In a similar fashion, NEPi alone promoted cell proliferation, while NEPi combined with BDKRB2i or both BDKRB1i and BDKRB2i inhibited cell proliferation (Fig. 1F).

Platelet Panx1 is Important for the Activation of Endothelial Cells

Abdominal aortic aneurysm (AAA) is a common and potentially lethal disease characterized by progressive aortic dilatation and chronic inflammation. Pannexins, particularly Panx1, play a crucial role in inflammation and remodeling. Metz et al. investigated the contribution of platelet Panx1 in AAA progression, given the role of platelets in cardiovascular diseases.

To see if platelet Panx1 affects endothelial cell activation, we tested the activation of MHEC5-T cells (an endothelial cell line) by platelets in vitro. Platelets were activated with CRP or thrombin, and the supernatant was collected. MHEC5-T cells were then incubated with platelet releasates from Panx1-deficient and control mice for 3 hours. They analyzed the gene expression of P-selectin and E-selectin in these cells (Fig. 2A, B). P-selectin expression in MHEC5-T cells changed only with resting, not activated, Panx1-deficient platelets (Fig. 2A). However, E-selectin gene expression was much lower when MHEC5-T cells were incubated with CRP-activated Panx1-deficient platelet supernatant compared to controls (Fig. 2B). This suggests that platelet Panx1 is important for endothelial cell activation, as shown by the expression of adhesion molecules like P- and E-selectin (Fig. 2A, B). They also checked plasma levels of soluble P-selectin and E-selectin in PPE mice using platelet-specific Panx1-deficient mice and controls. No differences were found 14 days after PPE infusion in mouse aorta between groups (Fig. 2C, D).