Neflamapimod stimulates vasodilation in resistance mesenteric arteries
To examine if acute neflamapimod treatment could regulate arterial contractility, we performed pressurized arterial myography on resistance 3rd to 5th order mesenteric arteries from male Sprague Dawley rats. Cannulated arterial segments were maintained in a temperature-controlled perfusion chamber (Living Systems), continuously perfused with 37°C PSS, gassed with a mixture of 21% O2, 5% CO2, and 74% N2. Intravascular pressure was gradually increased to 80mmHg to stimulate the development of myogenic tone. At 80mmHg, mesenteric arteries developed ~24% myogenic tone, after which a cumulative concentration response (0.0001–10µM) to neflamapimod was performed and diameter changes recorded. Our myography data shows that, within 2-3 minutes of application, neflamapimod produces a fully reversible and concentration-dependent vasodilation (Fig. 1a-d). The calculated EC50 was found to be ~0.2 µM (Fig. 1c). Of note, the Cmax reported in a preclinical study with rats is ~2 µM [11]. Neflamapimod at 10µM concentration potently relaxed mesenteric arteries by ~41µm, or by ~17% of arterial diameter from a baseline diameter at 80 mmHg (Fig. 1d). Considering the reported Cmax in a previous study, and for a better diameter read out in the presence of vasomotion, we used 10µM concentration for subsequent mechanistic studies. Altogether, our data for the first time demonstrate that acute neflamapimod application induces vasodilation in resistance-size mesenteric arteries.
Neflamapimod inhibits p38 MAPKα phosphorylation in mesenteric arteries
As neflamapimod is a selective inhibitor of p38 MAPKα, we asked if neflamapimod could inhibit p38 MAPKα phosphorylation and its kinase activity, in mesenteric arteries to stimulate vasodilation. To do so, we incubated mesenteric arteries for 30 minutes with either neflamapimod or SB203580, a non-selective inhibitor of p38 MAPK. Our Western blotting data reveals that mesenteric arteries have a basal p38 MAPKα phosphorylation, as detected by phospho-p38 MAPKα antibody (Fig. 2a, vehicle control, lane 1). Importantly, neflamapimod and SB203580 reduced p38 MAPKα phosphorylation to 33.74±0.3.7% and 32.25±0.1.8% of vehicle-treated control, respectively (Fig. 2a, 2b). This data suggests that both drugs are equally effective in reducing basal p38 MAPKα phosphorylation at the concentrations used. Overall, this data demonstrates that mesenteric arteries have baseline p38 MAPKα phosphorylation, and treatment with 10µM neflamapimod potently suppresses p38 MAPKα phosphorylation and, therefore, its activity in mesenteric arteries.
Neflamapimod reduces phosphorylation of actin-associated protein Hsp27
A critical downstream target of p38 MAPK is Hsp27 which, when phosphorylated, promotes actin filament formation and smooth muscle contraction. Earlier studies reported that reduction of Hsp27 phosphorylation by non-selective p38 MAPK inhibition with SB203580 stimulates vasodilation in mesenteric arteries[7, 23, 24]. This led us to test the hypothesis that neflamapimod-mediated selective inhibition of p38 MAPKα may also decrease Hsp27 phosphorylation to elicit vasodilation. To test this, we processed mesenteric arteries that had been treated with either neflamapimod or SB203580 for Western blotting. Our data revealed that neflamapimod-treated arteries had mean 37.75±1.19% phosphorylation of Hsp27, or 62.25% less compared to that in control arteries (Fig. 3a, 3b). Likewise, non-selective p38 MAPK inhibitor SB203580 also reduced arterial Hsp27 phosphorylation to 31.57±3.47% or by 69%. Combined application of neflamapimod and SB203580 reduced Hsp 27 phosphorylation to 29.5%±5.66%, or by 71% (Fig. 3a, 3b), which is similar to the inhibition produced by either drugs alone. These data suggest that neflamapimod-induced selective inhibition of p38 MAPKα reduces the phosphorylation of its downstream target Hsp27, an actin-associated protein that takes part in cytoskeletal reorganization and smooth muscle contraction.
We next asked if the inhibition of basal Hsp27 phosphorylation by selective p38 MAPKα inhibitor neflamapimod and non-selective p38 MAPK inhibitor SB203580 correlate with their vasodilatory actions in mesenteric arteries. Our pressure myography data showed that, like neflamapimod, SB203580 application relaxed mesenteric arteries by ~44 µm, which is slightly higher than that produced by neflamapimod (Fig 3c, 3d). When neflamapimod and SB203580 were applied together, they produced a mean arterial dilation of ~50 µm. Importantly, these data correlates with the magnitude of reduction of Hsp27 phosphorylation in mesenteric arteries. Overall, our data demonstrate that neflamapimod selectively inhibits p38 MAPKα and downstream Hsp27 phosphorylation to induce vasodilation in resistance mesenteric arteries.
Neflamapimod-induced vasodilation does not depend on endothelial signaling
Endothelium is an important regulator of arterial contractility. Endothelial cells induce vasodilation primarily through the production of vasodilators and their paracrine action on underlying smooth muscle cells. Therefore, we next assessed the role of endothelial signaling in neflamapimod-elicited vasodilation. To determine the role of endothelium, neflamapimod-induced vasodilatory responses in endothelium-intact and -denuded mesenteric arteries were compared. To this end, we performed endothelium denudation by slowing passing air bubbles through the vessel lumen and confirming the absence of 1µM acetylcholine (ACh)-mediated dilation as described previously (Fig 4a)[25-28]. Both endothelium-intact and -denuded vessels were preconstricted with 1µM phenylephrine (PE). Application of 1µM ACh fully reversed PE constriction in endothelium-intact arteries. In contrast, endothelium-denuded vessel showed only 5% reversal, which is 95% less than that shown by endothelium-denuded arteries (Fig 4a, 4b). However, application of 1µM sodium nitroprusside (SNP), a nitric oxide (NO) donor, fully reversed PE constriction in both endothelium-intact and -denuded vessels, suggesting that endothelium denudation does not affect smooth muscle responses to NO (Fig 4b). 60K-induced vasoconstriction was also similar between endothelium intact and denuded arteries (Fig 4c). Since ACh-induced vasodilation is primarily dependent on endothelial NO production, selective loss of ACh-evoked vasodilation demonstrates successful endothelium denudation. After validating endothelium denudation, we recorded neflamapimod-evoked vasorelaxation in both endothelium-intact and -denuded mesenteric arteries. Our myography data on endothelium-intact mesenteric arteries reveal that application of 10µM neflamapimod dilated mesenteric arteries by ~43 µm (Fig. 4d, 4e). On the other hand, neflamapimod application on endothelium-denuded mesenteric arteries produced a dilation of ~38 µm, which is ~89% or similar, compared to endothelium-intact mesenteric arteries (Fig 4d, 4e). This data suggests that endothelium denudation does not affect neflamapimod-evoked vasodilation.
Consistently, pharmacological inhibition of endothelial nitric oxide synthase (eNOS) with L-NNA and the production of the most important physiological vasodilator molecule NO do not reduce neflamapimod-evoked vasodilation (Fig. 5a, 5b), precluding the role of NO in this process. Likewise, neflamapimod-evoked vasodilation remained unchanged by the application of indomethacin which inhibits cyclooxygenase (COX) and synthesis of PGI2, another important endothelium-derived vasodilator (Fig. 5a, 5b). These data suggest that endothelial signaling has no role in acute neflamapimod-evoked vasodilation.
Neflamapimod-induced vasodilation is not mediated by smooth muscle cell K+ channels
Opening of arterial smooth muscle cell K+ channels and K+ efflux induce smooth muscle cell hyperpolarization, leading to vasodilation. Arterial smooth muscle cells express a variety of K+ channels including voltage-gated K+ channels (KV), large-conductance Ca2+-activated K+ channels (BKCa), and ATP-sensitive K+ channels (KATP). We therefore assessed the role of the major K+ channels in neflamapimod-induced mesenteric artery vasodilation. Application of 4-aminopyridine (4AP), an inhibitor of KV channels, produced a mean dilation of ~37 µm, which is 90% of that produced by neflamapimod (Fig. 6a, 6b). Similarly, application paxillin, a BKCa channel blocker, and glibenclamide, a blocker of KATP channels, both relaxed mesenteric arteries by ~35 µm (Fig. 6a, 6b). Application of TEA, a general K+ channel blocker, dilated mesenteric arteries by ~36 µm, which is ~88% of that produced by neflamapimod (Fig. 6a, 6b). Overall, these data suggest that smooth muscle cell K+ channels do not mediate neflamapimod-induced vasodilation in resistance mesenteric arteries.