Preeclampsia is a pregnancy-related disorder characterized by hypertension, vascular dysfunction and an increase in circulating inflammatory factors including the cytokine, tumor necrosis factor-α (TNF-α). Studies have shown that placental ischemia is associated with 1) increased circulating TNF-α, 2) attenuated pressure-induced cerebral vascular tone, and 3) suppression of β-epithelial Na+ channel (βENaC) protein in cerebral vessels. In addition to its role in epithelial Na+ and water transport, βENaC is an essential signaling element in transduction of pressure-induced (aka “myogenic”) constriction, a critical mechanism of blood flow autoregulation. While cytokines inhibit expression of certain ENaC proteins in epithelial tissue, it is unknown if the increased circulating TNF-α associated with placental ischemia mediates the loss of cerebrovascular βENaC and cerebral blood flow regulation. Therefore, the purpose of this study was to test the hypothesis that increasing plasma TNF-α in normal pregnant rats reduces cerebrovascular βENaC expression and impairs cerebral blood flow (CBF) regulation. In vivo TNF-α infusion (200 ng/day, 5 days) inhibited cerebrovascular expression of βENaC and impaired CBF regulation in pregnant rats. To determine the direct effects of TNF-α and underlying pathways mediating vascular smooth muscle cell βENaC reduction, we exposed cultured VSMCs (A10 cell line) to TNF-α (1–100 ng/mL) for 16–24 h. TNF-α reduced βENaC protein expression in a concentration-dependent fashion from 0.1 to 100 ng/mL, without affecting cell death. To assess the role of canonical MAPK signaling in this response, VSMCs were treated with p38MAPK or c-Jun kinase (JNK) inhibitors in the presence of TNF-α. We found that both p38MAPK and JNK blockade prevented TNF-α-mediated βENaC protein suppression. These data provide evidence that disorders associated with increased circulating TNF-α could lead to impaired cerebrovascular regulation, possibly due to reduced βENaC-mediated vascular function. NEW & NOTEWORTHY This manuscript identifies TNF-α as a possible placental-derived cytokine that could be involved in declining cerebrovascular health observed in preeclampsia. We found that infusion of TNF-α during pregnancy impaired cerebral blood flow control in rats at high arterial pressures. We further discovered that cerebrovascular β-epithelial sodium channel (βENaC) protein, a degenerin protein involved in mechanotransduction, was reduced by TNF-α in pregnant rats, indicating a potential link between impaired blood flow and this myogenic player. We next examined this effect in vitro using a rat vascular smooth muscle cell line. TNF-α reduced βENaC through canonical MAPK-signaling pathways and was not dependent on cell death. This study demonstrates the pejorative effects of TNF-α on cerebrovascular function during pregnancy and warrants future investigations to study the role of cytokines on vascular function during pregnancy.
Pressure-induced, but not agonist-induced, vasoconstriction is abolished in the middle cerebral artery (MCA) of TrpC6 null mice. TrpC6 localization in dissociated cerebral vascular smooth muscle cells is primarily cytoplasmic and not associated with the surface membrane where a mechanoelectrical coupler might be expected. These findings suggest that TrpC6 is required for transduction of pressure-induced constriction in the MCA; however, its role as a mechanoelectrical coupler or downstream signal amplifier remains unresolved.
Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. While Epithelial Na+ Channels (ENaC) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue resident macrophages in healthy humans using the Human Protein Atlas data base (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (~50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Further, we generated a cell line stably expressing an N-terminal truncated aENaC to interrupt normal channel trafficking, and found it suppressed migration. Prolonged exposure (48 hr) of RAW 264.7 cells to proinflammatory cytokines interferon gamma (IFNg) and/or tumor necrosis factor alpha (TNFa) inhibited RAW 264.7 migration and abolished the amiloride (1 µM) sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate aENaC protein expression, cells were exposed to proinflammatory cytokines IFNg (10 ng/mL, last 48 hr ) and TNFα (10 ng/mL, last 24 hr). By western blotting we found whole cell aENaC protein is reduced ≥ 50%. Immunofluorescence demonstrated heterogenous aENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.
Migration of monocytes‐macrophages play an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. While Epithelial Na+ Channels (ENaC) are required for normal migratory responses in glial, trophoblast, and vascular smooth muscle cells, their role in monocyte‐macrophage migration signaling is unknown. The Human Protein Atlas data base (http://www.proteinatlas.org/) shows ENaC message in peripheral blood mononuclear cells and tissue resident macrophages in human populations. We used an in vitro model to determine the importance of ENaC in chemotactic migration of monocytes‐macrophages using a standard Boyden chamber assay. Cells were suspended in 0.4% FBS ± amiloride (0.1‐10 µM) or benzamil (0.01‐1 µM) to inhibit ENaC channels, then migrated for 4 hr towards a 10% FBS gradient. In mouse RAW cells, migration responses were inhibited by amiloride (65±6 – 46±4% of control at 0.1 and 10 µM, p=0.0001) and benzamil (55±6 – 55±5% of control at 0.01 and 1.0 µM, p<0.0001). In human THP‐1 cells, migration responses were similarly inhibited by amiloride (73±5 ‐ 40±6% of control at 0.1 and 10 µM, p<0.0001) and benzamil (68±7 – 53±4% of control at 0.01 and 1.0 µM, p<0.0001). Since benzamil and amiloride are selective for ENaC at sub and low micromolar concentrations, respectively, our findings indicate ENaC signaling is required for migration of unstimulated monocyte‐macrophage cells. To determine if pro‐inflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interferon gamma (INFγ), regulate ENaC expression and function, we used TaqMan quantitative PCR, western blotting and chemotactic migration. Cells were exposed to a standard protocol to activate macrophages: INFγ for 48 hr plus TNFα (10 ng/mL each) during the last 24 hr. Expression of α and βENaC and GAPDH was calculated as fold change using the delta delta CT method (2‐∆∆CT). INFγ48hr + TNFα24hr increased α and βENaC message (9±2 and 5±1, n=4‐6, p=0.01). γENaC was undetectable. Despite increased message, whole cell αENaC protein expression was inhibited 50% by western blotting. The mechanism underlying message stimulation and protein inhibition remains to be determined. A similar effect on message upregulation and migration inhibition also occurred with 18 hr INFγ or TNFα alone (0‐10 ng/mL). Activation with INFγ/TNFα inhibited migration up to 70% and abolished the amiloride (1 µM) sensitive component. In unstimulated cells, amiloride (1 µM) coculture induced a rebound increase in migration following withdrawal, which was abolished by INFγ/TNFα treatment. This finding suggests cytokine induced ENaC loss mediates the migration inhibition. In summary, ENaC mediates monocyte migration, however, monocyte activation by INFγ/TNFα inhibits migration by inhibiting ENaC. Since pro‐inflammatory cytokines initiate monocyte differentiation and polarization to the phagocytic M1 phenotype, future studies will determine if ENaC is required for the phenotypic switch.
Degenerin proteins, such as βENaC and ASIC2, and Transient Receptor Potential Channel 6 (TrpC6) have been implicated in cardiovascular function. However, their roles and potential interaction in metabolic disease has not been studied. To begin to assess this interaction, we evaluated the impact of a high fat diet (HFD) on in mice lacking normal levels of ASIC2, βENaC and TrpC6. Twenty week old male and female mice were placed on a 60% HFD for 12 weeks. Body weight was measured weekly, body composition by non‐invasive ECHO MRI and fasting blood glucose were measured at 0, 4, 8 and 12 weeks. A glucose tolerance test was administered after 12 weeks. Differences between ASIC2/βENaC/TrpC6 and WT groups were compared using independent t‐tests within each sex. Data are presented as mean ± SEM, ASIC2/βENaC/TrpC6 vs. WT. At 20 weeks of age, female ASIC2/βENaC/TrpC6 mice (n=6) weighed less (22.7±1.0 vs 26.3±0.8g, p=0.029) and gained less weight (12.1±1.7 vs. 20.5±1.3g, p=0.004) than WT (n=5). Total body fat (16.2±2.0 vs 23.2±1.1g, p=0.017) and lean body masses (19.2±1.0 vs 24.8±0.7, p=0.0014) were reduced in female ASIC2/βENaC/TrpC6 mice. In contrast, male ASIC2/βENaC/TrpC6 (n=5) mice had similar body weight (34.1±0.8 vs. 36.8±1.8g, p=0.165) at 20 weeks and 12 week HFD weight gain (11.8±1.5 vs. 12.1±0.9g, p=0.881) compared to WT (n=4). Fasting blood glucoses were lower in female (166±6.4 vs.212±5.3 mg/dL, p=0.0004) and male (181±2.8 vs.210±9 mg/dL, p=0.006) ASIC2/βENaC/TrpC6 mice after 12 weeks HFD. The area under the curve for the glucose tolerance test was reduced in female (18263 ± 510 vs 34623±4719 min.mg/dL), but increased in male (26100±1639 vs. 19408±1364 min.mg/dL) ASIC2/βENaC/TrpC6 mice. Liver (0.83±0.05 vs. 1.93±0.34g, p=0.0066) and liver fat (0.011±0.007 vs.0.333±.158, p=0.05) masses, as well as percent liver fat (1.1±0.6 vs. 14.4±4.1%, p=0.006), were reduced in female ASIC2/βENaC/TrpC6 mice after HFD. While liver (1.77±0.13 vs. 2.49±0.15g, p=0.007) and liver fat (0.17±.06 vs. 0.37±.03g, p=0.044) masses were reduced in male ASIC2/βENaC/TrpC6 mice, percent liver fat was not statistically lower (8.8±2.6 vs. 14.7±0.7%, p=0.11). These highly novel findings suggest that ASIC2, βENaC and/or their interaction with TrpC6, protects against HFD induced‐metabolic disease in female, but not male, mice. The mechanisms underlying this response will be examined in future studies.Support or Funding InformationThis work was support by NIH P01HL051971, P20GM104357, P20GM121334 and R01HL136684.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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