Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.

Interleukin-6 Promotes Murine Estrogen Deficiency-Associated Cerebral Aneurysm Rupture.

BACKGROUND: Estrogen deficiency is associated with cerebral aneurysm rupture, but the precise mechanism is unknown. OBJECTIVE: To test the hypothesis that IL-6 is required for the increase in aneurysm rupture rate observed in estrogen-deficient mice. METHODS: We analyzed IL-6 expression in human cerebral aneurysms. We induced cerebral aneurysms in estrogen-deficient female C57BL/6 mice that had undergone 4-vinylcyclohexene diepoxide (VCD) treatment or bilateral ovariectomy (OVE). Mice were blindly randomized to selective IL-6 inhibition (IL-6 receptor [IL-6R] neutralizing antibody, n = 25) or control (isotype-matched IgG, n = 28). Murine cerebral arteries at the circle of Willis were assessed for aneurysm rupture and macrophage infiltration. RESULTS: IL-6 is expressed in human cerebral aneurysms, but not in control arteries. Serum IL-6 is elevated in ovariectomized female mice compared to sham control (14.3 ± 1.7 pg/mL vs 7.4 ± 1.5 pg/mL, P = .008). Selective IL-6R inhibition suppressed cerebral aneurysm rupture in estrogen-deficient mice compared with control (VCD: 31.6% vs 70.0%, P = .026; OVE: 28.6% vs 65.2%, P = .019). IL-6R inhibition had no effect on formation or rupture rate in wild-type mice. IL-6R neutralizing antibody significantly reduced macrophage infiltration at the circle of Willis (1.9 ± 0.2 vs 5.7 ± 0.6 cells/2500 µm2; n = 8 vs n = 15; P < .001). CONCLUSION: IL-6 is increased in the serum of estrogen-deficient mice and appears to play a role in promoting murine estrogen deficiency-associated cerebral aneurysm rupture via enhanced macrophage infiltration at the circle of Willis. Inhibition of IL-6 signaling via IL-6 receptor neutralizing antibody inhibits aneurysm rupture in estrogen-deficient mice. IL-6 receptor inhibition had no effect on aneurysm formation or rupture in wild-type animals.

Estrogen Deficiency Promotes Cerebral Aneurysm Rupture by Upregulation of Th17 Cells and Interleukin-17A Which Downregulates E-Cadherin.

BACKGROUND: Estrogen deficiency is associated with the development of cerebral aneurysms; however, the mechanism remains unknown. We explored the pathway of cerebral aneurysm development by investigating the potential link between estrogen deficiency and inflammatory factors. METHODS AND RESULTS: First, we established the role of interleukin-17 (IL-17)A. We performed a cytokine screen demonstrating that IL-17A is significantly expressed in mouse and human aneurysms (P=0.03). Likewise, IL-17A inhibition was shown to prevent aneurysm formation by 42% (P=0.02) and rupture by 34% (P<0.05). Second, we found that estrogen deficiency upregulates T helper 17 cells and IL-17A and promotes aneurysm rupture. Estrogen-deficient mice had more ruptures than control mice (47% versus 7%; P=0.04). Estradiol supplementation or IL-17A inhibition decreased the number of ruptures in estrogen-deficient mice (estradiol 6% versus 37%; P=0.04; IL-17A inhibition 18% versus 47%; P=0.018). Third, we found that IL-17A-blockade protects against aneurysm formation and rupture by increased E-cadherin expression. IL-17-inhibited mice had increased E-cadherin expression (P=0.003). E-cadherin inhibition reversed the protective effect of IL-17A inhibition and increased the rate of aneurysm formation (65% versus 28%; P=0.04) and rupture (12% versus 0%; P=0.22). However, E-cadherin inhibition alone does not significantly increase aneurysm formation in normal mice or in estrogen-deficient mice. In cell migration assays, E-cadherin inhibition promoted macrophage infiltration across endothelial cells (P<0.05), which may be the mechanism for the estrogen deficiency/IL-17/E-cadherin aneurysm pathway. CONCLUSIONS: Our data suggest that estrogen deficiency promotes cerebral aneurysm rupture by upregulating IL-17A, which downregulates E-cadherin, encouraging macrophage infiltration in the aneurysm vessel wall.

Local Delivery Is Critical for Monocyte Chemotactic Protein-1 Mediated Site-Specific Murine Aneurysm Healing.

BACKGROUND: Local delivery of monocyte chemotactic protein-1 (MCP-1/CCL2) via our drug-eluting coil has been shown to promote intrasaccular aneurysm healing via an inflammatory pathway. OBJECTIVE: In this study, we validate the importance of local MCP-1 in murine aneurysm healing. Whether systemic, rather than local, delivery of MCP-1 can direct site-specific aneurysm healing has significant translational implications. If systemic MCP-1 is effective, then MCP-1 could be administered as a pill rather than by endovascular procedure. Furthermore, we confirm that MCP-1 is the primary effector in our MCP-1 eluting coil-mediated murine aneurysm healing model. METHODS: We compare aneurysm healing with repeated intraperitoneal MCP-1 versus vehicle injection, in animals with control poly(lactic-co-glycolic) acid (PLGA)-coated coils. We demonstrate elimination of the MCP-1-associated tissue-healing response by knockout of MCP-1 or CCR2 (MCP-1 receptor) and by selectively inhibiting MCP-1 or CCR2. Using immunofluorescent probing, we explore the cell populations found in healed aneurysm tissue following each intervention. RESULTS: Systemically administered MCP-1 with PLGA coil control does not produce comparable aneurysm healing, as seen with MCP-1 eluting coils. MCP-1-directed aneurysm healing is eliminated by selective inhibition of MCP-1 or CCR2 and in MCP-1-deficient or CCR2-deficient mice. No difference was detected in M2 macrophage and myofibroblast/smooth muscle cell staining with systemic MCP-1 versus vehicle in aneurysm wall, but a significant increase in these cell types was observed with MCP-1 eluting coil implant and attenuated by MCP-1/CCR2 blockade or deficiency. CONCLUSION: We show that systemic MCP-1 concurrent with PLGA-coated platinum coil implant is not sufficient to produce site-specific aneurysm healing. MCP-1 is a critical, not merely complementary, actor in the aneurysm healing pathway.

Temporal cascade of inflammatory cytokines and cell-type populations in monocyte chemotactic protein-1 (MCP-1)-mediated aneurysm healing.

BACKGROUND: We have previously shown that monocyte chemotactic protein-1 (MCP-1) promotes aneurysm healing. OBJECTIVE: To determine the temporal cascade and durability of aneurysm healing. METHODS: Murine carotid aneurysms were treated with MCP-1-releasing or poly(lactic-co-glycolic) acid (PLGA)-only coils. Aneurysm healing was assessed by quantitative measurements of intraluminal tissue ingrowth on 5 µm sections by blinded observers. RESULTS: Aneurysm healing occurred in stages characteristic of normal wound healing. The 1st stage (day 3) was characterized by a spike in neutrophils and T cells. The 2nd stage (week 1) was characterized by an influx of macrophages and CD45+ cells significantly greater with MCP-1 than with PLGA (p<0.05). The third stage (week 2-3) was characterized by proliferation of smooth muscle cells and fibroblasts (greater with MCP-1 than with PLGA, p<0.05). The fourth stage (3-6 months) was characterized by leveling off of smooth muscle cells and fibroblasts. M1 macrophages were greater at week 1, whereas M2 macrophages were greater at weeks 2 and 3 with MCP-1 than with PLGA. Interleukin 6 was present early and increased through week 2 (p<0.05 compared with PLGA) then decreased and leveled off through 6 months. Tumour necrosis factor α was present early and remained constant through 6 months. MCP-1 and PLGA treatment had similar rates of tissue ingrowth at early time points, but MCP-1 had a significantly greater tissue ingrowth at week 3 (p<0.05), which persisted for 6 months. CONCLUSIONS: The sequential cascade is consistent with an inflammatory model of injury, repair, and remodeling.

Inactivation of TRPM7 kinase activity does not impair its channel function in mice.

Transient receptor potential (TRP) family channels are involved in sensory pathways and respond to various environmental stimuli. Among the members of this family, TRPM7 is a unique fusion of an ion channel and a C-terminus kinase domain that is highly expressed in immune cells. TRPM7 serves as a key molecule governing cellular Mg(2+) homeostasis in mammals since its channel pore is permeable to Mg(2+) ions and can act as a Mg(2+) influx pathway. However, mechanistic links between its kinase activity and channel function have remained uncertain. In this study, we generated kinase inactive knock-in mutant mice by mutagenesis of a key lysine residue involved in Mg(2+)-ATP binding. These mutant mice were normal in development and general locomotor activity. In peritoneal macrophages isolated from adult animals the basal activity of TRPM7 channels prior to cytoplasmic Mg(2+) depletion was significantly potentiated, while maximal current densities measured after Mg(2+) depletion were unchanged in the absence of detectable kinase function. Serum total Ca(2+) and Mg(2+) levels were not significantly altered in kinase-inactive mutant mice. Our findings suggest that abolishing TRPM7 kinase activity does not impair its channel activity and kinase activity is not essential for regulation of mammalian Mg(2+) homeostasis.