TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.Fil: Oda, Sayaka. Okazaki Institute for Integrative Bioscience; Japón. SOKENDAI; JapónFil: Numaga Tomita, Takuro. Okazaki Institute for Integrative Bioscience; Japón. SOKENDAI; JapónFil: Kitajima, Naoyuki. Okazaki Institute for Integrative Bioscience; Japón. Kyushu University; JapónFil: Tomizaki, Takashi. Okazaki Institute for Integrative Bioscience; Japón. Kyushu University; Japón. University of Tsukuba; JapónFil: Harada, Eri. Ajinomoto Co.; Japón. EA Pharma Co.; JapónFil: Shimauchi, Tsukasa. Okazaki Institute for Integrative Bioscience; Japón. Kyushu University; JapónFil: Nishimura, Akiyuki. Okazaki Institute for Integrative Bioscience; Japón. SOKENDAI; Japón. Ajinomoto Co.; JapónFil: Ishikawa, Tatsuya. Kyushu University; Japón. Ajinomoto Co.; Japón. EA Pharma Co.; JapónFil: Kumagai, Yoshito. University of Tsukuba; JapónFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nishida, Motohiro. Okazaki Institute for Integrative Bioscience; Japón. SOKENDAI; Japón. Kyushu University; Japón. PRESTO; Japó

TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6

Periodontal Tissue Regeneration Using Fibroblast Growth Factor -2: Randomized Controlled Phase II Clinical Trial

Background: The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings: We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ?3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC cotaining 0.1% FGF-2; and Group H, given HPC Containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 μL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attribute to the investigational drug were identified. Conclusions: Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis

<i>Porphyromonas gingivalis</i> Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

<div><p>The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, <i>Porphyromonas gingivalis</i>, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that <i>P</i>. <i>gingivalis</i> increased IL-33 expression in the cytoplasm of human gingival epithelial cells <i>in vitro</i>. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from <i>P</i>. <i>gingivalis</i> did not increase IL-33 expression. Specific inhibitors of <i>P</i>. <i>gingivalis</i> proteases (gingipains) suppressed IL-33 mRNA induction by <i>P</i>. <i>gingivalis</i> and the <i>P</i>. <i>gingivalis</i> gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by <i>P</i>. <i>gingivalis</i>. These results indicate that the PAR-2/IL-33 axis is promoted by <i>P</i>. <i>gingivalis</i> infection in human gingival epithelial cells through a gingipain-dependent mechanism.</p></div

<i>P</i>. <i>gingivalis</i> increases IL-33 mRNA expression in gingival epithelial cells.

<p>Ca9-22 cells were infected with fresh <i>P</i>. <i>gingivalis</i> W83 cultures at MOI of 0.5 for the indicated periods (A) or indicated MOI for 48 h (B). Ca9-22 cells were stimulated with 50 μg/ml (MOI of 0.1) of whole <i>P</i>. <i>gingivalis</i> W83 cells for the indicated periods (C) and indicated amounts of whole <i>P</i>. <i>gingivalis</i> W83 cells, fimbriae, PGTP2-RL, and LPS derived from <i>P</i>. <i>gingivalis</i> for 48 h (D). Cells were incubated with 10 μg/ml cycloheximide for 45 min (E), 1 μg/ml nocodazole for 1 h (F) or 0.5 μM cytochalasin D for 30 min (G) and then stimulated for 48 h with 50 μg/ml of <i>P</i>. <i>gingivalis</i> W83 whole cells with the respective inhibitors. Total cellular RNA was then extracted at the indicated times, and IL-33 transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistically significant differences are indicated. *, <i>P</i><0.05 compared with untreated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone.</p

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the NF-κB pathway.

<p>(A) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for indicated periods. (B) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with gingipain inhibitors (0.3 μM FPR-cmk plus 0.3 μM KYT-36) for 15 min at 37°C, and then used to stimulate Ca9-22 cells for 9 h. (C) Ca9-22 cells stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP136 cells for 9 h. (A-C) Cells were transiently transfected with pNFκB-<i>Metridia</i> luciferase reporter or control p<i>Metridia</i> luciferase reporter plasmids. Amount of secreted luciferase in culture supernatants were analyzed using a luminometer. (D) Ca9-22 cells incubated with indicated concentrations of PDTC (NF-κB inhibitor) for 1 h and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with respective unstimulated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (E) Ca9-22 cells were incubated with 25 μM PCTC for 1 h and then stimulated for 18 h with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Cell lysates were analyzed by Western blotting against anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to p38 and quantified by densitometry. Control media was adjusted to contain 0.1% (v/v) DMSO during incubation with inhibitor.</p

Induction of IL-33 mRNA expression by <i>P</i>. <i>gingivalis</i> requires activation of the p38 pathway.

<p>(A) Ca9-22 cells stimulated for indicated periods with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells in medium containing 1% FBS. Phosphorylation of p38 was detected in cell lysates by Western blotting against anti-phospho-p38 antibody (p-p38). Controls comprised antibody against total p38. Data are representative of three independent experiments. Relative expression of phosphorylated p38 was quantified using densitometry. Relative expression of phosphorylated p38 was normalized to that of p38. (B) Ca9-22 cells incubated with 10 μM PD98059, SP600125, or SB203580 for 30 min and then stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 48 h in medium containing 5% FBS. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with untreated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone). (C) Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with 0.3 μM FPR-cmk plus 0.3 μM KYT-36 for 15 min at 37°C and then used to stimulate Ca9-22 cells for 18 h in medium containing 1% FBS. (D) Ca9-22 cells were stimulated with 50 μg/ml of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP 136 whole cells for 18 h. Cell lysates were analyzed by Western blotting using anti-phospho-p38 and anti-p38 antibodies. Data are representative of three independent experiments. Relative expression levels of phosphorylated p38 were normalized to levels of p38 and quantified by densitometry. Controls were adjusted to contain 0.1% (v/v) DMSO in media during incubation with inhibitors.</p

<i>P</i>. <i>gingivalis</i> increases IL-33 protein expression in gingival epithelial cells.

<p>(A) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for the indicated periods. Cell lysates were analyzed by Western blotting with an anti-human IL-33 mAb. Expression levels of IL-33 were quantified by densitometry using ImageJ software and normalized to medium alone. (B) Ca9-22 cells were stimulated with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells for 4 d, and intracellular IL-33 protein was stained with PE-conjugated anti-human IL-33 mAb. Nuclei were stained with DAPI. Bar = 25 μm. Data are representative of three independent experiments.</p

Participation of gingipains in <i>P</i>. <i>gingivalis</i>-induced IL-33 mRNA expression in gingival/oral epithelial cells.

<p>Whole <i>P</i>. <i>gingivalis</i> W83 cells (50 μg/ml) were incubated with 0.3 μM FPR-cmk (Rgp inhibitor) or 0.3 μM KYT-36 (Kgp inhibitor) for 15 min at 37°C and then used to stimulate Ca9-22 (A) or primary oral epithelial (B) cells for 48 h. (C) Ca9-22 cells stimulated with 50 μg/ml of whole cells of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type, <i>P</i>. <i>gingivalis</i> KDP131 (Δ<i>rgpA</i>), KDP132 (Δ<i>rgpB</i>), KDP129 (Δ<i>kgp</i>), KDP133 (Δ<i>rgpA</i> Δ<i>rgpB</i>), or KDP136 (Δ<i>kgp</i> Δ<i>rgpA</i> Δ<i>rgpB</i>) gingipain-null mutant for 48 h. (D) Primary oral epithelial cells stimulated with 50 μg/ml of <i>P</i>. <i>gingivalis</i> ATCC 33277 wild-type or KDP136 for 48 h. (E) Ca9-22 cells cultured for 48 h with 50 μg/ml of whole <i>P</i>. <i>gingivalis</i> W83 cells after incubation with or without at 70°C for 1 h. Total cellular RNA was extracted and transcripts were analyzed by RT-qPCR. Data are representative of three independent experiments, and are shown as means ± SD of triplicate assays. Statistical significant differences are indicated (*, <i>P</i><0.05 compared with respective unstimulated control; §, <i>P</i><0.05 compared with <i>P</i>. <i>gingivalis</i> alone).</p