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Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown etiology. Although a number of investigators have attempted to determine biomarkers for diagnosing TAK, there exist no specific serological markers of this intractable disease. We undertook the exploration of novel serological markers which could be useful for an accurate diagnosis of TAK using an unbiased proteomics approach. The purified plasma samples from untreated patients with TAK and healthy individuals were separated by two-dimensional electrophoresis. The differentially expressed protein spots were detected by gel comparison and identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MS). Next, we validated plasma concentrations of identified proteins by enzyme-linked immunosorbent assay (ELISA). Two-dimensional electrophoresis and numerical analysis revealed 19 spots and 3 spot clusters whose sum of the sample averages was ≥ 0.01, and the average concentrations were ≥ 1.5 times in the patient group compared with the control group. Among them, 10 spots and spot clusters that met the condition of the average spot concentration being 2.5 times more than that in the control group were selected. After processing these spots using MS and conducting MS/MS ion search, we identified 10 proteins: apolipoprotein C-2 (ApoC-2), actin, apolipoprotein A-1, complement C3, kininogen-1, vitronectin, α2-macroglobulin, 14–3–3 protein ζ/δ, complement C4, and inter-α-trypsin inhibitor heavy chain H4 isoform 1 precursor. Finally, ELISA demonstrated that plasma ApoC-2 level was significantly elevated in patients with TAK compared with that in healthy individuals. Thus, ApoC-2 would be a promising candidate biomarker for TAK diagnosis.

Unfolded protein response (UPR) plays a critical role for preventing death of cardiomyocytes induced by excessive endoplasmic reticulum (ER) in response to cytotoxicity of anthracycline antineoplastic drugs through mitigating misfoled proteins accumulation in the hearts. Here, we demonstrate that ER-selective autophagy (ER-phagy) serves as a salvage system which enable cardiomyocytes to survive even if UPR fails to restore ER stress induced by doxorubicin (Dox), an anthracycline drug, by using in-house developed monitoring systems to evaluate the activity of ER-phagy in cardiomyocytes. Notably, ER-phagy contributes to relieving cardiac damage induced by Dox administration and is mediated by CCPG1, an ER-phagy receptor protein. Furthermore, ablation of CCPG1 abolished ER-phagy and worsened Dox-induced cardiac damage by inducing excessive ER stress-associated apoptosis. Our data highlight a compensatory role of CCPG1-driven ER-phagy in reducing Dox toxicity and pose a potential therapeutic target to intervene against Dox induced cardiomyopathy.

Visual Abstract, Highlights • Treatment with linagliptin, a DPP-4 inhibitor, alleviates not only EAM but also ICIM. • DPP-4 physically interacts with cathepsin G and enhances its activity. • Linagliptin promotes SerpinA3N activity, thereby suppressing cathepsin G activity. • Cathepsin G aggravates EAM through upregulating angiotensin II. • Linagliptin suppresses oxidative stress in EAM hearts., Summary This study sought to show the mechanism of how to ameliorate experimental autoimmune myocarditis (EAM) by administering dipeptidyl peptidase (DPP)-4 inhibitor linagliptin. The number of RAR-related orphan nuclear receptor gamma–positive Th17 cells infiltrated to the EAM myocardium was significantly attenuated by linagliptin treatment. Tandem mass spectrometry–based analysis demonstrated that DPP-4 binds to cathepsin G in EAM hearts, thereby protecting cathepsin G activity through inhibiting SerpinA3N activity. Linagliptin suppresses oxidative stress in EAM hearts as well. Thus, we found that DPP-4 plays a detrimental role in the progression of EAM by interacting with cathepsin G, which, in turn, suppresses SerpinA3N activity.

Background This study aimed to investigate the effect of periodontal disease (PD) on the outcomes of patients with coronary artery disease (CAD) treated with percutaneous coronary intervention (PCI).Methods and Results:The study included 77 consecutive non-smoking patients with de novo coronary lesions treated with a drug-eluting stent (DES). Periodontal measurements, including the community periodontal index (CPI), were performed by independent periodontists. A CPI score of ≥3 was used to define PD. The occurrence of major adverse cardiac events (MACEs), which were defined as a composite of cardiovascular death, non-fatal myocardial infarction, target lesion revascularization, or non-target lesion revascularization, was compared between patients with and without PD. Of the 77 patients, 49 (63.6%) exhibited a CPI score of 3 or 4 and were assigned to the PD group. The remaining 28 patients (36.4%) were assigned to the non-PD group. Baseline clinical characteristics and angiographic findings were comparable between the 2 groups. MACEs occurred in 13 (26.5%) of the PD patients and 2 (7.1%) of the non-PD patients. Kaplan-Meier analysis showed a significantly lower MACE-free survival rate in the PD group than for the non-PD group (P=0.034). Conclusions PD at baseline was associated with an increased risk of MACEs in CAD patients who were treated with a DES for de novocoronary lesions.

Introduction: Increasing lines of evidence suggest endoplasmic reticulum-selective autophagy (ER-phagy) as a cell-protective pathway to mediate degradation of damaged ER. However, the role of ER-phagy in cardiomyocytes and its molecular mechanisms remain unsolved. Hypothesis: Considering that various cardiac loads cause ER stress which contributes to the development of heart failure, we hypothesized that ER-phagy would be a compensatory system for protecting cardiomyocytes against ER stress. Methods and Results: To evaluate ER-phagy in cardiomyocytes, we generated lentivirus (LV) and cardiac-specific transgenic mice (Tg-mice) harboring ss-RFP-GFP-KDEL sequence, an ER-phagy reporter possessing an N-terminal ER signal sequence followed by tandem fluorescence sequences and ER retention signal. Fluorescent microscopic analyses and immunoblotting demonstrated that amino acid deprivation of LV-ss-RFP-GFP-KDEL-tranduced H9c2 (tfH9c2) cells markedly elevated the amount of RFP fragments, suggesting that ER-phagy is increased in tfH9c2 cells. 48 hours of food starvation markedly induced ER-phagy in Tg-mice hearts, as well. Treatment with Doxorubicin (Dox), a cardiotoxic compound inducing ER stress, activated ER-phagy in tfH9c2 cells as evidenced by the increase in RFP fragments detected by immunoblotting (Control vs. Dox: 1.0±0.2a.u. vs. 31.6±6.3a.u.; p Conclusions: These results suggest that ER-phagy plays a protective role in cardiomyocytes against Dox toxicity, possibly through CCPG1-mediated signaling.

Visual Abstract, Highlights • An appropriate dose of RIV to inhibit the activity of factor Xa significantly suppressed the atherosclerotic area in the aorta of HFD-fed ApoE–/– mice. • Macrophage autophagy induced by 7KC administration was suppressed by the presence of factor Xa, resulting in the acceleration of inflammasome formation through the PAR2-mediated pathway. • The administration of CQ, an autophagy inhibitor, to HFD-fed ApoE–/–/PAR2 knockout bigenic mice significantly aggravated atherosclerosis in the aorta compared with untreated mice., Summary The authors showed a mechanism for attenuating atherosclerosis by directly administering an oral factor Xa inhibitor (ie, rivaroxaban [RIV]). The autophagy activity of macrophages was significantly suppressed by factor Xa and was alleviated by the administration of RIV. However, factor Xa failed to inhibit 7-ketocholesterol–induced autophagy and inflammasome activation in protease-activated receptor 2 (PAR2) knockout macrophages. The atherosclerotic area of apolipoprotein E knockout mice was significantly reduced by the genetic ablation of PAR2, which was partially reversed by chloroquine. Thus, the authors found that RIV attenuates atherogenesis by inhibiting the factor Xa–PAR2-mediated suppression of macrophage autophagy and abrogating inflammasome activity.

Background There is a compelling need for establishing effective therapy for autoimmune myocarditis which primarily manifest as chest pain, heart failure or sudden death. Although our group have previously shown that dipeptidyl peptidase-4 (DPP-4) aggravates experimental autoimmune myocarditis (EAM), the detailed underlying mechanism remains to be unelucidated. Methods The effects of linagliptin, a xanthine-based dipeptidyl peptidase-4 inhibitor, on cardiac function were investigated by treating mouse EAM models and elucidated the role of DPP-4 on EAM using proteomic approaches. Results Immunohistochemical analyses demonstrated that the number of Th17 cells expressing high level of DPP-4 infiltrated to EAM myocardium was significantly attenuated by linagliptin treatment. MS/MS-based analyses demonstrated that DPP-4 binds to cathepsin-G in EAM hearts. DPP-4 also protects cathepsin-G activity by inhibiting the activity of SerpinA3N, a protease inhibitor that catalyzes cathepsin-G. The activity of cathepsin-G and the level of Angiotensin II were markedly elevated in EAM myocardium; this effect was reversed by linagliptin treatment. Furthermore, we found that linagliptin suppresses oxidative stress in EAM hearts. Conclusions DPP-4 physically interacts with cathepsin-G, which, in turn, suppresses SerpinA3N; this promotes angiotensin II accumulation in EAM hearts. Thus, DPP-4 derived from Th17 cells could aggravate cardiac dysfunction during EAM.

Background: Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown pathogenesis. Genome-wide association studies revealed that single-nucleotide polymorphisms in the MLX gene encoding the MLX (Max-like protein X) transcription factor are significantly associated with TAK in Japanese patients. MLX single-nucleotide polymorphism rs665268 is a missense mutation causing the Q139R substitution in the DNA-binding site of MLX. Methods: To elucidate the hypothesis that the single-nucleotide polymorphism of the MLX gene plays a critical role in the development of TAK, we conducted clinical and laboratory analyses. Results: We show that rs665268 significantly correlated with the severity of TAK, including the number of arterial lesions and morbidity of aortic regurgitation; the latter may be attributed to the fact that MLX mRNA expression was mostly detected in the aortic valve. Furthermore, the Q139R mutation caused structural changes in MLX, which resulted in enhanced formation of a heterodimer with MondoA, upregulation of TXNIP (thioredoxin-interacting protein) expression, and increase in the activity of the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome and cellular oxidative stress. Furthermore, autophagy, which negatively regulates inflammasome activation, was suppressed by the Q139R mutation in MLX. The MLX-Q139R mutant significantly induced macrophage proliferation and macrophage–endothelium interaction, which was abolished by the treatment with SBI-477, an inhibitor of MondoA nuclear translocation. Our findings suggest that the Q139R substitution in MLX plays a crucial role in the pathogenesis of TAK. Conclusions: MLX-Q139R mutation plays a crucial role in the pathogenesis of TAK through promoting inflammasome formation.