CXCR7 ameliorates myocardial infarction as a ß-arrestin-biased receptor.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through ß-arrestin as a biased receptor. As several ß-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a ß-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited ß-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a ß-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.

Critical Role of SREBP-1c Large-VLDL Pathway in Environment-Induced Hypertriglyceridemia of Apo AV Deficiency.

Objective- APOA5 variants are strongly associated with hypertriglyceridemia, as well as increased risks of cardiovascular disease and acute pancreatitis. Hypertriglyceridemia in apo AV dysfunction often aggravates by environmental factors such as high-carbohydrate diets or aging. To date, the molecular mechanisms by which these environmental factors induce hypertriglyceridemia are poorly defined, leaving the high-risk hypertriglyceridemia condition undertreated. Previously, we reported that LXR (liver X receptor)-SREBP (sterol regulatory element-binding protein)-1c pathway regulates large-VLDL (very low-density lipoprotein) production induced by LXR agonist. However, the pathophysiological relevance of the finding remains unknown. Approach and Results- Here, we reconstitute the environment-induced hypertriglyceridemia phenotype of human APOA5 deficiency in Apoa5-/- mice and delineate the role of SREBP-1c in vivo by generating Apoa5-/- ;Srebp-1c-/- mice. The Apoa5-/- mice, which showed moderate hypertriglyceridemia on a chow diet, developed severe hypertriglyceridemia on high-carbohydrate feeding or aging as seen in patients with human apo AV deficiency. These responses were nearly completely abolished in the Apoa5-/- ;Srebp-1c-/- mice. Further mechanistic studies revealed that in response to these environmental factors, SREBP-1c was activated to increase triglyceride synthesis and to permit the incorporation of triglyceride into abnormally large-VLDL particles, which require apo AV for efficient clearance. Conclusions- Severe hypertriglyceridemia develops only when genetic factors (apo AV deficiency) and environmental effects (SREBP-1c activation) coexist. We demonstrate that the regulated production of large-sized VLDL particles via SREBP-1c determines plasma triglyceride levels in apo AV deficiency. Our findings explain the long-standing enigma of the late-onset hypertriglyceridemia phenotype of apo AV deficiency and suggest a new approach to treat hypertriglyceridemia by targeting genes that mediate environmental effects.

Enhancing endogenous adenosine A2A receptor signaling induces slow-wave sleep without affecting body temperature and cardiovascular function.

Insomnia is one of the most common sleep problems with an estimated prevalence of 10%-15% in the general population. Although adenosine A2A receptor (A2AR) agonists strongly induce sleep, their cardiovascular effects preclude their use in treating sleep disorders. Enhancing endogenous A2AR signaling, however, may be an alternative strategy for treating insomnia, because adenosine levels in the brain accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A2AR positive allosteric modulator (PAM)-1, enhanced adenosine signaling at the A2AR and induced slow wave sleep (SWS) without affecting body temperature in wild-type male mice after intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid derivative was abolished in A2AR KO mice. In contrast to the A2AR agonist CGS 21680, the A2AR PAM-1 did not affect blood pressure or heart rate. These findings indicate that enhancing A2AR signaling promotes SWS without cardiovascular effects. Therefore, small molecules that allosterically modulate A2ARs could help people with insomnia to fall asleep.

Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury.

Accumulating data suggest that new cardiomyocytes in adults are generated from existing cardiomyocytes throughout life. To enhance the endogenous cardiac regeneration, we performed chemical screenings to identify compounds that activate pro-proliferative YES-associated protein and transcriptional enhancer factor domain activities in cardiomyocytes. We synthesized a novel fluorine-containing TT-10 (C11H10FN3OS2) from the biologically hit compound. TT-10 promoted cardiomyocyte proliferation and simultaneously exerted antioxidant and antiapoptotic effects in vitro. TT-10 treatment in mice ameliorated myocardial infarction-induced cardiac dysfunction at least in part via enhancing clonal expansion of existing cardiomyocytes with nuclear YES-associated protein expression. Stimulating cardiomyocyte proliferation and/or protection with TT-10 might complement current therapies for myocardial infarction.

A Novel Bioabsorbable Sheet That Delivers NF-κB Decoy Oligonucleotide Restrains Abdominal Aortic Aneurysm Development in Rats.

For the suppression of inflammation in the aneurysm development, we focused on inhibition of an important transcription factor, nuclear factor-kappa B (NF-κB), using a decoy strategy. We newly developed a novel bioabsorbable sheet that delivers NF-κB decoy oligodeoxynucleotide (ODN).We treated 5-week-old SD rats that were induced with abdominal aortic aneurysm (AAA) using 0.5 M CaCl2 with an NF-κB decoy sheet. Four weeks after AAA induction, aortic tissue was excised for further examinations. We showed that this bioabsorbable sheet could deliver the decoy ODN into the target tissues and dissolve within a week. Treatment with the NF-κB decoy sheet reduced the aneurysm size compared with the controls. It also suppressed inflammation due to the effect of NF-κB decoy ODN. Immunohistochemistry revealed that the expression of CD31, CD4, and CD11b in the NF-κB decoy sheet group was significantly lower than in the control sheet group. The NF-κB decoy sheet was absorbed on the target tissue.We have revealed that the bioabsorbable sheet mediated decoy ODN is effective for transfection into target organs. We have also indicated that NF-κB decoy ODN transfection using this sheet has the potential to suppress the dilatation of aneurysm. The bioabsorbable sheet mediated transfection of the decoy ODN can be beneficial for the clinical treatment of AAA and other NF-κB-related cardiovascular diseases.

Impact of Pathogenic FBN1 Variant Types on the Progression of Aortic Disease in Patients With Marfan Syndrome.

BACKGROUND: Marfan syndrome can cause life-threatening aortic complications. We investigated the relationship between FBN1 genotype and severe aortopathy (aortic root replacement, type A dissections, and related death). METHODS: We evaluated 248 patients with pathogenic or likely pathogenic FBN1 variants. The variants were classified as haploinsufficient type (HI, n=93) or dominant-negative type (DN, n=155) based on their location and predicted amino acid alterations, and we examined the effects of the FBN1 genotype on severe aortic events (aortic root replacement, type A dissections, and related death). RESULTS: The cumulative event-free probability was significantly lower in the HI group than in the DN group (adjusted hazard ratio, 2.1; 95% confidence interval, 1.4 -3.2; P<0.001). CONCLUSIONS: DN-CD+HI patients should be monitored more carefully than DN-nonCD patients for rapid development of aortic root aneurysms.

Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys-Dietz syndrome or multiple self-healing squamous epithelioma.

Variants in TGFBR1 have been reported to induce two completely distinct diseases, namely Loeys-Dietz syndrome (LDS) and multiple self-healing squamous epithelioma (MSSE). However, detailed mechanisms underlying this effect remain unknown. We report a Japanese familial case of LDS with a novel splice donor site variant in TGFBR1 gene (c.973 + 1 G > A; NG_007461.1). The intronic variant was predicted to mediate in-frame exon 5 skipping within the serine/threonine kinase (STK) domain, which may also be mediated by a similar TGFBR1 variant of a splice acceptor site in intron 4 (c.806-2 A > C), identified in a British familial case of MSSE. Therefore, ex vivo splicing and functional assays were performed in mammalian cells to evaluate the effect of these sequence variants. The MSSE variant activated a cryptic acceptor site at 76 bp downstream of the 3' natural splice acceptor site, which produced an out-of-frame transcript (r.807_882del, p.Asn270Thrfs*8). In contrast, the LDS variant generated two types of in-frame transcription products, r.[806_973del, 965_973 del], and produced two functionally inactivated proteins, p.[Asp269_Gln324del, Thr323_Gly325del], as a result of exon 5 skipping and the activation of a cryptic donor splice site at 9 bp upstream of the 5' natural splice donor site, respectively. Our results support the previously proposed but not yet approved mechanism that dominant-negative and truncating variants in STK domain induce LDS and MSSE, respectively.

Japanese Cardiovascular Disease Patients with Diabetes Mellitus Suffer Increased Tooth Loss in Comparison to Those without Diabetes Mellitus -A Cross-sectional Study.

Objective Tooth loss is an irreversible condition that reflects the end-stage of oral diseases, including periodontitis. Although periodontitis is a major factor in the progression of diabetes mellitus (DM) and cardiovascular disease (CVD), no previous studies have compared tooth loss in CVD patients with and without DM. Methods The subjects included CVD patients with (n=94) and without (n=145) DM who attended Tokyo Medical and Dental University Hospital. Blood examinations and periodontal measurements were performed. Results The oral and periodontal examinations revealed that the numbers of missing teeth in the DM group were increased in comparison to the non-DM group. There was no significant difference between the groups with regard to the incidence of edentulism, the probing pocket depth, the clinical attachment level or the incidence of bleeding on probing. Conclusion We showed that the numbers of missing teeth among CVD patients with DM was significantly higher than that among CVD patients without DM.

Specific periodontopathic bacterial infection affects hypertension in male cardiovascular disease patients.

Hypertension (HT) is a systemic disorder that results in the decline of quality of life and death. While patients with periodontitis are at a high risk of HT, little causal information has been provided to date. To clarify the relationship, periodontopathic bacterial infection in cardiovascular patients with or without HT was evaluated. The subjects were patients with (n = 412) or without (n = 199) HT who attended Tokyo Medical and Dental University hospital. Blood examinations and periodontal measurements were performed. Three periodontopathic bacteria existence and antibody titers were evaluated. We found that specific periodontopathic bacteria, Aggregatibacter actinomycetemcomitans and Prevotella intermedia, were highly detected in male subjects with HT compared to non-HT subjects, while they were comparable in the female patients. Mean probing pocket depth of elderly male patients with HT was higher compared to non-HT patients. The rates of obesity, dyslipidemia, and diabetes showed partial statistical difference between the two groups. Specific periodontopathic bacterial infection may affect HT in male cardiovascular patients.

Detrimental effects of specific Periodontopathic bacterial infection on tachyarrhythmia compared to Bradyarrhythmia.

BACKGROUND: Tachyarrhythmia (TA) and bradyarrhythmia (BA) are cardiac rhythm disorders that result in the decline of quality of life. While patients with periodontitis are at a high risk of cardiovascular disease (CVD), little causal information between TA and BA has been provided to date. To assess the relationship, periodontal bacterial infection in patients with TA or BA was evaluated. METHODS: The subjects were patients with TA (n = 98) or BA (n = 40) who attended Tokyo Medical and Dental University hospital. Periodontal and blood examinations were performed. Periodontopathic bacterial existence in saliva was evaluated. RESULTS: We found that specific periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, were highly detected in saliva from TA patients compared to BA subjects. The rates of hypertension and dyslipidemia were comparable between the two groups. CONCLUSION: Specific periodontal bacterial infection might affect TA progression.

Periodontitis deteriorates peripheral arterial disease in Japanese population via enhanced systemic inflammation.

Peripheral arterial disease (PAD) is a common manifestation of arterial stenosis of the extremity that reduces arterial flow. While patients with periodontitis are at a high risk of PAD, little causal information has been provided to date. To clarify the relationship, we conducted this cross-sectional study. The oral condition of patients with or without PAD, who attended Tokyo Medical and Dental University Hospital, was evaluated. Blood examinations and dental clinical measurements, including number of teeth, probing pocket depth (PPD), bleeding on probing (BOP) and clinical attachment level (CAL) were performed. Chi-square test was performed to compare gender, smoker rate, prevalence of DM, hypertension and dyslipidemia and edentulous rate. Wilcoxon test was used to compare bacterial counts and anti-bacterial antibodies and Student's t test was used to compare the other numerical values. The subjects were patients with (n = 34) or without (n = 956) PAD. We revealed that the PAD patients had more missing teeth (17.5 ± 11.0), a higher rate of edentulism (18%), and higher serum inflammatory factor levels than non-PAD patients (10.9 ± 8.7, 5%, respectively). On the other hand, there was no significant difference between hypertension, dyslipidemia, smoking status, HbA1c, bacterial antibody titers, and bacterial counts between the groups. In conclusion, we clarified that PAD patients had decreased tooth number and worsened oral and periodontal condition with enhanced systemic inflammation.

Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2.

Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.

Role of Hormone-sensitive Lipase in Leptin-Promoted Fat Loss and Glucose Lowering.

AIM: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action. METHODS: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. RESULTS: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. CONCLUSIONS: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.

Dysbiosis and compositional alterations with aging in the gut microbiota of patients with heart failure.

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

A peptide vaccine targeting angiotensin II attenuates the cardiac dysfunction induced by myocardial infarction.

A peptide vaccine targeting angiotensin II (Ang II) was recently developed as a novel treatment for hypertension to resolve the problem of noncompliance with pharmacotherapy. Ang II plays a crucial role in the pathogenesis of cardiac remodeling after myocardial infarction (MI), which causes heart failure. In the present study, we examined whether the Ang II vaccine is effective in preventing heart failure. The injection of the Ang II vaccine in a rat model of MI attenuated cardiac dysfunction in association with an elevation in the serum anti-Ang II antibody titer. Furthermore, any detrimental effects of the Ang II vaccine were not observed in the rats that underwent sham operations. Treatment with immunized serum from Ang II vaccine-injected rats significantly suppressed post-MI cardiac dysfunction in MI rats and Ang II-induced remodeling-associated signaling in cardiac fibroblasts. Thus, our present study demonstrates that the Ang II vaccine may provide a promising novel therapeutic strategy for preventing heart failure.

A Periodontal pathogen Porphyromonas gingivalis deteriorates Isoproterenol-Induced myocardial remodeling in mice.

Heart failure is a serious disease induced by several conditions, including hypertrophic cardiomyopathy. Although many reports suggest that there is an association between periodontal disease and cardiovascular disease, the mechanisms have yet to be elucidated. The purpose of this study was to clarify the relationship between periodontal disease and heart disease, especially in cardiac hypertrophy. We used C57BL/6J mice and implanted two types of subcutaneous chambers. First, we subcutaneously implanted a coil-shaped chamber into the back of a mouse. Porphyromonas gingivalis (P.g.), a major periodontal pathogen, was injected into the chamber. Then, an osmotic pump was implanted to infuse isoproterenol. Four weeks after the ISO infusion, we performed echocardiography and harvested the heart and blood. We measured the serum level of anti-P.g.-IgG using ELISA. The mRNA levels of several factors were measured using PCR. We found stronger cardiomyocyte hypertrophy in the ISO(+)/P.g.(+) mice compared with the ISO(+)/P.g.(-) mice. The total square of randomly selected cardiomyocytes was 23% larger in the ISO(+)/P.g.(+) mice than in the ISO(+)/P.g.(-) mice. We detected a higher level of mRNA expression in Toll-like receptor 2 and NADPH oxidase 4 in the ISO(+)/P.g.(-) mice compared with the control group. We revealed that a periodontal pathogen affected ISO-induced cardiac hypertrophy via oxidative stress.

Periodontitis and myocardial hypertrophy.

There is a deep relationship between cardiovascular disease and periodontitis. It has been reported that myocardial hypertrophy may be affected by periodontitis in clinical settings. Although these clinical observations had some study limitations, they strongly suggest a direct association between severity of periodontitis and left ventricular hypertrophy. However, the detailed mechanisms between myocardial hypertrophy and periodontitis have not yet been elucidated. Recently, we demonstrated that periodontal bacteria infection is closely related to myocardial hypertrophy. In murine transverse aortic constriction models, a periodontal pathogen, Aggregatibacter actinomycetemcomitans markedly enhanced cardiac hypertrophy with matrix metalloproteinase-2 activation, while another pathogen Porphyromonas gingivalis (P.g.) did not accelerate these pathological changes. In the isoproterenol-induced myocardial hypertrophy model, P.g. induced myocardial hypertrophy through Toll-like receptor-2 signaling. From our results and other reports, regulation of chronic inflammation induced by periodontitis may have a key role in the treatment of myocardial hypertrophy. In this article, we review the pathophysiological mechanism between myocardial hypertrophy and periodontitis.