Abstract Background Pulmonary arterial hypertension (PAH) is a disease with poor prognosis that causes right heart failure due to progressive pulmonary artery remodeling. Although existing pulmonary vasodilators contribute to pulmonary artery reverse remodeling by reducing share stress, the development of therapeutic agents that directly target pulmonary vascular remodeling is desired. Nerve growth factor receptor (Ngfr) is involved in the inflammatory reaction and repair process of damaged tissues. We have previously reported that Ngfr-positive cells are increased in the peripheral blood (PB) of PAH patients and are associated with disease progression. We also found that Ngfr inhibited the pathogenetic progression of pulmonary hypertension in a model of hypoxia-induced pulmonary hypertension. However, it remains unclear how Ngfr positive cells are involved in the pathology of PAH. Purpose In this study, we investigate how Ngfr-positive cells affect the pathogenesis of PAH. Methods & Results To evaluate the localization of Ngfr-positive cells in lung tissue, WT mice (C57BL/6) transplanted with bone marrow (BM) of GFP-Tg mice were kept under hypoxia for 3 weeks to create hypoxia-induced pulmonary hypertension (PH) model. Immunostaining of lung tissue sections indicated that double positive cells (Ngfr+GFP+) were present in the interstitial tissue around the small pulmonary artery. To examine the cellular characteristics of Ngfr-positive cells, RNA sequencing of Ngfr-positive and Ngfr-negative cells in PB was performed. Significantly 78 genes were upregulated in Ngfr-positive cells, including 11 secreted proteins. Among these, we focused on pigment epithelium-derived factor (PEDF), a secreted protein involved in angiogenesis. PEDF mRNA expression was decreased in the lung of Ngfr-KO mice compared to WT mice. We next examined the effect of PEDF on pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF)-induced cell proliferation of PASMC was inhibited by PEDF stimulation. Furthermore, PEDF stimulation significantly inhibited PDGF-induced mRNA expressions of TNF and MMP9 in PASMCs. Conclusion In this study, we show that PEDF expression is upregulated in Ngfr-positive cells and that PEDF inhibits PDGF-induced hPASMC proliferation, suggesting that NGFR-positive cells may suppress pulmonary vascular remodeling through PEDF-mediated paracrine effects. PEDF might be a new therapeutic target for PH.

Abstract Introduction Standing places a significant hemodynamic burden on the cardiovascular system. In healthy individuals, neurohumoral reflex mechanisms, particularly the arterial baroreflex, maintain blood pressure during standing. However, in some cases, the counterregulatory response can be excessive, resulting in a condition known as orthostatic hypertension (OrthoHT), which appears to be linked to an increased cardiovascular risk. Epidemiologically, orthostatic hypertension has been reported in 5-30% of hypertensive patients. Furthermore, in recent years, it has garnered attention as a responder group in renal denervation therapy. Purpose In hypertensive patients, sympathetic hyperactivity is expected to exacerbate cardiovascular risk. However, the relationship between OrthoHT and sympathetic nerve activity remains poorly understood. This study aims to assess the differences in sympathetic nerve activity between OrthoHT and conventional hypertensive patients using muscle sympathetic nerve activity (MSNA) measurements. Methods We conducted an examination of hypertensive patients undergoing drug treatment at our clinic between April 1, 2023, and June 30, 2023, to identify cases of OrthoHT. Only patients with essential HT were included in the HT group; patients diagnosed with secondary HT were excluded. HT patients with other active CV diseases (i.e., symptomatic cerebral/coronary artery diseases or heart failure) and/or severe renal dysfunction (estimated glomerular filtration rate <30 mL/min/1.73 m2) were also excluded. OrthoHT was defined as a sustained increase in systolic blood pressure ≥20 mmHg and/or diastolic blood pressure ≥10 mmHg within 3 minutes of standing. MSNA was recorded from the left peroneal nerve using a tungsten microelectrode at supine position. We measured MSNA in the cases for which consent was obtained. Statistical analyses were performed using SPSS software. Results Among the 183 cases, 31 were diagnosed with OrthoHT, and 7 of them consented to MSNA measurement. Of the 11 cases of hypertensive patients who consented, two groups were well age-matched. We assessed body mass index, resting systolic blood pressure, diastolic blood pressure, heart rate, blood glucose level, creatinine, cholesterol, brain natriuretic hormone, and ejection fraction measured by echocardiography, but found no significant differences between the two groups. OrthoHT patients exhibited elevated MSNA burst frequency (28.3 +/- 5.7 vs. 38.4 +/- 12.2 bursts/min, P < 0.05) and burst incidence (44.6 +/- 11.1 vs. 61.1 +/- 21.3 bursts/100 heartbeats, P < 0.05) at rest. Conclusions OrthoHT is associated with sympathetic hyperactivity compared to normal hypertensive patients, contributing to an increased cardiovascular risk.

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

Abstract Introduction Standing places a significant hemodynamic burden on the cardiovascular system. In healthy individuals, neurohumoral reflex mechanisms, particularly the arterial baroreflex, maintain blood pressure during standing. However, in some cases, the counterregulatory response can be excessive, resulting in a condition known as orthostatic hypertension (OHT), which appears to be linked to an increased cardiovascular risk. In recent years, OHT has been recognized as an indicator for assessing the effectiveness of renal denervation. It is suggested that abnormalities in sympathetic nervous system regulation, particularly related to the renal sympathetic nerves, may play a role in influencing RDN outcomes, however, the relationship between OHT and sympathetic nerve activity remains poorly understood. This study aims to assess the differences in sympathetic nerve activity between OHT patients, conventional hypertensive(cHT) patients and healthy individuals using muscle sympathetic nerve activity (MSNA) measurements. Methods We conducted an examination of hypertensive patients undergoing drug treatment at our clinic between April 1, 2023, and June 30, 2023, to identify cases of OHT. OHT was defined as a sustained increase in systolic blood pressure ≥20 mmHg and/or diastolic blood pressure ≥10 mmHg within 3 minutes of standing. We measured MSNA in the cases for which consent was obtained. Results Among the 138 cases, 31 were diagnosed with OHT, and 7 of them consented to MSNA measurement. Of the 11 cases of cHT patients who consented, two groups were well age-matched. And it was also compared with 11 cases of healthy individuals who underwent MSNA measurement during the same period. Both of the MSNA burst frequency and burst incidence in OHT (38.4 +/- 12.2 bursts/min, 61.1 +/- 21.3 bursts/100 heartbeats) was significantly greater (p<0.05) than that in cHT (28.3 +/- 5.7 bursts/min, 44.6 +/- 11.1 bursts/100 heartbeats), and all these were significantly greater (p<0.05) than in healthy individuals (21.8 ± 7.3 bursts/min, 35.8 +/- 11.3 bursts/100 heartbeats) at rest. Conclusions OHT is associated with sympathetic hyperactivity compared to normal hypertensive patients, contributing to an increased cardiovascular risk. These results indicate that therapy for OHT in hypertensive patients would be required to prevent sympathetic augmentation.

Abstract Background Anemia is a common comorbidity in older patients with heart failure (HF)1 and atrial fibrillation (AF)2, associated with an increased risk of adverse events. This study evaluated the prognostic effects of longitudinal changes in anemia status on clinical outcomes in patients with AF, as limited data are available on this aspect. Methods and Results We prospectively evaluated data of 1,388 patients with AF obtained from the Hokuriku-Plus AF registry (1,010 men, 72.3±9.7 years) and recorded the incidence of cardiovascular death (CVD), HF, thromboembolism, and major bleeding. Finally, 1,233 patients with AF with baseline and first year of follow up hemoglobin levels were evaluated. Patients were categorized into 3 groups based on the longitudinal changes in 1-year anemia status: AF without anemia (group 1), AF with improved anemia (group 2), and AF with sustained or new-onset anemia (group 3). During 1–5-year follow up, the incidences of CVD, HF, thromboembolism, and major bleeding were significantly higher in patients with AF and anemia than in those without (Figure1). Additionally, the incidence of CVD or HF was significantly higher in group 3 than in groups 1 and 2 (Figure 2). Multivariate analysis revealed no anemia or improvement from anemia in 1 year as an independent predictor for reducing CVD and HF. Conclusions Recovery from anemia may be associated with a favorable clinical course in AF.

Abstract Background Peripheral blood mononuclear cells (MNCs) contribute to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is present in peripheral blood and the ischemic coronary artery. NGFR transduces neurotrophin signals towards cell survival or apoptosis in different cell types dependent on coupling co-receptors. However, the role of NGFR-positive (NGFR+) MNCs in arterial remodelling is unknown. Purpose To investigate the functional mechanisms under which NGFR+ MNCs are involved in arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -bone marrow (BM)-specific (KO) mice were subjected to unilateral carotid artery ligation. The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human NGFR+ MNCs from a healthy volunteer were sorted using fluorescence-activated cell sorting and characterised using RNA sequencing. The cell apoptosis (AnnexinV+7AAD-) and chemotaxis in response to the ligands, NGF and its precursor proNGF, were assessed using flow cytometry and a Transwell migration. Results In WT mice, BM-derived NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly greater in the BM-specific depletion of NGFR than in WT mice. NGFR+ cells in the neointima exhibited apoptosis (TUNEL+ and cleaved caspase-3+) accompanied by promoted F4/80+ macrophage and anti-inflammatory IL-10. By contrast, in the BM-specific NGFR-KO model, non-BM-derived SMCs increased in the neointima with augmented proliferation, and the accumulation of BM-derived cells and the expression of IL-10 were decreased. The expressions of proNGF/NGF and inflammatory cytokines, including IL-6, were not changed in a ligated artery, irrespective of NGFR+ depletion. Human NGFR+ MNCs expressed TrkB, TrkC, and sortilin co-receptors but not macrophage markers. Human NGFR+ MNCs, but not NGFR- MNCs, co-cultured with artery SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated proNGF/NGF expression in SMCs, and proNGF/NGF did not affect the proliferation of SMCs in vitro. Furthermore, NGFR+ MNCs, but not NGFR- MNCs, increased migration toward NGF and did not show chemotaxis toward proNGF. Conclusions These data imply that local NGF might draw NGFR+ MNCs to the injured artery, in which proNGF induces NGFR+ MNCs apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis to resolve inflammation and decrease neointimal formation. Thus, we propose the proNGF/NGF-NGFR axis as potentially contributing to the suppression of arterial remodelling.

Abstract Background In heart failure, the failure of the pump function results in reduced blood flow and dysfunction of energy metabolism in tissues and organs throughout the body. The liver is the largest organ in the human body and plays a central role in lipid and glucose metabolism. Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Inhibition of SeP protects the heart from ischemia reperfusion injury and serum levels of SeP are elevated in patients with heart failure with reduced ejection fraction. Objective We investigated the role of SeP in the regulation of cardiac remodeling in response to pressure overload. Methods and Results To examine the role of SeP in cardiac remodeling, transverse aortic constriction (TAC) was subjected to SeP knockout (KO) and wild-type (WT) mice for 2 weeks. LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice. Lung weight/TL was significantly smaller in SeP KO than in WT mice. TAC-induced cardiac upregulation of the fetal type genes, including atrial and brain natriuretic factors, was significantly attenuated in SeP KO compared to WT. Furthermore, azan staining revealed that there was significantly less interstitial fibrosis in hearts after TAC in SeP KO than in WT mice. Expression of SeP in the liver of WT mice was significantly increased by TAC, while expression in the heart was unchanged. Hepatocyte-specific SeP KO mice were generated using the albumin-Cre-LoxP system. LV weight/TL was significantly smaller in hepatocyte-specific SeP KO than in WT mice. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL after TAC compared to that in other SeP KO mice. Conclusions These results suggest that cardiac pressure overload induced hepatic expression of SeP and the absence of endogenous SeP attenuated cardiac hypertrophy, dysfunction and fibrosis in response to pressure overload in mice. SeP possibly plays a maladaptive role against progression of heart failure through the cardiohepatic interaction.

Objective: The early diagnosis and treatment initiation for children with familial hypercholesterolemia (FH) has been recommended in guidelines. However, there is limited data on the impact of early treatments on the prognosis of children with FH. To investigate if the early initiation of lipid-lowering therapies among Japanese pediatric patients with FH reduced the occurrence of cardiovascular disease (CVD) events in them. Methods: We retrospectively investigated the occurrence of CVD events (myocardial infarction, unstable angina, or coronary artery revascularization) in patients with FH (N = 1050, male/female = 490/560), including 106 children below 20 years. We compared a variety of phenotypes, including genetic backgrounds, other complications, LDL cholesterol, medical therapies, and their prognoses between the patients’ diagnoses before the age of 20 years (children, mean age = 15 years) and after that age (adults, mean age = 52 years). Overall, 290 patients (27.6%) had a history of prior CVD events. Results: The median follow-up duration was 12.6 [9.5–17.9] years. The baseline LDL cholesterol level, 239 mg/dL, dropped to 112 mg/dL with the treatments. The Achilles tendon thickness was significantly lower in children than that of adults (7.2 vs. 8.9 mm, P < 0.001). Over the follow-up duration, 119 CVD events were observed. Importantly, no CVD event was observed in children despite their median LDL cholesterol level at follow-up being significantly higher than that of adults (122 vs. 111 mg/dL, P < 0.001). Conclusion: The likelihood of CVD events in those with FH diagnosed and treated in childhood is low.

Abstract Aims This study was designed to examine the association between coronary artery calcium (CAC) and major adverse cardiac events (MACEs) and to estimate CAC progression in patients with familial hypercholesterolemia (FH) in primary prevention settings. Methods and results Data of patients with FH admitted to our university hospital between 2000 and 2020, who underwent CAC measurement and were followed up (N = 622, male = 306), were retrospectively reviewed. Risk factors for MACEs were determined using the Cox proportional hazard model. The median follow-up duration was 13.2 years (interquartile range: 9.8–18.4 years). We observed 132 MACEs during the follow-up period. The event rate per 1,000 person-years for CAC scores of 0 (N = 283 [45.5%]), 1–100 (N = 260 [41.8%]), and >100 (N = 79 [12.7%]) was 1.2, 17.0, and 78.8, respectively. Log (CAC score+1) was a significant predictor of the occurrence of MACEs (hazard ratio: 3.24; 95% confidence interval: 1.68–4.80; p = 5.6×10−5) in the multivariate Cox regression analysis, independent of other factors. The risk discrimination of MACEs was enhanced by adding CAC information to other conventional risk factors (C-statistics: 0.833–0.934; p = 1.4×10−15). The regression equations between age and the CAC score in male and female were Y = 0.074X–1.52 and Y = 0. 087X–3.21, respectively. Conclusion CAC score helps in further risk stratification in patients with FH. Furthermore, the average age of CAC onset was 21 years in male and 37 years in females.

Abstract Background Bone marrow (BM) contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in BM stroma and is present in peripheral blood and the ischemic coronary artery. NGFR transduces signals towards cell survival or apoptosis in different cell types according to the coupling co-receptors. The previous clinical study demonstrated that low gene expression of NGFR in peripheral leucocytes in patients with acute coronary syndrome predicted 5-year repetitive coronary interventions at a de novo lesion. Purpose To investigate the hypothesis that BM-derived NGFR-positive (NGFR+) cells are associated with arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -knockout (KO) mice were subjected to unilateral carotid artery ligation after BM transplantation (BMT) from green fluorescent protein-positive (GFP+) NGFR-WT or -KO mice (N = 4-6). The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human peripheral blood NGFR+ mononuclear cells (MNCs) from a healthy volunteer were sorted using FACS Aria II and co-cultured with smooth muscle cells (SMCs) to examine early apoptosis (AnnexinV+7AAD-) in response to the NGFR ligands; proNGF and NGF. Also, human NGFR+MNCs from three healthy volunteers underwent RNA sequencing analysis and were immunophenotypically characterised by flow cytometry. Results In WT mice, NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly larger in the mice with BM-specific depletion of NGFR than that in WT mice. After BMT from GFP+NGFR-WT mice, NGFR+GFP+ cells accumulated in the neointima, exhibited apoptosis (TUNEL+ and cleaved caspase-3+), and promoted F4/80+GFP+ macrophage accumulation. By contrast, in the BM-specific NGFR-KO model, medial SMCs (αSMA+GFP-) occupied the neointima with augmented proliferation, and GFP+ cells were rare. Notably, apoptotic NGFR+ cells were accompanied by the accumulation of macrophages (F4/80+NGFR-) and expressed IL-10, which were abolished by BM-specific depletion of NGFR. Furthermore, human NGFR+, but not NGFR- cells, co-cultured with SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated NGF expression in SMCs in vitro, and proNGF was expressed in a ligated artery, implying that local proNGF might cause NGFR+ cell apoptosis. Meanwhile, RNA sequencing and flow cytometric analysis demonstrated that human NGFR+MNCs resemble dendritic cells (DCs) and predominantly express the plasmacytoid DCs markers. Consistently, NGFR+ cells in the neointima were positive for HLA-DR and CD11c. Conclusions BM-derived NGFR+ cells expressed dendritic cell markers and contributed to the suppression of arterial remodelling mediated by apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis and coordinately inhibited medial SMC migration to, and proliferation in, the neointima.

Background and aims: No previous study has investigated the association between attainment of low-density li-poprotein (LDL) cholesterol treatment target and better prognosis in patients with familial hypercholesterolemia (FH). The current research aimed to examine the association between attainment of LDL cholesterol treatment target and major adverse cardiac events (MACEs) in patients with FH to validate the current LDL cholesterol treatment targets in primary (<100 mg/dL) and secondary (<70 mg/dL) prevention settings.Methods: The data of patients with FH who were admitted to Kanazawa University Hospital between 2000 and 2020 and who were followed-up were retrospectively reviewed. The number of MACEs, including mortality associated with cardiovascular disease, unstable angina, and myocardial infarction per 1000 person-years, was calculated for each stratum for the attainment of LDL cholesterol target.Results: The median follow-up duration was 12.6 years. In total, 132 MACEs were recorded during the follow-up period. The numbers of patients who attained the LDL cholesterol target in the primary and secondary prevention groups were 228 (31.9%) and 40 (11.9%), respectively. The event rates per 1000 person-years for LDL cholesterol levels of <100 and >= 100 mg/dL in the primary prevention group were 2.6 and 4.4, respectively. The event rates per 1000 person-years for LDL cholesterol levels of <70 and >= 70 mg/dL in the secondary prevention group were 15.3 and 27.5, respectively.Conclusions: Attainment of the LDL cholesterol target is associated with better prognosis in patients with FH. However, the attainment rate is currently inadequate among Japanese.

Abstract Background Therapeutic angiogenesis mediated by stem/progenitor cells is an attractive therapeutic option against cardiovascular disease (CVD). Adipose tissue (AT) can be safely obtained even in CVD patients with anti-platelet medications, and it is a readily available source of culture-expanded adipose-derived stem cells (ADSCs) for transplantation. Single-cell transcriptome enables us to screen all the surface markers at once, while conventional strategies have been limited for the number of target markers. Furthermore, gene profiling at single-cell resolution can be used for the quantification of each marker by how many favorable cells can be purified without mixing of detrimental cells. Purpose We aimed to identify and characterize a cell population with in vivo angiogenic potential by single-cell RNA sequencing (scRNA-seq) analysis and xenograft experiments. Methods We revisited scRNA-seq datasets of single cell fraction from AT, bone-marrow (BM), and umbilical-cord blood (UCB, n=6/organ) to find cell populations with pro-angiogenic potential. Next, we collected AT from CVD patients (n=23) and used multicolor flow cytometry to quantify and sort the specific populations. PBS, the specific marker-negative and unsorted ADSCs were used as controls. Xenograft models of PKH26 pre-labeled human ADSC transplantation in limb ischemia were used to evaluate the lectin capillary density, PKH+ engrafted ADSCs, and blood flow recovery. Results Clustering divided CD45–CD31–CD34+ progenitor fraction into 3 clusters. We identified pro-/anti-angiogenic clusters based on the expressions of well-known pro-/anti-angiogenic factors. All genes encoding cell-surface proteins were compared in this functional clustering, resulted in 17 markers screened (Fig. 1A, B). Taken together with enrichment analysis, CD271+ cells showed predominant and pro-angiogenic gene profile from the other top candidates including CD36 and CD54 (Fig. 1C, D). Next, we evaluated the number and gene profile of CD271+ cells in well-known stem cell sources including BM and UCB. Surprisingly, the number of CD271 expressing cells were significantly lower and did not show angiogenic gene profile in BM and UCB (Fig. 2A). In analysis of AT from 23 CVD patients, CD271+ cells were significantly decreased by donor insulin resistance (Fig. 2B). Cell therapy using CD271+ ADSCs demonstrated in vivo angiogenic capacity compared to those of CD271– ADSCs and PBS in limb ischemia model. Furthermore, CD271+ ADSC transplantation showed enhanced efficacy compared to unsorted ADSCs from the same donors (Fig. 2C–E). Conclusion In this study, we identified CD271+ cell population in AT as an angiogenic cell population through scRNA-seq analysis and cell therapy experiments. AT obtained from donors without insulin resistance would be the most suitable for CD271+ ADSC isolation. CD271+ ADSC transplantation with a promising angiogenic capacity could contribute better cell-based therapy tackling CVD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science (JSPS) KAKENHI (Tokyo, Japan)

Abstract Background Genetic testing for inherited arrhythmias and discriminating pathogenic from benign variants are integral for the gene-based medicine. However, the high throughput in vivo functional analysis for the rare variants of the KCNQ1 potassium channel is scarce. Purpose We tested the utility of the in vivo zebrafish cardiac assay for determining the pathogenicity of the KCNQ1 variants identified in patients with long QT syndrome (LQTS) and atrial fibrillation (AF). Methods We generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the KCNQ1 homolog in zebrafish (kcnq1+/+). To test the utility of the cardiac assay, we used five KCNQ1 variants identified in patients with LQTS or familial AF. Human wild-type or mutant KCNQ1 cRNA (Q1) was co-injected with human KCNE1 cRNA (E1) into the F3 generation embryos with homozygous deletions. We dissected the hearts from the thorax at 72 hour-post-fertilization and measured transmembrane potential in zebrafish heart using the disrupted patch technique. Action potential duration was calculated as the time interval between the peak maximum upstroke velocity and 90% of repolarization (APD90). We compared the APD90s with patients' clinical phenotype and IKs density measured by patch-clamp technique in heterologous system. Results The mean APD90 of embryos with kcnq1del/del was 279±48 ms, which was restored by injecting Q1 WT and E1 (159±29 ms) to that with kcnq1+/+ (167±28 ms). We tested if the mean APD90 of embryos with kcnq1del/del was restored (shortened) by injecting the KCNQ1 variants. First we tested the dominant negative variant p.S277L and the trafficking deficient variant p.T587M. Patients with these variants showed significant prolonged QT intervals, and patch clamp study showed both variants caused the non-functional channels. Zebrafish cardiac assay showed the mean APD90 of embryos with kcnq1del/del+ Q1 S277L+E1 or Q1 T587M+E1 was significantly longer than that with kcnq1del/del+Q1 WT+E1 (Table). Next we tested in-frame variant c.1472_1473 ins GGACCT, which was identified from a patient with AF and normal QT interval. Patch clamp study showed the current density of the mutant KCNQ1 channel with KCNE1 was comparable to that of wild-type KCNQ1 channel with KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del shortened by injecting Q1 insACCTGG +E1 (Table). Finally we tested a missense variant p.R451Q, which was identified from a patient with LQTS. Patch clamp study showed the currents in the cells transfected with R451Q+KCNE1 were similar to those with WT+KCNE1. Zebrafish assay showed the mean APD90 of embryos with kcnq1del/del+Q1 R451Q+E1 was longer than that with kcnq1del/del+Q1 WT+E1 (Table). Conclusions Functional analysis of in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with LQTS. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Grant-in-Aid for Scientific Research (C)

Abstract Background The incidence of Brugada syndrome (BrS) varies among racial groups. Several studies reported Glycerol-3-Phosphate Dehydrogenase 1-Like (GPD1-L) gene is associated with BrS. However, most of these studies were reported from Western countries, so the evidence about GPD1-L mutation is limited especially among Asian BrS patients. This study aimed to search for rare variants in GPD1-L among Japanese BrS patients and to investigate the pathogenicity. Method We performed whole-exome sequencing for patients with Brugada type 1 ECG pattern from Japanese multicenter BrS cohort consisting of SCN5A-negative BrS probands (n=288) and controls (n=372). We conducted patch-clamp study in human embryonic kidney (HEK) 293 cells cotransfected with the wild-type sodium channel (SCN5A) and wild-type or mutant GPD1-L expression plasmid. Results We identified a rare variant in GPD1-L, p.D262N (c.784g>a) in 2 of 288 BrS probands, which was not identified in 372 controls. The minor allele frequency of the variant is 0.0014% in the Genome Aggregation Database. One proband was a 49-year-old man and the other was 34-year-old man who both developed a ventricular fibrillation. ECGs of both probands showed Brugada Type 1 pattern after administration of the pilsicainide. In functional study, coexpression of D262N GPD1-L with SCN5A in HEK293 cells significantly reduced inward sodium currents compared with wild-type GPD1-L. Additionally, inward sodium currents with D262N were similar to those with A280V GPD1-L, which was associated with BrS in previous reports (Figure). Also, several pathogenicity prediction programs, such as SIFT (score: 0.031) and PolyPhen2 (score: 0.937) predicted deleterious effects of GPD1-L D262N. Conclusion We identified a rare variant in GPD1-L at the rate of 0.7% in Japanese BrS patients without SCN5A mutations. GPD1-L, p.D262N reduces inward sodium currents and may be a novel susceptible variant for BrS in the Japanese population. Funding Acknowledgement Type of funding sources: None. Figure 1. Current–voltage curve

Abstract Background Pulmonary arterial hypertension (PAH) causes congestive liver due to right heart failure. There are few cases of PAH that lead to liver cirrhosis, and little attention is paid to liver function in PAH patients. However, it is certain that long-term congestion due to right heart failure in PAH causes a gradual exacerbation of liver dysfunction and affects metabolic function. Purpose The purpose of this study is to investigate liver fibrosis associated with the severity and prognosis of PAH. Methods This retrospective observational study was included 57 PAH patients and 22 control subjects. PAH patients were assigned to three risk variables according to the simplified risk stratification proposed at the 6thWSPH 2018 after measuring hemodynamic parameters using right heart catheterization, WHO functional class, 6- minutes walking distance (6MWD), and BNP plasma levels. The Fibrosis-4 (FIB4)-index, a liver fibrosis marker, was calculated using the formula: FIB-4 = Age (years) × AST (U/L) / [PLT (109/L) × ALT1/2 (U/L)], and assessed for association with severity of PAH. PAH patients were followed up for 12 years to assess the occurrence of major adverse event, such as death or lung transplantation. Next, adult 8-week-old C57BL/6 mice were exposed to chronic hypoxia (10% O2) or normoxia for 6 weeks. Then, mice were anesthetized and performed right heart catheterization. Liver tissue was collected for histological assessment by Hematoxylin and eosin and Azan staining, and evaluated RNA expression involved in liver fibrosis by real-time PCR. Results The levels of FIB4-index in intermediate and high risk groups of PAH patients had significantly increased compared to those in control group. In PAH patients, FIB4-index was not obviously correlated with hemodynamic parameters, BNP, or 6MWD. Major adverse events occurred in 18 PAH patients (32%): death in 18 (100%) and lung transplant in none (0%). Kaplan-Meier curves for PAH patients with and without major adverse events were constructed based on a cut-off frequency of 2.001 for FIB4-index. During the 12-years follow-up period, major-event-free survival was significantly better in PAH patients with FIB4-index <2.001 than in patients with FIB4-index >2.001 (hazard ratio, 3.3; P=0.038). In a PAH model mice, hemodynamic parameters showed that chronic hypoxia significantly increased the right ventricular systolic pressure. In histological analysis, there was no significantly difference in liver fibrosis in hypoxia or normoxia group. However, the RNA expression such as αSMA and TGFβ1 associated with liver fibrosis in PAH model mice was increased compared to control mice. Conclusion This study showed that the liver fibrosis gradually progressed subsurfacely with severity of PAH. Even the slight liver dysfunction may affect metabolism and cause exacerbation of PAH, so it might be necessary to pay attention to liver fibrosis as one of the risk factors of PAH. Funding Acknowledgement Type of funding sources: None.

Abstract Introduction Left ventricular diastolic dysfunction (LVDD) is the main cause of heart failure with preserved ejection fraction (HFpEF). LVDD is related not only to arterial stiffness but also sympathetic nerve activity (SNA). Recent study demonstrated that increased muscle sympathetic nerve activity (MSNA) may be one of contributing factor for arterial stiffness. In clinical practice, Cardio-ankle vascular index (CAVI) provides a reproducible index of arterial stiffness, independent of blood pressure (BP). Recently, Arterial Velocity pulse Index (AVI), which is an index of arterial reflected waves, have been proposed as new index of arterial stiffness. We reported that AVI was associated with MSNA in hypertensive (HT) patients. However, it is still uncertain the effect of LVDD on the association between AVI and SNA in HT patients. Thus, we tested the hypothesis that AVI would be increased and related to MSNA in HT patients with LVDD. Methods Patients with essential HT subjects were included in this study. HT was diagnosed as systolic blood pressure (SBP) ≥140mmHg or diastolic blood pressure (DBP) ≥90mmHg. Patients with secondary HT was excluded. AVI was measured from left upper arm by NAS-1000 (Nihon Koden, Japan). CAVI was measured by VaSera VS-1500A (Fukuda Denshi, Japan). Transthoracic echocardiography was performed by trained sonographers. SNA was evaluated by direct recording of MSNA from peroneal nerves. Results 25 HT patients were included (age 63±14 years, Male/Female 9/16). They were divided into two groups according to E/e' (no LVDD group, E/e' ≤9, N=12; LVDD group, E/e' >9, N=13). There were no significant differences between no LVDD and LVDD groups in age (63±9 vs 69±9 years p=0.205), body mass index (23±3 vs 24±4 p=0.355), BP (SBP 139±16 vs 144±20mmHg p=0.524, DBP 87±15 vs 78±14mmHg p=0.167). LV Ejection Fraction (EF) and Stroke Volume (SV) did not differ between two groups (EF 66±7 vs 69±6% p=0.471, SV 58±7 vs 62±14ml p=0.599). MSNA had tendency to increase in LVDD group compared to no LVDD group (MSNA 53±10 vs 44±12 bursts/100 heartbeats, p=0.052). Contrary to our hypothesis, AVI and CAVI did not differ between two groups (AVI 27±7 vs 29±7 p=0.398, CAVI 8.7±1.4 vs 8.6±1.4 p=0.894). However, a significant correlation was seen between AVI and MSNA in no LVDD group (r=0.57, p<0.05), but no correlation in LVDD group. There is no correlation between CAVI and MSNA in no LVDD and LVDD group. Significant relationship was observed between AVI and CAVI in LVDD group (r=0.61, p<0.05), but no relationship in no LVDD group. Conclusion AVI was significantly associated with MSNA in HT patients without LVDD, but not with LVDD. CAVI was related to AVI in HT patients with LVDD, but not without LVDD. MSNA was slightly increased in HT patients with LVDD compared to without LVDD. These results indicate that augmented SNA could contribute to the increase in arterial stiffness in HT patients without LVDD, however, this contribution might be attenuated in HT patients with LVDD. Funding Acknowledgement Type of funding sources: None.

Abstract Background Increased arterial stiffness characterize by aging. It is reported that age-related increases in muscle sympathetic nerve activity (MSNA) may be one of contributing factor for arterial stiffness. Arterial reflected wave was composed of SNA and aging. Increased arterial reflected wave partly plays an important role in blood pressure. Recently, we reported that arterial velocity pulse index (AVI), a novel index of arterial reflected waves, was associated with MSNA in hypertensive patients. It is still uncertain the effect of age on the association between AVI and SNA in hypertensive patients. Method Patients with essential HT and matched non-hypertensive control subjects were included in this study. HT was diagnosed as systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg. AVI was measured from left upper arm by NAS-1000. SNA was evaluated by direct recording of muscle sympathetic nerve activity (MSNA) from peroneal nerves. Results 45 HT patients and 46 control subjects were included. Age, SBP and DBP were significantly increased in HT group compared to control (Age 63±14 vs 42±16 years, p&lt;0.001; SBP 144±16 vs 115±9 mmHg, p&lt;0.001; DBP 80±14 vs 67±9 mmHg, p&lt;0.001). MSNA and AVI were significantly increased in HT group compared to control (MSNA 34±10 vs 25±8 bursts/min, p&lt;0.05; AVI 28±9 vs 17±5, p&lt;0.05). AVI was significantly correlated with MSNA, age, and SBP in HT group. HT group was divided into two groups according to their age (group 1, age ≤63 N=21, group 2, age ≥64 N=26). AVI in group 1 showed correlation with MSNA (r=0.59, p&lt;0.05), but no correlation was seen in group 2. However excluded SBP&gt;160 mmHg subjects in group 2, significant correlation was clarified between AVI and MSNA (r=0.62, p&lt;0.05). Conclusion The relationship between AVI and MSNA in HT patients is preserved regardless of aging, however, high blood pressure over 160mmHg might obscure its correlation. These results indicate that AVI is useful to estimate sympathetic nerve activity in high aging HT patient treated &lt;160 blood pressure. Funding Acknowledgement Type of funding sources: None.

Abstract Background The genetic bases of cardiac conduction-system disease (CCSD) range from ion channelopathies to mutations in many other genes. Genome-wide association studies have shown common variants in SCN10A influence cardiac conduction. However, it has not yet to be determined whether vulnerability to CCSD is associated with rare coding sequence variation in the SCN10A gene. Purpose We sought to determine the clinical impact of rare variants in SCN10A in patients with CCSD and classified the variants according to the 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines. Methods We performed screening for rare variants (minor allele frequency ≤0.001) in SCN10A in CCSD patients with an onset at a young age under 65 or those who had a family history of pacemaker implantation (PMI) (n=40; 18 female; mean age, 41±18 years). We transiently expressed engineered variants in ND 7/23 cells, and conducted whole-cell voltage clamp experiments to clarify the functional properties of the Nav1.8 current. Results We identified nine rare variants in SCN10A in 7 patients. Two patients were carriers of two rare variants in SCN10A and 5 were carriers of one rare variant in SCN10A. Four patients were affected with sinus node dysfunction, 1 were atrioventricular block, and 2 were both dysfunctions. We performed electrophysiological study for 8 of 9 rare variants. It demonstrated that 2 rare variants showed gain-of-function, and 3 rare variants showed loss-of-function. We finally determined 5 likely pathogenic variants in SCN10A in 5 patients (12.5%) according to the ACMG standards and guidelines. All 5 patients underwent a pacemaker implantation at an average age of 43±16. Conclusions These results demonstrate that SCN10A variants play a pivotal role in enhanced susceptibility of CCSD. We suggest the importance for screening SCN10A variants in clinical settings. Funding Acknowledgement Type of funding source: None

Abstract Background Arterial reflected wave is determined by not only atherosclerosis but also sympathetic nerve activity. Recently, Arterial Velocity pulse Index (AVI), which is an index of arterial reflected waves, and Arterial Pressure volume Index (API), which is an index of volume of a conductive blood vessel, have been proposed as new index of arterial stiffness. However, it is unclear whether API and AVI would be associated with muscle sympathetic nerve activity (MSNA) in hypertensive subjects. Purpose The purpose of this study was to evaluate the correlation between AVI, API and MSNA in hypertensive subjects. Method 41 hypertensive patients and 40 non-hypertensive subjects were included in this study. We performed a cross-sectional, observational study. Hypertension (HT) was defined as systolic blood pressure (SBP) ≥140 mmHg, diastolic blood pressure (DBP) ≥90 mmHg or medical treatment for HT. AVI and API was measured by NAS-1000 (Nihon Koden, Japan). MSNA, central sympathetic outflow to peripheral muscle, was recorded directly from peroneal nerve. MSNA was expressed by burst frequency (bursts/minute) and burst incidence (bursts/100heartbeats). Blood pressure, heart rate and MSNA were recorded simultaneously. Results Age, systolic and diastolic pressure were significantly higher in hypertensive patients compared to control (40±15 vs 61±13 years, p<0.001; 142±16 vs 113±9 mmHg, p<0.001; 81±14 vs 67±9 mmHg, p<0.001). MSNA and AVI were significantly augmented in hypertensive patients compared to control (34±11 vs. 23±6 bursts/min, p<0.05; 26±7 vs. 16±4, p<0.05). AVI was correlated with MSNA in each group (hypertension: r=0.59, P<0.001, non-hypertension: r=0.51, p<0.001). However, no correlation was shown between API and MSNA in each group (hypertension: r=0.22, p=0.15, non-hypertension: r=0.07, p=0.63). Multiple regression analysis also showed MSNA was significantly related with AVI but was not with API. Conclusion Our finding showed that AVI relates to MSNA independent of API in patients with hypertension. It suggested that Novel index of arterial reflected waves, AVI, is helpful to estimate augmented SNA in hypertensive subjects regardless of volume of a conductive blood vessel. Funding Acknowledgement Type of funding source: None