The effects of romosozumab combined with active vitamin D3 on fracture healing in ovariectomized rats.

BACKGROUND: In this study, we investigated the potential acceleration of fracture healing and bone mineral density-increasing effects of romosozumab and active vitamin D3 combination therapy for fractures in ovariectomized rats. METHODS: Ovariectomy was performed on 40 24-week-old female Sprague-Dawley rats. After 8 weeks, the rats were subjected to periosteum removal and osteotomy of the femoral shaft followed by osteosynthesis with intramedullary nailing to create fracture models. The rats were then divided into four groups: C group (control), R group (receiving romosozumab at 25 mg/kg once a month via subcutaneous injection), VD group (receiving active vitamin D3 at 0.2 µg/kg twice a week via subcutaneous injection), and R + VD group. Further, 10 rats were included in a sham group. At 10 weeks after the intervention, both femurs were removed and blood samples were collected from all rats. Soft X-ray imaging was used to evaluate bone union, and microcomputed tomography (micro-CT) was used for bone morphometric evaluation. Toluidine blue staining was used for the histopathological evaluation of the undecalcified specimens, and bone turnover marker levels were measured using enzyme-linked immunosorbent assay. RESULTS: Bone morphometry analysis via micro-CT revealed increased mineral density of the trabecular bone in the R + VD group femurs, demonstrating the effectiveness of romosozumab plus active vitamin D3 combination therapy. However, there were no differences in bone union evaluated using soft X-ray imaging, indicating no acceleration of fracture healing. CONCLUSIONS: Although romosozumab and active vitamin D3 combination therapy increased trabecular bone volume, there was no evidence on its ability to accelerate fracture healing.

Clinical Significance of the Left Atrial Appendage Orifice Area.

Objective The left atrial appendage (LAA) is one of the major sources of cardiac thrombus formation. Three-dimensional transesophageal echocardiography (TEE) made it possible to perform a detailed evaluation of the LAA morphologies. This study aimed to evaluate the clinical implications of the LAA orifice area. Methods A total of 149 patients who underwent TEE without significant valvular disease were studied. The LAA orifice area was measured using three-dimensional TEE. The patients were divided into two groups according to the LAA orifice area (large LAA orifice group, ≥median value, and small LAA orifice group). The clinical characteristics and echocardiographic findings were evaluated. Results The median LAA orifice area among all patients was 4.09 cm2 (interquartile range 2.92-5.40). The large LAA orifice group were older (67.2±10.4 vs. 62.4±15.3 years, p=0.02), more often had hypertension (66.7% vs. 44.6%, p=0.007), and atrial fibrillation (70.7% vs. 39.2%, p<0.001) than the small LAA orifice group. Regarding the TEE findings, the LAA flow velocity was significantly lower (33.7±20.0 vs. 50.2±24.3, p<0.001) and spontaneous echo contrast was more often observed (21.3% vs. 8.1%, p=0.02) in the large LAA orifice group. Multivariate models demonstrated that atrial fibrillation was an independent predictor of the LAA orifice area. In the analysis of atrial fibrillation duration, the LAA orifice area tended to be larger as patients had a longer duration of atrial fibrillation. Conclusion Our findings indicated that a larger LAA orifice area was associated with the presence of atrial fibrillation and high thromboembolic risk based on TEE findings. A continuation of the atrial fibrillation rhythm might lead to the gradual expansion of the LAA orifice.

The Influence of Eicosapentaenoic Acid to Arachidonic Acid Ratio on Long-term Cardiovascular Events Following Percutaneous Coronary Intervention.

Objective The relationship between cardiovascular disease and the serum polyunsaturated fatty acid parameters has been reported. The aim of the present study was to investigate the association between the eicosapentaenoic acid and arachidonic acid (EPA/AA) ratio and long-term cardiovascular events in patients with coronary artery disease. Methods We identified a total of 831 patients who underwent percutaneous coronary intervention and whose EPA/AA ratio was available. The patients were divided into two groups according to their serum EPA/AA ratio (median, 0.29; interquartile range 0.19-0.47): those in the lower quartile of EPA/AA ratios (Low EPA/AA group; n=231) and all other subjects (High EPA/AA group; n=600). The primary endpoints included a composite of cardiovascular death, myocardial infarction, and ischemic stroke. Results Patients in the Low EPA/AA group were significantly younger (66.0±12.6 years vs. 69.9±9.3 years, p<0.001), current smokers (33.3% vs. 22.7%, p=0.002), and had a history of myocardial infarction (20.3% vs. 12.3%, p=0.003). During the follow-up (median, 1,206 days; interquartile range, 654-1,910 days), the occurrence of the primary endpoint was significantly higher in the Low EPA/AA group than in the High EPA/AA group. Of note, the rate of cardiovascular death was significantly higher in the Low EPA/AA group, and the rates of myocardial infarction and stroke tended to be higher. Conclusion A low EPA/AA ratio was associated with long-term adverse cardiovascular events in Japanese patients with coronary artery disease.

Relationship between epicardial adipose tissue volume and coronary artery spasm.

BACKGROUND: Epicardial adipose tissue (EAT) is considered to play a critical role in vascular endothelial function. Coronary artery spasm has been postulated to be a causal factor in vascular endothelial abnormalities and atherosclerosis. This study aimed to investigate the relationship between coronary artery spasm and EAT volume, total abdominal adipose tissue (AAT) area, and abdominal visceral adipose tissue (AVAT) area. METHOD: Among patients undergoing coronary computed tomography (CT) to evaluate coronary artery disease, we identified 110 patients who did not have significant coronary artery stenosis and underwent a coronary spasm provocation test with cardiac catheterization. They were divided into two groups according to the results of the spasm provocation test: spasm-positive and spasm-negative. EAT volume, total AAT area, and AVAT area were evaluated using CT images. RESULTS: Seventy-seven patients were included in the spasm-positive group and 33 patients in the spasm-negative group. There were no significant differences in baseline clinical characteristics between the two groups, except for the prevalence of current smoking (48% vs. 27%, p = 0.04). EAT volume was significantly higher in the spasm-positive group (108 ±â€¯38 mL vs. 87 ±â€¯34 mL, p = 0.007), while no significant difference was seen in total AAT area (280 ±â€¯113 cm2 vs. 254 ±â€¯128 cm2, p = 0.32) or AVAT area (112 ±â€¯54 cm2 vs. 98 ±â€¯55 cm2, p = 0.27). Multivariate logistic analysis indicated that EAT volume (per 10 cm3) (odds ratio, 1.198; 95% confidence interval, 1.035-1.388; p = 0.016) was a significant predictor of coronary artery spasm. CONCLUSION: Our results suggest that EAT has a strong association with coronary artery spasm, while AAT may not.

Clinical outcomes following percutaneous coronary intervention for bifurcation lesions: kissing balloon inflation vs. sequential dilation.

Percutaneous coronary intervention for bifurcation lesions remains challenging, with there being several debatable issues, including the requirement for kissing balloon inflation (KBI). The objective of this study was to assess the clinical outcomes following single crossover stent implantation with KBI or sequential dilation alone. Data were examined for 255 non-left main bifurcation lesions (246 patients) treated with single crossover stent implantation, followed by side branch (SB) strut dilation with KBI (n = 74 lesions) or sequential dilation (n = 181 lesions) in three hospitals. Target lesion revascularization (TLR) was the primary endpoint. There was no significant difference in the pre-procedural reference diameter of both the main vessel (MV) and SB between the KBI and sequential dilation groups. However, MV post-dilation balloon size was smaller with lower pressure and post-procedural minimal lumen diameter was significantly smaller in the KBI group. During the median follow-up period of approximately 3 years, TLR incidence was significantly higher in the KBI group than in the sequential dilation group; in particular, the TLR rate at the distal MV was higher in the former. For bifurcation lesions treated with single crossover stent implantation, the TLR rate was higher after KBI than after sequential dilation; this was mainly due to higher revascularization in the distal MV. For bifurcation lesions treated with KBI, MV post-dilation balloon diameter tended to be smaller with lower pressure, which might lead to poorer stent expansion and a higher TLR rate.

Lipid-rich large plaques in a non-culprit left main coronary artery and long-term clinical outcomes.

BACKGROUND: An integrated backscatter (IB) intravascular ultrasound (IVUS) provides an information about tissue components and vulnerability of coronary plaques. The presence of vulnerable plaque in non-culprit lesion is associated with future clinical events. The purpose of this study was to assess the association between the characteristics of non-culprit left main coronary artery (LMCA) plaques evaluated by IB-IVUS and long-term clinical outcomes in patients undergoing percutaneous coronary intervention (PCI). METHODS: Among the patients who underwent non-LMCA PCI, we studied 366 patients with adequate LMCA IVUS images. Conventional and IB-IVUS analyses of the LMCA segment were performed. Lipid-rich large plaque was defined as the presence of both a lager plaque volume and a higher percentage of the lipid component than the obtained median values. Major adverse cardiovascular events (MACE) included cardiac death, myocardial infarction, and unplanned revascularization. RESULTS: The mean age of the patients was 68.5 ± 10.2 years, 79.8% were men. Median follow-up period was 6.0 years (IQR: 4.2-8.1 years). The incidence of MACE was significantly higher in patients with lipid-rich large plaques (P = .006). The incidence rates of cardiac death, myocardial infarction, and unplanned revascularization were significantly higher in patients with lipid-rich large plaques (P = .02, 0.004, and 0.02, respectively). Multivariate Cox regression analysis showed that the presence of a lipid-rich large plaque was significantly associated with MACE (HR: 1.74; 95%CI: 1.17-2.58; P = .006). CONCLUSION: The presence of lipid-rich large plaques in a non-culprit LMCA can be associated with the long-term MACE in patients who have undergone PCI.

Impact of Diabetes Mellitus on the Aortic Wall Changes as Atherosclerosis Progresses: Aortic Dilatation and Calcification.

AIM: An inverse association between diabetes mellitus (DM) and aortic dilatation has recently been reported. However, little is known about the association between DM and the progression of aortic dilatation/calcification as atherosclerosis progresses. METHODS: We identified 216 patients who had undergone percutaneous coronary intervention (PCI) and abdominal computed tomography (CT) during the PCI and follow-up phases. The patients were classified into two groups: those with DM (DM+ group; n=107) and those without DM (DM- group; n=109). The infrarenal aortic diameter and aortic calcification index (ACI) were measured, and annual changes were calculated using measurement results obtained during the PCI and follow-up phases. RESULTS: Infrarenal aortic diameters were significantly shorter in the DM+ group than in the DM- group during the PCI phase, and no significant ACI differences were observed between the DM+ and DM- groups. The median duration between the PCI and follow-up phase CT was 3.0 years. The growth rate of the infrarenal aortic dilatation from the PCI phase in the DM+ group was similar to that in the DM- group. Annual ACI changes were significantly larger in the DM+ group than in the DM- group. Multivariate logistic regression analysis indicated that the prevalence of DM was an independent predictor of rapid aortic calcification progression (odds ratio: 2.51; 95% confidence interval: 1.23-5.14; p=0.01). CONCLUSION: Our findings suggest that DM negatively affects aortic dilatation during an earlier phase of atherosclerosis progression and positively affects the progression of aortic calcification in a later phase.

Prognostic impact of recanalizing chronic total occlusion in non-infarct related arteries on long-term clinical outcomes in acute myocardial infarction patients undergoing primary percutaneous coronary intervention.

Although chronic total occlusion (CTO) in non-infarct-related arteries (non-IRAs) negatively affects long-term mortality in patients with acute myocardial infarction (AMI) who are undergoing primary percutaneous coronary intervention (PCI), the prognostic impact of successful CTO-PCI has not been completely addressed. Among 1855 consecutive patients with AMI who underwent primary PCI, those who were treated for CTO with either PCI or medical therapy were included. We evaluated the association between recanalization of CTO and long-term cardiac mortality. Of the 172 included patients, 88 underwent CTO-PCI, and the procedures were successful in 65 patients. Thus, the successfully recanalized CTO (SR-CTO) group included 65 patients; and the no recanalized CTO (NR-CTO) group, 107 patients. During the follow-up, 72 patients died, and of whom 56 (77.8%) died because of cardiac causes. The cumulative 10-year, 30-day, and 30-day to 10-year incidences of cardiac mortality were lower in the SR-CTO group than in the NR-CTO group (19.0% vs. 51.9% p = 0.004; 4.6% vs. 14.0%, p = 0.05; 15.0% vs. 44.1%, p = 0.003, respectively). After adjusting for confounding factors, the benefits of SR-CTO for the 10-year cardiac mortality remained significant compared with those of NR-CTO (hazard ratio 0.37; 95% confidence interval 0.17-0.75; p = 0.004). In conclusion, patients with SR-CTO in non-IRAs after AMI was associated with reduced long-term cardiac mortality compared with those with NR-CTO.

Trends in physiological coagulation factors in Japanese patients receiving novel oral anticoagulants.

BACKGROUND: Little is known about physiological anticoagulation effects via antithrombin III (AT III) and protein C/S (PC/PS) in patients using new oral anticoagulants (NOACs). METHODS: We evaluated 120 consecutive patients with non-valvular atrial fibrillation (AF) receiving NOACs. Patients were randomly divided into three groups: a dabigatran group (DG, N=40), a rivaroxaban group (RG, N=40) or an apixaban group (AG, N=40). A warfarin group (WG, N=40) was matched with NOAC groups for age, sex and type of AF during the same time period. Blood samples were obtained in pretreatment, trough and peak phases to measure the activity of physiological coagulation inhibitors, including AT III and PC/PS or thrombus formation markers such as D-dimer and thrombin-antithrombin complex (TAT). RESULTS: D-dimer, TAT and AT III values for the NOAC groups were equivalent in the peak and trough phases. PC/PS activity in both phases was equally maintained in the pretreatment phase in the NOAC groups, while the activity in the WG was significantly suppressed in steady state. Moreover, no differences in trends for PC/PS activity were observed among NOAC groups. CONCLUSIONS: PC/PS activity was constant in both peak and trough phases in the patients on NOACs compared with activity of those on warfarin. In addition, there was no difference in the findings among NOACs.

Increased expression of the adipocytokine omentin in the epicardial adipose tissue of coronary artery disease patients.

BACKGROUND AND AIMS: Omentin, an adipocytokine secreted by visceral adipose tissue, protects against obesity-linked cardiovascular complications. However, little is known about its role in epicardial adipose tissue (EAT) and coronary artery disease (CAD). We investigated the expression of omentin in EAT from CAD subjects. METHODS: EAT, subcutaneous adipose tissue (SCAT), and plasma samples were collected from CAD (n = 15; 23.3 ± 3.1 kg/m(2)) and non-CAD patients (n = 10; 20.8 ± 3.9 kg/m(2)). Omentin mRNA expression was measured using real-time PCR, while plasma concentrations were measured using an ELISA. EAT volume was determined with 64-slice computed tomography. RESULTS: Omentin expression in EAT and EAT volume were higher in CAD patients compared with controls (2.49 ± 2.6 vs. 0.85 ± 0.3, p = 0.002 and 113 ± 58 ml vs. 92.4 ± 30 ml, p = 0.045, respectively). Omentin expression in SCAT was similar between CAD and control patients (1.37 ± 0.84 vs. 1.07 ± 0.55, p = 0.267). Plasma omentin levels were lower in CAD patients compared with controls (343 ± 158 ng/ml vs. 751 ± 579 ng/ml, p = 0.025), and were negatively associated with the expression of omentin in EAT, in patients with CAD (ß = -0.78, p = 0.049). On the other hand, there was no association between omentin in EAT and clinical variables in patients with non-CAD. CONCLUSIONS: Omentin expression increases in the EAT of non-obese CAD patients, despite a decrease in plasma levels, suggesting that omentin may play a role in the pathogenesis of CAD.

Prognostic Value of Albuminuria on Cardiovascular Outcomes After Elective Percutaneous Coronary Intervention.

Albuminuria is the most widely evaluated marker of kidney damage. Many previous studies have demonstrated an association between the presence of albuminuria and increased cardiovascular events. However, there are limited data regarding the impact of albuminuria in patients requiring coronary revascularization. This study investigated whether the urinary albumin excretion rate could predict cardiovascular events in such a population. We enrolled 698 consecutive patients who underwent elective percutaneous coronary intervention. The baseline urinary albumin-to-creatinine ratio (ACR; mg/gCr) was measured and patients were divided into those with normoalbuminuria (ACR <30 mg/gCr), microalbuminuria (ACR 30 to 300 mg/gCr), or macroalbuminuria (ACR >300 mg/gCr). We collected data on the incidences of cardiac death and/or nonfatal myocardial infarction. We identified 389, 230, and 79 patients with normoalbuminuria, microalbuminuria, and macroalbuminuria, respectively. During follow-up (median: 1,564 days), 41 events occurred. The event-free survival rate was 89% in patients with macroalbuminuria, 92% in those with microalbuminuria, and 97% in those with normoalbuminuria, respectively (log-rank test p = 0.002). After adjustment for conventional risk factors, Cox analysis revealed hazard ratios for cardiac death and/or nonfatal myocardial infarction were 2.56 (95% CI 1.23 to 5.32, p = 0.01) in those with microalbuminuria and 4.02 (95% CI 1.59 to 10.12, p = 0.003) in those with macroalbuminuria compared with those with normoalbuminuria. In conclusion, an elevated urinary albumin excretion rate independently predicted adverse cardiovascular outcomes, with a gradual risk increase that progressed from microalbuminuria to macroalbuminuria in patients undergoing elective percutaneous coronary intervention.

Impact of abdominal and epicardial fat on the association between plasma adipocytokine levels and coronary atherosclerosis in non-obese patients.

OBJECTIVE: Ectopic fat accumulation is associated with coronary artery disease. Visceral adipose tissue has paracrine and systemic effects and is a source of adipocytokines. It has been implicated in the pathogenesis of coronary atherosclerosis; however, nothing is known about whether increases in epicardial fat have the same effect on coronary atherosclerosis as increases in abdominal visceral fat. METHODS: We examined 216 consecutive patients suspected to have coronary artery disease. Individuals with acute coronary syndrome and inadequate computed tomography (CT) imaging were excluded. We enrolled 164 patients (65 ± 10 years old; 70% men; body mass index [BMI], 23.8 ± 3.6 kg/m(2)). The plasma concentrations of adiponectin, interleukin-6 (IL-6), plasminogen activator inhibitor-1, and vascular endothelial growth factor were measured. The characteristics of coronary plaque, abdominal visceral fat area, and epicardial fat volume (EFV) were determined by 64-slice CT imaging. RESULTS: EFV was greater in subjects with noncalcified plaque than in those with no plaque or with calcified plaque (126 ± 39 mL vs. 98 ± 34 mL and 97 ± 45 mL, respectively; P = 0.010). EFV was significantly correlated with BMI, triglycerides, and the triglyceride/high-density lipoprotein cholesterol ratio (r = 0.51, 0.19, and 0.20, respectively) but not with plasma levels of adipocytokines. The plasma adiponectin and IL-6 concentration was significantly correlated with abdominal visceral fat area in coronary plaque patients (r = -0.49 and 0.20). CONCLUSIONS: In non-obese Japanese patients, epicardial fat may have unique mechanisms affecting the development of coronary atherosclerosis, which is different from abdominal visceral fat.

Developmental stages and germ cell lineage of the loach (Misgurnus anguillicaudatus).

The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20 degrees C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus.

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.

Lack of histamine alters gastric mucosal morphology: comparison of histidine decarboxylase-deficient and mast cell-deficient mice.

Histamine plays an important role in the regulation of gastric acid secretion; however, its role in maintenance of gastric morphology remains unclear. To clarify the necessity of histamine for gastric mucosal development and maintenance, we evaluated two different kinds of mice that lacked either mast cells (one of the gastric histamine-producing cell types) or histidine decarboxylase (HDC; a histamine-synthesizing enzyme). Measurements of stomach weight, intragastric pH, mucosal histamine levels, as well as serum gastrin and albumin levels were performed in mice. Gastric mucosal appearance was examined by immunohistochemical techniques. Although gastric mucosal histamine levels in mast cell-deficient mice were half of those observed in the wild-type mice, intragastric pH, serum gastrin levels, and gastric morphology at 12 mo were unchanged compared with the wild-type mice. In contrast, HDC-deficient mice possessed no detectable gastric histamine, but did exhibit hypergastrinemia, as well as marked increases in intragastric pH and stomach weight compared with the wild-type mice. Histological analysis revealed that 9-mo-old HDC-deficient mice demonstrated hyperplasia in the oxyntic glandular base region, as well as increased numbers of parietal and enterochromaffin-like cells. These results indicate that enterochromaffin-like cell-derived histamine is potentially involved in gastric mucosal morphology regulation.