C-Type Natriuretic Peptide Analogue Therapy in Children with Achondroplasia.

BACKGROUND: Achondroplasia is a genetic disorder that inhibits endochondral ossification, resulting in disproportionate short stature and clinically significant medical complications. Vosoritide is a biologic analogue of C-type natriuretic peptide, a potent stimulator of endochondral ossification. METHODS: In a multinational, phase 2, dose-finding study and extension study, we evaluated the safety and side-effect profile of vosoritide in children (5 to 14 years of age) with achondroplasia. A total of 35 children were enrolled in four sequential cohorts to receive vosoritide at a once-daily subcutaneous dose of 2.5 µg per kilogram of body weight (8 patients in cohort 1), 7.5 µg per kilogram (8 patients in cohort 2), 15.0 µg per kilogram (10 patients in cohort 3), or 30.0 µg per kilogram (9 patients in cohort 4). After 6 months, the dose in cohort 1 was increased to 7.5 µg per kilogram and then to 15.0 µg per kilogram, and in cohort 2, the dose was increased to 15.0 µg per kilogram; the patients in cohorts 3 and 4 continued to receive their initial doses. At the time of data cutoff, the 24-month dose-finding study had been completed, and 30 patients had been enrolled in an ongoing long-term extension study; the median duration of follow-up across both studies was 42 months. RESULTS: During the treatment periods in the dose-finding and extension studies, adverse events occurred in 35 of 35 patients (100%), and serious adverse events occurred in 4 of 35 patients (11%). Therapy was discontinued in 6 patients (in 1 because of an adverse event). During the first 6 months of treatment, a dose-dependent increase in the annualized growth velocity was observed with vosoritide up to a dose of 15.0 µg per kilogram, and a sustained increase in the annualized growth velocity was observed at doses of 15.0 and 30.0 µg per kilogram for up to 42 months. CONCLUSIONS: In children with achondroplasia, once-daily subcutaneous administration of vosoritide was associated with a side-effect profile that appeared generally mild. Treatment resulted in a sustained increase in the annualized growth velocity for up to 42 months. (Funded by BioMarin Pharmaceutical; ClinicalTrials.gov numbers, NCT01603095, NCT02055157, and NCT02724228.).

Increased serum myonectin and irisin levels with myonectin and FNDC5 expressions in polycystic ovary syndrome: a case control study.

The aim of this study is to assess the FNDC5 and myonectin expressions and serum levels of myonectin and irisin in women with PCOS. 90 participants were included in this case-control study. 45 of these participants were with PCOS, and 45 of them were healthy volunteers matched for age and body mass index (BMI). Serum irisin and myonectin levels were measured with commercially available enzyme-linked immune sorbent assay (ELISA) kits. Expression of the myonectin and FNDC5 genes were determined by RT-PCR analysis. It was found out that FSI, HOMA-IR, LH, LH/FSH, TT, serum irisin and serum myonectin levels, myonectin mRNA expression, and FNDC5 mRNA expression were higher in the PCOS group, whereas HDL-C level was lower in the PCOS group (p < .05). When the groups were compared, it was detected that IR and HA were significantly higher in the PCOS group (p < .05). Serum irisin and myonectin levels, and myonectin and FNDC5 mRNA expressions were increased in women with PCOS. These molecules can be target molecules in PCOS pathophysiology and treatment.IMPACT STATEMENTWhat is already known on this subject? Although the aetiology of PCOS is not fully understood, it is thought that insulin resistance may play a critical role. In recent studies, the relationship of cytokines secreted from skeletal muscle with insulin resistance has been shown. The effects of irisin and myonectin, which are members of the myokine family, on lipid and glucose metabolism are known.What do the results of this study add? Although there are many studies in the literature regarding serum irisin levels in women with PCOS, their results are confusing. There is a study in the literature investigating the relationship between myonectin and PCOS. In our study, we evaluated myonectin and FNDC mRNA expressions in addition to serum irisin and myonectin levels. As a result, we found that markers and their mRNA expressions were lower in patients with PCOS compared to controls.What are the implications of these findings for clinical practice and/or further research? We think that the results of our study will shed light on future studies. Due to their effects on adipose tissue, these markers may play a role in the aetiology of long-term complications of PCOS. Moreover, they can become pharmacological targets in preventing these complications.

Characterization of the 'White' Appearing Clots that Cause Acute Ischemic Stroke.

OBJECTIVES: Most clots retrieved from patients with acute ischemic stroke are 'red' in color. 'White' clots represent a less common entity and their histological composition is less known. Our aim was to investigate the composition, imaging and procedural characteristics of 'white' clots retrieved by mechanical thrombectomy. MATERIALS AND METHODS: Seventy five 'white' thrombi were selected by visual inspection from a cohort of 760 clots collected as part of the RESTORE registry. Clots were evaluated histopathologically. RESULTS: Quantification of Martius Scarlett Blue stain identified platelets/other as the major component in 'white' clots' (mean of 55% of clot overall composition) followed by fibrin (31%), red blood cells (6%) and white blood cells (3%). 'White' clots contained significantly more platelets/other (p<0.001*) and collagen/calcification (p<0.001*) and less red blood cells (p<0.001*) and white blood cells (p=0.018*) than 'red' clots. The mean platelet and von Willebrand Factor expression was 43% and 24%, respectively. Adipocytes were found in four cases. 'White' clots were significantly smaller (p=0.016*), less hyperdense (p=0.005*) on computed tomography angiography/non-contrast CT and were associated with a smaller extracted clot area (p<0.001*) than 'red' clots. They primarily caused the occlusion of middle cerebral artery, were less likely to be removed by aspiration and more likely to require rescue-therapy for retrieval. CONCLUSIONS: 'White' clots represented 14% of our cohort and were platelet, von Willebrand Factor and collagen/calcification-rich. 'White' clots were smaller, less hyperdense, were associated with significantly more distal occlusions and were less successfully removed by aspiration alone than 'red' clots.

Exploring the role of extracellular matrix proteins to develop biomarkers of plaque vulnerability and outcome.

Cardiovascular disease (CVD) is the most common cause of death in industrialized countries. One underlying cause is atherosclerosis, which is a systemic disease characterized by plaques of retained lipids, inflammatory cells, apoptotic cells, calcium and extracellular matrix (ECM) proteins in the arterial wall. The biologic composition of an atherosclerotic plaque determines whether the plaque is more or less vulnerable, that is prone to rupture or erosion. Here, the ECM and tissue repair play an important role in plaque stability, vulnerability and progression. This review will focus on ECM remodelling in atherosclerotic plaques, with focus on how ECM biomarkers might predict plaque vulnerability and outcome.

Circulating levels of C1q/TNF-α-related protein 6 (CTRP6) in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting females of reproductive age. It has been associated with cardiometabolic disorders including diabetes mellitus and cardiovascular disorders, and increases the risk of developing fecundity pathologies including recurrent pregnancy loss (RPL) and infertility. C1q/tumor necrosis factor-α-related protein-6 (CTRP6) is a novel adipokine involved in glucose and lipid metabolism, host inflammation, and organogenesis. In the present study, we aimed to determine the association of serum CTRP6 levels with some components of metabolic syndrome in PCOS patients (infertile PCOS [inf-PCOS] and PCOS-RPL). This case-control study included 120 PCOS patients (60 inf-PCOS and 60 PCOS-RPL) and 60 healthy controls. Serum high-sensitivity C-reactive protein (hs-CRP) and homocysteine were measured using commercial kits, while adiponectin and CTRP6 levels were assessed using ELISA technique. Inf-PCOS and PCOS-RPL individuals had higher levels of serum CTRP6 than controls (546.15 ± 125.02 ng/ml and 534.04 ± 144.19 ng/ml vs. 440.16 ± 159.24 ng/ml; both p < .001). Moreover, serum adiponectin levels were significantly reduced, while fasting insulin, homeostasis model assessment of insulin resistance, free testosterone, and hs-CRP levels were significantly elevated in PCOS group, when compared with controls. Furthermore, serum CTRP6 positively associated with body mass index in all subjects. It showed an inverse correlation with adiponectin in PCOS group and subgroups. However, it had a direct association with hs-CRP in PCOS group and inf-PCOS subgroup, but not PCOS-RPL subgroup. These findings unravel a probable role of CTRP6 in PCOS pathogenesis, which poses a possibility to be a good diagnostic target. However, further investigation is needed.

Reduced model to predict thrombin and fibrin during thrombosis on collagen/tissue factor under venous flow: Roles of γ'-fibrin and factor XIa.

During thrombosis, thrombin generates fibrin, however fibrin reversibly binds thrombin with low affinity E-domain sites (KD = 2.8 µM) and high affinity γ'-fibrin sites (KD = 0.1 µM). For blood clotting on collagen/tissue factor (1 TF-molecule/µm2) at 200 s-1 wall shear rate, high µM-levels of intraclot thrombin suggest robust prothrombin penetration into clots. Setting intraclot zymogen concentrations to plasma levels (and neglecting cofactor rate limitations) allowed the linearization of 7 Michaelis-Menton reactions between 6 species to simulate intraclot generation of: Factors FXa (via TF/VIIa or FIXa), FIXa (via TF/FVIIa or FXIa), thrombin, fibrin, and FXIa. This reduced model [7 rates, 2 KD's, enzyme half-lives~1 min] predicted the measured clot elution rate of thrombin-antithrombin (TAT) and fragment F1.2 in the presence and absence of the fibrin inhibitor Gly-Pro-Arg-Pro. To predict intraclot fibrin reaching 30 mg/mL by 15 min, the model required fibrinogen penetration into the clot to be strongly diffusion-limited (actual rate/ideal rate = 0.05). The model required free thrombin in the clot (~100 nM) to have an elution half-life of ~2 sec, consistent with measured albumin elution, with most thrombin (>99%) being fibrin-bound. Thrombin-feedback activation of FXIa became prominent and reached 5 pM FXIa at >500 sec in the simulation, consistent with anti-FXIa experiments. In predicting intrathrombus thrombin and fibrin during 15-min microfluidic experiments, the model revealed "cascade amplification" from 30 pM levels of intrinsic tenase to 15 nM prothrombinase to 15 µM thrombin to 90 µM fibrin. Especially useful for multiscale simulation, this reduced model predicts thrombin and fibrin co-regulation during thrombosis under flow.

Non-targeted and targeted analysis of collagen hydrolysates during the course of digestion and absorption.

Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate. Graphical abstract.

Association of decreased myonectin levels with metabolic and hormonal disturbance in polycystic ovary syndrome.

Myonectin is a myokine involving in glucose and lipid metabolisms. Polycystic ovary syndrome (PCOS) is a metabolic and reproductive disorder associated with insulin resistance. Our aims were to discover whether myonectin levels were altered in PCOS women comparing to controls and to determine the link of myonectin with hormonal-metabolic parameters in PCOS women. The current research was designed as a case-control study. Seventy-two subjects with PCOS and 72 age- and body mass index (BMI)-matched subjects as controls were enrolled into the study. Circulating myonectin levels were measured by ELISA. Myonectin levels were significantly lower in PCOS subjects compared to controls (6.77 ± 1.96 vs. 9.14 ± 2.87 ng/ml, p< .001). Myonectin exhibited an inverse association with BMI, insulin resistance, free androgen index (FAI) and triglycerides whereas it showed a positive association with high-density lipoprotein cholesterol (HDL-C) in women with PCOS. Logistic regression analysis revealed that decreased myonectin levels were parallel with increased probability of having PCOS risk. Decreased myonectin levels were associated with metabolic and hormonal disturbances in PCOS women, suggesting that myonectin may play a role in pathophysiology of PCOS.

Biomarkers of collagen synthesis predict progression in the PROFILE idiopathic pulmonary fibrosis cohort.

Idiopathic pulmonary fibrosis (IPF) is characterised by excessive extracellular matrix (ECM) deposition and remodelling. Measuring this activity provides an opportunity to develop tools capable of identifying individuals at-risk of progression. Longitudinal change in markers of ECM synthesis was assessed in 145 newly-diagnosed individuals with IPF.Serum levels of collagen synthesis neoepitopes, PRO-C3 and PRO-C6 (collagen type 3 and 6), were elevated in IPF compared with controls at baseline, and progressive disease versus stable disease during follow up, (PRO-C3 p < 0.001; PRO-C6 p = 0.029). Assessment of rate of change in neoepitope levels from baseline to 3 months (defined as 'slope to month 3': HIGH slope, slope > 0 vs. LOW slope, slope < =0) demonstrated no relationship with mortality for these markers (PRO-C3 (HR 1.62, p = 0.080); PINP (HR 0.76, p = 0.309); PRO-C6 (HR 1.14, p = 0.628)). As previously reported, rising concentrations of collagen degradation markers C1M, C3M, C6M and CRPM were associated with an increased risk of overall mortality (HR = 1.84, CI 1.03-3.27, p = 0.038, HR = 2.44, CI 1.39-4.31, p = 0.002; HR = 2.19, CI 1.25-3.82, p = 0.006; HR = 2.13 CI 1.21-3.75, p = 0.009 respectively).Elevated levels of PRO-C3 and PRO-C6 associate with IPF disease progression. Collagen synthesis and degradation biomarkers have the potential to enhance clinical trials in IPF and may inform prognostic assessment and therapeutic decision making in the clinic.

Circulating C1q/TNF-related protein isoform 15 is a marker for the presence of metabolic syndrome.

BACKGROUND: C1q/TNF-related protein isoform 15 (CTRP15) has been reported to be related to glucose and lipid metabolism, but the results are inconsistent. Metabolic syndrome (MetS) is a cluster of metabolic disorders. The aim of this study is to determine circulating CTRP15 levels in individuals with MetS and investigate the association among circulating CTRP15 and markers for MetS as well as insulin resistance. METHODS: A total of 341 individuals were recruited for this cross-sectional study. These subjects were screened for MetS. Serum CTRP15 concentrations were measured by ELISA. RESULTS: Serum CTRP15 levels were significantly higher in MetS individuals relative to those of the healthy individuals. Circulating CTRP15 correlated positively with WHR, BMI, SBP, FAT %, 2 h-BG, FIns, 2 h-Ins, TG, FFA, HbA1c, HOMA-IR, and AUCglucose , while negatively with HDL-C and ISI. Multiple linear regression showed that HOMA-IR and HDL-C are independently related factors influencing serum CTRP15 concentrations. In addition, binary logistic regression analysis showed that serum CTRP15 concentrations were significantly related to MetS. When the mean concentrations of circulating CTRP15 in MetS subjects were stratified by the number of components of the MetS, circulating CTRP15 was found to increase progressively with increasing number of the MetS components. Finally, ROC curve analysis showed that the best cutoff values for circulating CTRP15 to predict MetS and insulin resistance were 63.6 and 64.0 µg/L. CONCLUSIONS: Serum CTRP15 concentrations were associated with the key components of MetS and insulin resistance.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

BACKGROUND: Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. METHODS: Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n = 13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22 h/d, 7 d/wk, for a total of 12 months. Control animals (n = 9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3 days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. RESULTS: Monkeys in the EtOH group consumed an average of 2.8 ± 0.2 (mean ± SE) g/kg/d of EtOH (30 ± 2% of total calories), resulting in an average blood EtOH concentration of 88.3 ± 8.8 mg/dl 7 hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). CONCLUSIONS: Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Correlation between Serum IL-35 Levels and Bone Loss in Postmenopausal Women with Rheumatoid Arthritis.

OBJECTIVE: IL-35 was reported as a crucial anti-inflammatory cytokine and could efficiently regulate bone metabolism in murine collagen-induced arthritis model. However, the relationship between IL-35 and bone health in human rheumatoid arthritis (RA) has not been clarified. In this study, the aim was to explore the correlations between IL-35 and bone loss in postmenopausal women with RA. METHODS: The study included 76 postmenopausal women with RA and 53 healthy postmenopausal women as healthy controls (HCs). Serum IL-35 levels were detected by enzyme-linked immunosorbent assay. Bone mineral density (BMD) at lumbar spine 1-4 and at total hip was measured using dual-energy X-ray absorptiometry. Alkaline phosphatase (ALP), ß-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (ß-CTX), and 25-(OH) VitD3 were measured by turbidimetric inhibition immunoassay. RESULTS: Serum IL-35 levels were increased compared with HCs, and it positively correlated with BMD and 25-(OH) VitD3 and negatively correlated with ß-CTX in postmenopausal women with RA. Furthermore, serum IL-35 levels in the increased ALP group were higher than those in the normal ALP group. CONCLUSIONS: IL-35, an important anti-inflammatory cytokine, may participate in the pathogenesis of bone loss in postmenopausal women with RA.

Isolation and Characterisation of Major and Minor Collagens from Hyaline Cartilage of Hoki (Macruronus novaezelandiae).

The composition and properties of collagen in teleost (bony fish) cartilage have never been studied. In this study, we aimed to identify and characterise all collagen species in the nasal cartilage of hoki (Macruronus novaezelandiae). Four native collagen species were extracted using two techniques, and isolated with differential salt precipitation. We were able to assign the identity of three of these collagen species on the basis of solubility, SDS-PAGE and amino acid analyses. We found that hoki cartilage contains the major collagen, type II, and the minor collagens, type IX and type XI, which are homologous to those found in mammal and chicken cartilage. Using these extraction protocols, we also isolated a full-length type IX collagen from cartilage for the first time. In addition, we detected a 90 kDa, highly glycosylated collagen that has not been identified in any other species. For each isolate, structural and biochemical characterisations were performed using circular dichroism and Fourier transform infrared spectroscopy analyses, and the thermal denaturation properties were determined. Our results showed that the properties of hoki cartilage-derived collagens are similar to those of collagens in mammalian cartilage, indicating that teleost cartilage could provide biological ingredients for the development of biomaterials to treat cartilage-related illnesses.

Aerobic exercise training-induced changes in serum C1q/TNF-related protein levels are associated with reduced arterial stiffness in middle-aged and older adults.

Adiponectin regulates endothelial nitric oxide synthase in endothelial cells, and body fat loss by aerobic exercise training promotes adiponectin secretion. Recently, C1q/tumor necrosis factor-related proteins (CTRPs) have been identified as novel adipokines and are paralogs of adiponectin, but the association between exercise training-induced reduction of arterial stiffness and circulating CTRPs levels remains unclear. This study aimed to clarify whether the reduction of arterial stiffness in middle-aged and older adults is associated with the change in serum levels of CTRPs induced by exercise training. A total of 52 middle-aged and older participants were randomly divided into two groups: a training group ( n = 26) and a sedentary control group ( n = 26). Participants in the training group completed 8 wk of aerobic exercise training (60-70% peak oxygen uptake for 45 min, 3 days/wk). The reduction of percent whole body fat, abdominal visceral fat area, and carotid-femoral pulse-wave velocity (cfPWV) was significantly greater in the training group than in the control group ( P < 0.05). Moreover, the increase in serum adiponectin, CTRP3, and CTRP5 from baseline to 8 wk was significantly higher in the training group compared with the control group ( P < 0.05). Additionally, the training-induced change in cfPWV was negatively correlated with the training-induced change in serum adiponectin, CTRP3, and CTRP5 levels ( r = -0.51, r = -0.48, r = -0.42, respectively, P < 0.05), and increased plasma nitrite/nitrate level by exercise training was correlated only with adiponectin levels ( r = 0.41, P < 0.05). These results suggest that the exercise training-induced increase in serum CTRPs levels may be associated with the reduction of arterial stiffness in middle-aged and older adults.

Abnormal bone turnover in individuals with low serum alkaline phosphatase.

The clinical spectrum of hypophosphatasia (HPP) is broad and variable within families. Along severe infantile forms, adult forms with mild manifestations may be incidentally discovered by the presence of low alkaline phosphatase (ALP) activity in serum. However, it is still unclear whether individuals with persistently low levels of ALP, in the absence of overt manifestations of HPP, have subclinical abnormalities of bone remodeling or bone mass. The aim of this study was to obtain a better understanding of the skeletal phenotype of adults with low ALP by analyzing bone mineral density (BMD), bone microarchitecture (trabecular bone score, TBS), and bone turnover markers (P1NP and ß-crosslaps). We studied 42 individuals with persistently low serum ALP. They showed lower levels of P1NP (31.4 ± 13.7 versus 48.9 ± 24.4 ng/ml; p = 0.0002) and ß-crosslaps (0.21 ± 0.17 versus 0.34 ± 0.22 ng/ml, p = 0.0015) than individuals in the control group. There were no significant differences in BMD, bone mineral content, or TBS. These data suggest that individuals with hypophosphatasemia have an overall reduction of bone turnover, even in the absence of overt manifestations of HPP or low BMD. We evaluated bone mineral density (BMD), bone microarchitecture, and bone turnover markers in patients with low serum levels of alkaline phosphatase. Our results show that these patients have low bone remodeling even in the absence of BMD abnormalities, thus supporting the recommendation of avoiding antiresorptives such as bisphosphonates in these subjects.

Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase.

OBJECTIVE: Class III phosphoinositide 3-kinase, also known as VPS34 (vacuolar protein sorting 34), is a highly conserved enzyme regulating important cellular functions such as NADPH oxidase (NOX) assembly, membrane trafficking, and autophagy. Although VPS34 is expressed in platelets, its involvement in platelet activation remains unclear. Herein, we investigated the role of VPS34 in platelet activation and thrombus formation using VPS34 knockout mice. APPROACH AND RESULTS: Platelet-specific VPS34-deficient mice were generated and characterized. VPS34 deficiency in platelets did not influence tail bleeding time. In a ferric chloride-induced mesenteric arteriolar thrombosis model, VPS34-/- mice exhibited a prolonged vessel occlusion time compared with wild-type mice (42.05±4.09 versus 18.30±2.47 minutes). In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen under arterial shear was significantly reduced for VPS34-/- platelets. VPS34-/- platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin. VPS34 deficiency delayed clot retraction but did not influence platelet spreading on fibrinogen. We also demonstrated that VPS34 deficiency altered the basal level of autophagy in resting platelets and hampered NOX assembly and mTOR (mammalian target of rapamycin) signaling during platelet activation. Importantly, we identified the NOX-dependent reactive oxygen species generation as the major downstream effector of VPS34, which in turn can mediate platelet activation. In addition, by using a specific inhibitor 3-methyladenine, VPS34 was found to operate through a similar NOX-dependent mechanism to promote human platelet activation. CONCLUSIONS: Platelet VPS34 is critical for thrombosis but dispensable for hemostasis. VPS34 regulates platelet activation by influencing NOX assembly.

[Effect of valsartan on the expression of leptin, leptin receptor and collagen in rats with hepatic fibrosis].

Objective: To investigate the effects of angiotensin II type 1 receptor antagonist valsartan on leptin, leptin receptor and collagen in rats with hepatic fibrosis. Methods: Thirty-six male wistar rats were randomly divided into control group, model group and drug-treated group, with 12 rats in each group. Liver fibrosis models were made by subcutaneous injection of carbon tetrachloride on the dorsal of the rats, simultaneously gastric gavage with Valsartan and were killed at the end of 8th week. The degree of liver fibrosis was observed by HE and Masson staining. The serum leptin (LP) and TGFß1 were determined by ELISA. Liver LP mRNA and leptin receptor mRNA (OB-R mRNA) were detected by RT-PCR. Liver LP, OB-R and collagen I were detected by Western blot. The data of multiple groups were analyzed by one-way analysis variance (ANOVA), and linear correlation was performed between serum LP and TGF ß1. Results: After the intervention of valsartan, HE and Masson staining showed that the degree of liver fibrosis was significantly reduced. The levels of serum LP and TGFß1 in the control group were (18.92 ± 7.10) ng/ml and (9.13 ± 1.58) pg/ml respectively, which were significantly lower than those in the model group (46.92 ± 28.54) ng/ml and (16.39 ± 3.56) pg/ml, And (29.27 ± 7.27) ng/ml and (12.24 ± 2.94) pg/ml in the drug-treated group, respectively. The F values were 7.864 and 20.057 respectively. The P values were < 0.05. The differences were statistically significant. The relative expression levels of LP and OB-R mRNA in the control group were 0.35 ± 0.18 and 0.62 ± 0.18, respectively, which were significantly lower than those in the model group (1.79 ± 1.79 and 1.52 ± 1.44, and drug-treated group 0.48 ± 0.34 and 0.75 ± 0.26, respectively), F values = 6.914,3.894, P values were < 0.05, the differences were statistically significant. The relative expression levels of LP, OB-R and collaten I in liver were 0.71 ± 0.13, 0.81 ± 0.11 and 0.76 ± 0.13 in the model group, 0.97 ± 0.06, 1.04 ± 0.06, and 1.05 ± 0.04 respectively in the drug-treated group and 0.74 ± 0.05, 0.93 ± 0.05 and 0.91 ± 0.05. The F values were 15.425, 13.757 and 19.130 respectively in three groups (P < 0.001), the difference was statistically significant. Conclusion: Valsartan, an angiotensin II type 1 receptor antagonist, can reduce the expression of leptin and leptin receptor, reduce the production of TGFß1 and collaten I, and play an anti-hepatic fibrosis effect.

Adipocyte C1QTNF5 expression is BMI-dependently related to early adipose tissue dysfunction and systemic CTRP5 serum levels in obese children.

BACKGROUND/OBJECTIVES: The recently identified adipocytokine C1QTNF5 (encodes for CTRP5) has been demonstrated to inhibit pro-metabolic insulin signaling in adipocytes. We hypothesized that adipocyte C1QTNF5 expression in subcutaneous (sc) adipose tissue (AT) would correlate with the degree of obesity, systemic CTRP5 serum levels, and early AT and metabolic dysfunction in children. SUBJECTS/METHODS: Sc AT samples were obtained from 33 healthy Caucasian lean children aged 10.06±4.84 years and 42 overweight and obese children aged 13.34±3.12 years. C1QTNF5 expression in sc AT as well as in investigated cell lines was assessed by quantitative real-time PCR. Systemic CTRP5 levels were assessed by ELISA. RESULTS: C1QTNF5 expression in sc adipocytes increased with body mass index (BMI) standard deviation score (SDS; R=0.48, P<0.001), body fat percentage (R=0.4, P=0.004), adipocyte number (R=0.69, P<0.001) and systemic CTRP5 serum levels (R=0.28, P=0.025) whereas expression in the stromal vascular fraction (SVF) was inversely correlated with BMI SDS (R=-0.24, P=0.04). Multiple regression analysis confirmed that BMI SDS was the strongest independent predictor for C1QTNF5 expression in sc adipocytes. SVF C1QTNF5 levels strongly correlated with SVF CD31 expression (R=0.54, P<0.001) indicating expression by endothelial cells. Primary human endothelial cells demonstrated stronger expression compared with human Simpson-Golahbi-Behmel syndrome pre-adipocytes and adipocytes. Adipocyte C1QTNF5 expression levels were BMI-dependently related to fasting insulin (R=0.3, P=0.03) and leptin serum levels (R=0.5, P<0.001). Sc AT samples containing crown-like structures (CLS) demonstrated increased adipocyte C1QTNF5 expression compared to CLS-negative samples (P=0.03). Functionally, tumor necrosis factor (TNF)α caused a fourfold induction of C1QTNF5 in human adipocytes (P<0.001) and a 50% reduction in primary human endothelial cells (P<0.001). CONCLUSIONS: In children adipocyte C1QTNF5 expression is already strongly related to the degree of obesity and is associated with obesity-related AT alterations, systemic CTRP5 serum levels as well as circulating markers of metabolic disease and is positively regulated by TNFα in vitro.

Collagen Turnover Markers in Relation to Future Cardiovascular and Noncardiovascular Disease: The Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Sustained remodeling of extracellular matrix can compromise organs and tissues. Procollagen type III N-terminal propeptide (PIIINP) and collagen type I carboxy-terminal telopeptide (ICTP) reflect collagen synthesis and degradation. We studied their predictive value for future death and disease. METHODS: A total of 3068 men and women in the Multi-Ethnic Study of Atherosclerosis who were free of cardiovascular disease (CVD) and in generally good health had a baseline blood sample taken for ICTP and PIIINP. Median follow-up was 13.0 years. Among 4 primary outcomes, CVD events (n = 697) were adjudicated, death (n = 571) was by death certificate, and chronic inflammatory-related severe hospitalization and death (ChrIRD, n = 726) and total cancer (n = 327) were classified using International Classification of Diseases codes. We used Poisson regression to study baseline ICTP and PIIINP relative to these outcomes. RESULTS: Mean (SD) PIIINP was 5.47 (1.95) µg/L and ICTP was 3.37 (1.70) µg/L. PIIINP and ICTP were highly correlated with each other and with estimated glomerular filtration rate (eGFR). Adjustment for age and eGFR attenuated relative risks, remaining 20%-30% per SD of both PIIINP and ICTP in prediction for total death and ChrIRD, and of PIIINP for cancer, with little additional attenuation by adjusting for risk factors and inflammatory biomarkers. CVD outcome was generally unrelated to PIIINP but became marginally inversely related to ICTP in the most adjusted model. CONCLUSIONS: The collagen biomarkers PIIINP and ICTP, in part through pathophysiologically parallel associations with renal function, predicted ChrIRD and total death. Moreover, PIIINP predicted future cancer. These collagen markers may help differentiate healthy from unhealthy aging.

Effect of vitamin D3 on bone turnover markers in critical illness: post hoc analysis from the VITdAL-ICU study.

In this post hoc analysis of the VITdAL-ICU study, an RCT in critically ill adults with 25-hydroxyvitamin D levels ≤20 ng/ml, vitamin D3 did not have a significant effect on ß-Crosslaps and osteocalcin. INTRODUCTION: Observational studies have shown accelerated bone loss in ICU survivors. A reversible contributor is vitamin D deficiency. In a post hoc analysis of the VITdAL-ICU study, we evaluated the effect of high-dose vitamin D3 on the bone turnover markers (BTM) ß-Crosslaps (CTX) and osteocalcin (OC). METHODS: The VITdAL-ICU study was a randomized, double-blind, placebo-controlled trial in critically ill adults with 25-hydroxyvitamin D levels ≤20 ng/ml who received placebo or high-dose vitamin D3 (a loading dose of 540,000 IU and starting 1 month after the loading dose five monthly maintenance doses of 90,000 IU). In this analysis on 289 survivors (209 telephone, 80 personal follow-up visits), BTM were analyzed on days 0, 3, 7, 28, and 180; self-reported falls and fractures were assessed. Bone mineral density (BMD) was measured after 6 months. RESULTS: At baseline, CTX was elevated; OC was low in both groups-after 6 months, both had returned to normal. There were no differences between groups concerning BTM, BMD, falls, or fractures. In linear mixed effects models, CTX and OC showed a significant change over time (p < 0.001, respectively), but there was no difference between the vitamin D and placebo group (p = 0.688 and p = 0.972, respectively). CONCLUSIONS: Vitamin D supplementation did not have a significant effect on BTM. Further studies should assess the effectiveness of vitamin D on musculoskeletal outcomes in ICU survivors.