Proteomic analysis of serum extracellular vesicles reveals Fibulin-3 as a new marker predicting liver-related events in MASLD.

BACKGROUND: There is a need for novel noninvasive markers for metabolic dysfunction-associated steatotic liver disease (MASLD) to stratify patients at high risk for liver-related events including liver cancer and decompensation. In the present study, we used proteomic analysis of proteins in extracellular vesicles (EVs) to identify new biomarkers that change with fibrosis progression and can predict the development of liver-related events. METHODS: We analyzed serum EVs from 50 patients with MASLD assessed for liver fibrosis by biopsy and identified proteins that altered with advanced fibrosis. A further evaluation was conducted on another cohort of 463 patients with MASLD with biopsy. RESULTS: Eight candidate proteins were identified by proteomic analysis of serum EVs. Among them, serum levels of Fibulin-3, Fibulin-1, and Ficolin 1 correlated with their EV levels. In addition, serum Fibulin-3 and serum Fibulin-1 levels changed significantly with advanced fibrosis. Using another cohort with biopsy, we found that the serum Fibulin-3 concentration was significantly greater in those with advanced fibrosis but that the serum Fibulin-1 concentration was not significantly different. Multivariate Cox proportional hazards analysis revealed that a higher Fibrosis-4 (FIB-4) index and higher serum Fibulin-3 concentration were independent risk factors for liver-related events. When the cutoff value for the serum Fibulin-3 concentration was 6.0 µg/mL according to the Youden index of AUROCs, patients with high serum Fibulin-3 significantly more frequently developed liver-related events than did other patients. Validation using another cohort of 226 patients with clinically diagnosed MASLD confirmed that high serum Fibulin-3 levels are associated with a greater frequency of liver-related events. CONCLUSIONS: Serum Fibulin-3 was identified as a biomarker for predicting liver-related events in patients with MASLD.

The Usefulness of Vitamin K-Dependent Proteins in the Diagnosis of Colorectal Carcinoma.

Colorectal cancer (CRC) is one of the most common neoplasms in developed countries, with increasing incidence and mortality, even in young people. A variety of serum markers have been associated with CRC (CEA, CA 19-9), but neither should be used as a screening tool for the diagnosis or evolution staging of CRC. The sensitivity and specificity of these markers are not as good as is required, so new ones need to be found. Matrix Gla protein and PIVKA II are involved in carcinogenesis, but few studies have evaluated their usefulness in predicting the presence and severity of CRC. Two hundred patients were divided into three groups: 80 patients were included in the control group; 80 with CRC and without hepatic metastasis were included in Group 1; 40 patients with CRC and hepatic metastasis were included in Group 2. Vitamin K-dependent proteins (VKDPs) levels in plasma were determined. Patients with CRC without methastasis (Group 1) and CRC patients with methastasis (Group 2) presented significantly higher values of CEA, CA 19-9, PIVKA II (310.05 ± 38.22 vs. 430.13 ± 122.13 vs. 20.23 ± 10.90), and ucMGP (14,300.00 ± 2387.02 vs. 13,410.52 ± 2243.16 vs. 1780.31 ± 864.70) compared to control group (Group 0). Interestingly, Group 1 presented the greatest PIVKA II values. Out of all the markers, significant differences between the histological subgroups were found only for ucMGP, but only in non-metastatic CRC. Studying the discrimination capacity between the patients with CRC vs. those without, no significant differences were found between the classical tumor markers and the VKDP AUROC curves (PIVKA II and ucMGP AUROCs = 1). For the metastatic stage, the sensitivity and specificity of the VKDPs were lower in comparison with those of CA 19-9 and CEA, respectively (PIVKA II AUROC = 0.789, ucMGP AUROC = 0.608). The serum levels of these VKDPs are significantly altered in patients with colorectal carcinoma; it is possible to find additional value of these in the early stages of the disease.

Extracellular matrix turnover proteins as risk markers in people with type 2 diabetes and microalbuminuria.

BACKGROUND: This post-hoc study investigated whether biomarkers reflecting extracellular matrix (ECM) turnover predicted cardiovascular disease (CVD), mortality, and progression of diabetic kidney disease (DKD) in individuals with type 2 diabetes (T2D) and microalbuminuria. METHODS: Serum levels of specific ECM turnover biomarkers were assessed in 192 participants with T2D and microalbuminuria from an observational study conducted at Steno Diabetes Center Copenhagen from 2007 to 2008. Endpoints included CVD events, mortality, and DKD progression, defined as decline in estimated glomerular filtration rate (eGFR) of >30 %. RESULTS: Participants had a mean age of 59 years, with 75 % males. Over a median follow-up of 4.9 to 6.3 years, the study recorded 38 CVD events, 24 deaths, and 40 DKD events. Elevated levels of a degradation fragment of collagen type I (C1M) were associated with an increased risk of >30 % eGFR decline, although this association was not independent of other risk factors. No significant associations were found between other ECM turnover biomarkers and DKD progression, mortality, or CVD risk. CONCLUSION: Elevated C1M levels were linked to DKD progression in individuals with T2D and microalbuminuria, but not independently of other risk factors. None of the ECM turnover biomarkers were associated with CVD or mortality.

Use of data-independent acquisition mass spectrometry to identify an objective serum indicator of the need for osteoporotic therapeutic intervention.

Osteoporosis is characterized by weakened bone microstructure and loss of bone mass. Current diagnostic criteria for osteoporosis are based on the T-score, which is a measure of bone mineral density. However, osteoporotic fragility fractures can occur regardless of the T-score, underscoring the need for additional criteria for the early detection of patients at fracture risk. To identify indicators of reduced bone strength, we performed serum proteomic analysis using data-independent acquisition mass spectrometry with serum samples from two patient groups, one with osteoporosis but no fractures and the other with osteopenia and fragility fractures. Collective evaluation of the results identified six serum proteins that changed to a similar extent in both patient groups compared with controls. Of these, extracellular matrix protein 1 (ECM1), which contributes to bone formation, showed the most significant increase in serum levels in both patient groups. An ELISA-based assay suggested that ECM1 could serve as a serum indicator of the need for therapeutic intervention; however, further prospective studies with a larger sample size are necessary to confirm these results. The present findings may contribute to the provision of early and appropriate therapeutic strategies for patients at risk of osteoporotic fractures. SIGNIFICANCE: This study aimed to identify objective serum indicators of the need for therapeutic intervention in individuals at risk of osteoporotic fracture. Comprehensive proteome analyses of serum collected from patients with osteoporosis but no fractures, patients with osteopenia and fragility fractures, and controls were performed by data-independent acquisition mass spectrometry. Collective evaluation of the proteome analysis data and ELISA-based assays identified serum ECM1 as a potential objective marker of the risk of fragility fractures in patients with osteoporosis or osteopenia. The findings are an important step toward the development of appropriate bone health management methods to improve well-being and maintain quality of life.

Matrix Gla protein and the long-term incidence and progression of coronary artery and aortic calcification in the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND AND AIMS: Matrix Gla protein (MGP) is an inhibitor of calcification that requires carboxylation by vitamin K for activity. The inactive form of MGP, dephosphorylated-uncarboxylated matrix Gla protein (dp-ucMGP), has been associated with increased calcification. However, it is not known whether there is a longitudinal relationship between dephosphorylated-uncarboxylated matrix Gla protein levels and coronary and aortic calcification in large population cohorts. METHODS: The Multi-Ethnic Study of Atherosclerosis (MESA) followed participants with serial cardiac computed tomography (CT) measures of vascular calcification. Dp-ucMGP was measured at baseline in a subset of participants who completed baseline and follow-up CTs approximately 10 years later and had available plasma specimens (n = 2663). Linear mixed effects models (LMMs) were used to determine the association of dp-ucMGP with the simultaneous incidence and progression of coronary artery, ascending thoracic aortic, or descending thoracic aortic calcification (CAC, ATAC, DTAC)]. RESULTS: For every one standard deviation (SD, 178 pmol/L) increment in dp-ucMGP, CAC increased by 3.44 ([95% CI = 1.68, 5.21], p < 0.001) Agatston units/year (AU/year), ATAC increased by 0.63 ([95% CI = 0.27, 0.98], p = 0.001) AU/year, and DTAC increased by 8.61 ([95% CI = 4.55, 12.67], p < 0.001) AU/year. The association was stronger for DTAC in those ≥65 years and with diabetes. CONCLUSIONS: We found a positive association of the inactive form of matrix Gla protein, dp-ucMGP, and long-term incidence/progression of CAC, ATAC, and DTAC. Future studies should investigate dp-ucMGP as a calcification regulator and MGP as a possible therapeutic target to slow progression of calcification in the vasculature.

The role of serum asprosin levels in predicting the severity of coronary artery disease in patients with diabetes mellitus.

BACKGROUND: Asprosin is an emerging biomarker that plays a role in metabolic diseases. This study investigates asprosin as a predictive marker for coronary artery disease (CAD) severity in diabetic patients. METHODS: Diabetic patients (n = 181) and healthy controls (n = 60) were analyzed. CAD severity was assessed using SYNTAX score. Diabetic patients were divided into 3 groups. Group 1 = patients without CAD, group 2 = patients with low SYNTAX score, and group 3 = patients with moderate-high SYNTAX score. Asprosin levels were measured for all participants using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Asprosin levels were significantly higher in patient group compared to control group (p < 0.001). Asprosin levels were significantly higher in group 3 compared to group 1 and group 2 (p = 0.002). In logistic regression analysis, asprosin levels independently predicted patients with moderate-high SYNTAX scores. According to this analysis, 1 ng/mL increase in asprosin level was found to increase the risk of having moderate-high SYNTAX score by 14.1%. When the threshold value of asprosin level was set as 22.17 ng/mL, it predicted patients with moderate-high SYNTAX score with 63.6% sensitivity and 62.6% specificity. In multivariate regression analysis, SYNTAX score independently correlated with asprosin level. CONCLUSION: This is the first study in the literature to demonstrate a positive correlation between asprosin levels and SYNTAX scores in diabetic patients with CAD. More comprehensive studies with larger groups are needed.

Association of serum secreted protein acidic and rich in cysteine-like protein 1 with metabolic measures and dyslipidemia among Chinese adults.

Objectives: Recent studies found that secreted protein acidic and rich in cysteine-like protein 1 (Sparcl1) could inhibit lipid droplets accumulation by peroxisome proliferator-activated receptor-gamma (PPARγ) signal pathway. However, the associations of serum Sparcl1 level with lipids profiles and other metabolic phenotypes remain unknown in human population study. Methods: We determined serum Sparcl1 using sandwich enzyme-linked immunosorbent assays among 1750 adults aged 40 years and older from a community in Shanghai, China. Generalized linear regression models were used to evaluate the association between Sparcl1 and metabolic measures. Multivariable-adjusted logistic regression analyses were performed to evaluate the relationship of serum Sparcl1 with prevalent dyslipidemia. Results: With the increment of serum Sparcl1, participants tended to have lower level of triglycerides, and higher level of high-density lipoprotein cholesterol (all P for trend < 0.01). No significant associations between serum Sparcl1 and glucose, blood pressure, or body size were observed. The generalized linear regression models suggested that per standard deviation (SD) increment of serum Sparcl1 was significantly inversely associated with triglycerides (ß= -0.06, P=0.02). The prevalence of dyslipidemia decreased across the sparcl1 quartiles (P for trend <0.01). After controlling the potential confounders, participants in the highest quartile of sparcl1 concentration had the lowest prevalence of dyslipidemia (odds ratio [OR], 0.69; 95% confidence interval [CI], 0.52-0.91), compared with the lowest quartile. Per SD increment of Sparcl1 was associated with 20% (OR, 0.80; 95%CI, 0.69-0.94) lower prevalence of hypertriglyceridemia and 12% (OR, 0.88; 95%CI, 0.79-0.97) lower prevalence of dyslipidemia. The association between serum Sparcl1 and dyslipidemia were generally consistent across subgroups (all P for interaction > 0.05). Conclusion: Serum Sparcl1 was significantly associated with decreased risk of prevalent dyslipidemia in Chinese population. Further studies are warranted to confirm this association.

A potential serological biomarker for inner ear pathologies: OTOLIN-1.

Objective: The goal of the study was to determine whether the level of OTOLIN-1, a protein whose expression is highly restricted to the inner ear,is increased in the body fluids of patients with inner ear disorders in comparison to healthy subjects. Methods: In the preliminary part of the study, OTOLIN-1 levels were measured in the serum, urine, and saliva of patients with an acute onset of Ménière´s disease and in healthy individuals. Subsequently, only serum OTOLIN-1 levels were taken into account and were compared between patients with acute onset of Ménière´s disease, sudden hearing loss, vestibular neuritis and healthy subjects. Results: The most reliable diagnostic parameter was OTOLIN-1 levels in serum. Serum samples of patients with Ménière's disease and sudden hearing loss showed significantly higher OTOLIN-1 levels than those from healthy individuals. In addition, there was no significant difference between the serum concentration of OTOLIN-1 in patients with vestibular neuritis and the control group. Conclusions: Serum levels of OTOLIN-1 can potentially be used as a biomarker for acute onset of inner ear disorders due to its significant increase in patients with acute Meniere´s disease and sudden hearing loss in comparison to healthy individuals.

Human platelets release TGFBIp in acute myocardial infarction.

Transforming growth factor-ß-induced protein (TGFBIp) is released from activated platelets and promotes pro-thrombotic complications like pulmonary embolism. The role of TGFBIp in acute coronary syndrome, especially with a focus on platelets, has not been investigated so far. Using ELISA and immunoblotting, we demonstrate platelet TGFBIp release in patients with myocardial infarction (MI). We investigated TGFBIp-induced platelet adhesion and rolling by flow chamber and chemotactic effects of TGFBIp in transwell experiments. Immunochemistry staining of arterial vessels detected TGFBIp and the platelet-specific protein GPVI in the vessel wall.We demonstrate for the first time that platelet TGFBIp release is significantly increased in MI and correlates with the severity of acute coronary syndromes (STEMI, NSTEMI). After activation with TRAP, platelets release TGFBIp and TGFBIp itself activates platelets. Under flow, TGFBIp-mediated platelet rolling and adherence similarly to collagen. TGFBIp significantly increased platelet transmigration and we demonstrate TGFBIp deposits in the wall of human arteries. In this study, we add novel aspects to the role of TGFBIp in acute coronary syndrome by demonstrating that TGFBIp is partially released from platelets during MI and has activating, pro-adhesive and pro-migratory effects on platelets that could contribute to the disease development of coronary vascular inflammation and MI.

Plasma MIA, CRP, and Albumin Predict Cognitive Decline in Parkinson's Disease.

OBJECTIVE: Using a multi-cohort, discovery-replication-validation design, we sought new plasma biomarkers that predict which individuals with Parkinson's disease (PD) will experience cognitive decline. METHODS: In 108 discovery cohort PD individuals and 83 replication cohort PD individuals, we measured 940 plasma proteins on an aptamer-based platform. Using proteins associated with subsequent cognitive decline in both cohorts, we trained a logistic regression model to predict which patients with PD showed fast (> = 1 point drop/year on Montreal Cognitive Assessment [MoCA]) versus slow (< 1 point drop/year on MoCA) cognitive decline in the discovery cohort, testing it in the replication cohort. We developed alternate assays for the top 3 proteins and confirmed their ability to predict cognitive decline - defined by change in MoCA or development of incident mild cognitive impairment (MCI) or dementia - in a validation cohort of 118 individuals with PD. We investigated the top plasma biomarker for causal influence by Mendelian randomization (MR). RESULTS: A model with only 3 proteins (melanoma inhibitory activity protein [MIA], C-reactive protein [CRP], and albumin) separated fast versus slow cognitive decline subgroups with an area under the curve (AUC) of 0.80 in the validation cohort. The individuals with PD in the validation cohort in the top quartile of risk for cognitive decline based on this model were 4.4 times more likely to develop incident MCI or dementia than those in the lowest quartile. Genotypes at MIA single nucleotide polymorphism (SNP) rs2233154 associated with MIA levels and cognitive decline, providing evidence for MIA's causal influence. CONCLUSIONS: An easily obtained plasma-based predictor identifies individuals with PD at risk for cognitive decline. MIA may participate causally in development of cognitive decline. ANN NEUROL 2022;92:255-269.

Application of collagen triple helix repeat containing-1 and mitotic spindle apparatus antibody in small cell lung cancer diagnosis.

BACKGROUND: The clinical significance of serum collagen triple helix repeat protein-1 (CTHRC1) and mitotic spindle apparatus antibody (MSA) in the diagnosis of small cell lung cancer (SCLC). METHODS: Of the 229 lung tumor patients selected, 62 patients were divided into SCLC, 94 patients with non-small cell lung cancer (NSCLC), and 73 patients with benign lung disease (BLD). The health controls (HC) had a span of 66 cases with normal physical condition. The serum extracted from each participator and enzyme-linked immunosorbent assay was adopted for measuring the serum CTHRC1 and MSA; in the meantime, automatic electrochemiluminescence immunoassay was used for the quantitative determination of serum NSA and CEA. And then, the differences in serum CTHRC1, MSA, NSE, and CEA were compared among involved groups. RESULTS: ① Compared with other groups, the concentrations of CTHRC1, MSA, and NSE showed a marked increase in the group of SCLC (all p < 0.01). Especially for SCLC patients with lymph node metastasis, CTHRC1 provided a notably higher level than those without metastasis. ② CTHRC1 and MSA established a diagnostic criterion with the specificity of 90.99% and 86.27% for SCLC, respectively. ③ In series, the specificity of CTHRC1 and NSE was the highest (99.30%), while MSA and NSE had the highest sensitivity (96.72%) in parallel. ④ Both CTHRC1 and MSA were hazardous factors interconnected with SCLC. CONCLUSION: Serum CTHRC1 and MSA had a more exciting prospect of application. When used in conjunction with NSE and CEA, they could optimize the clinical diagnosis value of SCLC.

Proteomic analysis of the umbilical cord in fetal growth restriction and preeclampsia.

Fetal growth restriction (FGR) is associated with adverse perinatal outcomes. Pre-eclampsia (PreE) increases the associated perinatal morbidity and mortality. The structure of the umbilical cord in the setting of FGR and PreE is understudied. This study aimed to examine changes in the umbilical cord (UC) composition in pregnancies complicated by FGR and FGR with PreE. UC from gestational age-matched pregnancies with isolated FGR (n = 5), FGR+PreE (n = 5) and controls (n = 5) were collected, and a portion of the UC was processed for histologic and proteomic analysis. Manual segmentation analysis was performed to measure cross-section analysis of umbilical cord regions. Wharton's Jelly samples were analyzed on a tims-TOF Pro. Spectral count and ion abundance data were analyzed, creating an intersection dataset from multiple mass spectrometry search and inference engines. UCs from FGR and FGR with PreE had lower cross-sectional area and Wharton's Jelly area compared with control (p = 0.03). When comparing FGR to control, 28 proteins were significantly different in abundance analysis and 34 in spectral count analysis (p < 0.05). Differential expression analysis between PreE with FGR vs controls demonstrated that 48 proteins were significantly different in abundance and 5 in spectral count. The majority of changes occurred in proteins associated with extracellular matrix, cellular process, inflammatory, and angiogenesis pathways. The structure and composition of the UC is altered in pregnancies with FGR and FGR with PreE. Future work in validating these proteomic differences will enable identification of therapeutic targets for FGR and FGR with PreE.

N-extended photoprotein obelin to competitively detect small protein tumor markers.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

Biological Role of Vitamin K-With Particular Emphasis on Cardiovascular and Renal Aspects.

Vitamin K (VK) plays many important functions in the body. The most important of them include the contribution in calcium homeostasis and anticoagulation. Vascular calcification (VC) is one of the most important mechanisms of renal pathology. The most potent inhibitor of this process-matrix Gla protein (MGP) is VK-dependent. Chronic kidney disease (CKD) patients, both non-dialysed and hemodialysed, often have VK deficiency. Elevated uncarboxylated matrix Gla protein (ucMGP) levels indirectly reflected VK deficiency and are associated with a higher risk of cardiovascular events in these patients. It has been suggested that VK intake may reduce the VC and related cardiovascular risk. Vitamin K intake has been suggested to reduce VC and the associated cardiovascular risk. The role and possibility of VK supplementation as well as the impact of anticoagulation therapy on VK deficiency in CKD patients is discussed.

Impact of Fetuin-A, Lp(a), matrix gla protein and macrophage density on calcific aortic valve disease: a clinical study.

BACKGROUND: Calcific aortic valve disease (CAVD) has a substantial and increasing burden in the ageing population with occult onset.Present study aimed to assess association of clinical characteristics of these patients and occurrence of CAVD. METHODS: Patients diagnosed with CAVD and those receiving healthy medical examination in our hospital from January 2019 to February 2021 were enrolled in this retrospective study. Clinical characteristics, ultrasonic indicators, serological indicators and histology of CAVD were collected and compared among different groups. Logistic regression and Pearson correlation analysis was used to explore relationship between these indexes and occurrence of CAVD. RESULTS: DBP, SBP, LVESD, LVEDD, IVS, PW, AV Vmax, TC, TG, LDL-C, Fetuin-A, Lp(a) in severe group were higher than mild, moderate and control groups (P<0.05), while those indexes of patients in moderate group were higher than that in mild and controlled groups (P<0.05). Besides, theses indexes of patients in mild group were also higher than that of controlled one (P<0.05). However, LVEF, HDL-C and MGP of patients in severe group was the lowest (P<0.05), while those in moderate group were lower than mild and controlled groups. Moreover, these indexes in mild group were also lower than control group (P<0.05). In Logistic regression analysis, MGP, Fetuin-A and Lp(a) were all independently associated with occurrence of CAVD (P<0.05). In Pearson correlation analysis, Fetuin-A and Lp(a) were positively correlated with progression of the disease, while MGP and macrophage density were negatively correlated with it. CONCLUSIONS: Fetuin-A, MPG and Lp(a) were independently associated with the occurrence of CAVD, and they might be potential predictors for diagnosis of this disease.

Matrix Gla Protein Levels Are Associated With Arterial Stiffness and Incident Heart Failure With Preserved Ejection Fraction.

OBJECTIVE: Arterial stiffness is a risk factor for cardiovascular disease, including heart failure with preserved ejection fraction (HFpEF). MGP (matrix Gla protein) is implicated in vascular calcification in animal models, and circulating levels of the uncarboxylated, inactive form of MGP (ucMGP) are associated with cardiovascular disease-related and all-cause mortality in human studies. However, the role of MGP in arterial stiffness is uncertain. Approach and Results: We examined the association of ucMGP levels with vascular calcification, arterial stiffness including carotid-femoral pulse wave velocity (PWV), and incident heart failure in community-dwelling adults from the Framingham Heart Study. To further investigate the link between MGP and arterial stiffness, we compared aortic PWV in age- and sex-matched young (4-month-old) and aged (10-month-old) wild-type and Mgp+/- mice. Among 7066 adults, we observed significant associations between higher levels of ucMGP and measures of arterial stiffness, including higher PWV and pulse pressure. Longitudinal analyses demonstrated an association between higher ucMGP levels and future increases in systolic blood pressure and incident HFpEF. Aortic PWV was increased in older, but not young, female Mgp+/- mice compared with wild-type mice, and this augmentation in PWV was associated with increased aortic elastin fiber fragmentation and collagen accumulation. CONCLUSIONS: This translational study demonstrates an association between ucMGP levels and arterial stiffness and future HFpEF in a large observational study, findings that are substantiated by experimental studies showing that mice with Mgp heterozygosity develop arterial stiffness. Taken together, these complementary study designs suggest a potential role of therapeutically targeting MGP in HFpEF.

Sparcl1 promotes nonalcoholic steatohepatitis progression in mice through upregulation of CCL2.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of chronic liver disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms of NASH progression remain incompletely understood. White adipose tissue (WAT) has emerged as an important endocrine organ and contributes not only to the initial stage of NAFLD, but also to its severity. In the current study, through transcriptomic analysis we identified increased expression of Sparcl1, a secreted glycoprotein, in the WAT from NASH mice. Plasma Sparcl1 levels were similarly elevated and positively correlated with hepatic pathological features in NASH patients. Functional studies showed that both chronic injection of recombinant Sparcl1 protein and overexpression of Sparcl1 exaggerated hepatic inflammation and liver injury in mice. In contrast, genetic ablation of Sparcl1, knockdown of Sparcl1 in WAT, and treatment with a Sparcl1-neutralizing antibody dramatically alleviated diet-induced NASH pathogenesis. Mechanistically, Sparcl1 promoted the expression of C-C motif chemokine ligand 2 (CCL2) in hepatocytes through binding to Toll-like receptor 4 (TLR4) and activation of the NF-κB/p65 signaling pathway. Genetically or pharmacologically blocking the CCL2/CCR2 pathway attenuated the hepatic inflammatory response evoked by Sparcl1. Thus, our results demonstrated an important role for Sparcl1 in NASH progression, suggesting a potential target for therapeutic intervention.

Hepatic and Vascular Vitamin K Status in Patients with High Cardiovascular Risk.

Vitamin K dependent proteins (VKDP), such as hepatic coagulation factors and vascular matrix Gla protein (MGP), play key roles in maintaining physiological functions. Vitamin K deficiency results in inactive VKDP and is strongly linked to vascular calcification (VC), one of the major risk factors for cardiovascular morbidity and mortality. In this study we investigated how two vitamin K surrogate markers, dephosphorylated-undercarboxylated MGP (dp-ucMGP) and protein induced by vitamin K absence II (PIVKA-II), reflect vitamin K status in patients on hemodialysis or with calcific uremic arteriolopathy (CUA) and patients with atrial fibrillation or aortic valve stenosis. Through inter- and intra-cohort comparisons, we assessed the influence of vitamin K antagonist (VKA) use, vitamin K supplementation and disease etiology on vitamin K status, as well as the correlation between both markers. Overall, VKA therapy was associated with 8.5-fold higher PIVKA-II (0.25 to 2.03 AU/mL) and 3-fold higher dp-ucMGP (843 to 2642 pM) levels. In the absence of VKA use, non-renal patients with established VC have dp-ucMGP levels similar to controls (460 vs. 380 pM), while in HD and CUA patients, levels were strongly elevated (977 pM). Vitamin K supplementation significantly reduced dp-ucMGP levels within 12 months (440 to 221 pM). Overall, PIVKA-II and dp-ucMGP showed only weak correlation (r2 ≤ 0.26) and distinct distribution pattern in renal and non-renal patients. In conclusion, VKA use exacerbated vitamin K deficiency across all etiologies, while vitamin K supplementation resulted in a vascular VKDP status better than that of the general population. Weak correlation of vitamin K biomarkers calls for thoughtful selection lead by the research question. Vitamin K status in non-renal deficient patients was not anomalous and may question the role of vitamin K deficiency in the pathogenesis of VC in these patients.

Intravenous Vitamin K1 for the Correction of Prolonged Prothrombin Times in Non-Bleeding Critically Ill Patients: A Prospective Observational Study.

The aim of this study was to evaluate the effects of vitamin K1 on various vitamin K-dependent proteins in critically ill patients with prolonged Owren PT. We included critically ill non-bleeding adult patients without liver failure or anticoagulation treatment, with Owren PT > 1.2, who were prescribed intravenous vitamin K1. Blood was drawn at baseline and at 20-28 h after vitamin K1 administration. At both time points, we measured various vitamin K-dependent proteins and coagulation assays. ClinicalTrials.gov; Identifier: NTC3782025. In total, 52 patients were included. Intravenous vitamin K1 reduced Owren PT, Quick PT, protein induced by vitamin K absence/antagonist-II and desphospho-uncarboxylated matrix Gla protein (dp-ucMGP), but not to normal levels. Concomitantly, there were increases in thrombin generation and the activity of coagulation factors II, VII, IX and X that was only counteracted with a small increase in Protein C activity. In conclusion, the results suggest that vitamin K1 strengthens coagulation as measured by PT decrease and increases in the activity of vitamin K-dependent clotting factors and thrombin generation. The decreased dp-ucMGP, and its potential positive short- and long-term non-coagulative effects, merits further research.

Development of the BioHybrid Assay: Combining Primary Human Vascular Smooth Muscle Cells and Blood to Measure Vascular Calcification Propensity.

BACKGROUND: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). METHODS: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. RESULTS: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. CONCLUSION: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.