ADAMTSL3 knock-out mice develop cardiac dysfunction and dilatation with increased TGFß signalling after pressure overload.

Heart failure is a major cause of morbidity and mortality worldwide, and can result from pressure overload, where cardiac remodelling is characterized by cardiomyocyte hypertrophy and death, fibrosis, and inflammation. In failing hearts, transforming growth factor (TGF)ß drives cardiac fibroblast (CFB) to myofibroblast differentiation causing excessive extracellular matrix production and cardiac remodelling. New strategies to target pathological TGFß signalling in heart failure are needed. Here we show that the secreted glycoprotein ADAMTSL3 regulates TGFß in the heart. We found that Adamtsl3 knock-out mice develop exacerbated cardiac dysfunction and dilatation with increased mortality, and hearts show increased TGFß activity and CFB activation after pressure overload by aortic banding. Further, ADAMTSL3 overexpression in cultured CFBs inhibits TGFß signalling, myofibroblast differentiation and collagen synthesis, suggesting a cardioprotective role for ADAMTSL3 by regulating TGFß activity and CFB phenotype. These results warrant future investigation of the potential beneficial effects of ADAMTSL3 in heart failure.

The extracellular matrix glycoprotein ADAMTSL2 is increased in heart failure and inhibits TGFß signalling in cardiac fibroblasts.

Fibrosis accompanies most heart diseases and is associated with adverse patient outcomes. Transforming growth factor (TGF)ß drives extracellular matrix remodelling and fibrosis in the failing heart. Some members of the ADAMTSL (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs-like) family of secreted glycoproteins bind to matrix microfibrils, and although their function in the heart remains largely unknown, they are suggested to regulate TGFß activity. The aims of this study were to determine ADAMTSL2 levels in failing hearts, and to elucidate the role of ADAMTSL2 in fibrosis using cultured human cardiac fibroblasts (CFBs). Cardiac ADAMTSL2 mRNA was robustly increased in human and experimental heart failure, and mainly expressed by fibroblasts. Over-expression and treatment with extracellular ADAMTSL2 in human CFBs led to reduced TGFß production and signalling. Increased ADAMTSL2 attenuated myofibroblast differentiation, with reduced expression of the signature molecules α-smooth muscle actin and osteopontin. Finally, ADAMTSL2 mitigated the pro-fibrotic CFB phenotypes, proliferation, migration and contractility. In conclusion, the extracellular matrix-localized glycoprotein ADAMTSL2 was upregulated in fibrotic and failing hearts of patients and mice. We identified ADAMTSL2 as a negative regulator of TGFß in human cardiac fibroblasts, inhibiting myofibroblast differentiation and pro-fibrotic properties.

Etiology-Dependent Impairment of Diastolic Cardiomyocyte Calcium Homeostasis in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Whereas heart failure with reduced ejection fraction (HFrEF) is associated with ventricular dilation and markedly reduced systolic function, heart failure with preserved ejection fraction (HFpEF) patients exhibit concentric hypertrophy and diastolic dysfunction. Impaired cardiomyocyte Ca2+ homeostasis in HFrEF has been linked to disruption of membrane invaginations called t-tubules, but it is unknown if such changes occur in HFpEF. OBJECTIVES: This study examined whether distinct cardiomyocyte phenotypes underlie the heart failure entities of HFrEF and HFpEF. METHODS: T-tubule structure was investigated in left ventricular biopsies obtained from HFrEF and HFpEF patients, whereas cardiomyocyte Ca2+ homeostasis was studied in rat models of these conditions. RESULTS: HFpEF patients exhibited increased t-tubule density in comparison with control subjects. Super-resolution imaging revealed that higher t-tubule density resulted from both tubule dilation and proliferation. In contrast, t-tubule density was reduced in patients with HFrEF. Augmented collagen deposition within t-tubules was observed in HFrEF but not HFpEF hearts. A causative link between mechanical stress and t-tubule disruption was supported by markedly elevated ventricular wall stress in HFrEF patients. In HFrEF rats, t-tubule loss was linked to impaired systolic Ca2+ homeostasis, although diastolic Ca2+ removal was also reduced. In contrast, Ca2+ transient magnitude and release kinetics were largely maintained in HFpEF rats. However, diastolic Ca2+ impairments, including reduced sarco/endoplasmic reticulum Ca2+-ATPase activity, were specifically observed in diabetic HFpEF but not in ischemic or hypertensive models. CONCLUSIONS: Although t-tubule disruption and impaired cardiomyocyte Ca2+ release are hallmarks of HFrEF, such changes are not prominent in HFpEF. Impaired diastolic Ca2+ homeostasis occurs in both conditions, but in HFpEF, this mechanism for diastolic dysfunction is etiology-dependent.

A Privacy Attack on Multiple Dynamic Match-key based Privacy-Preserving Record Linkage.

INTRODUCTION: Over the last decade, the demand for linking records about people across databases has increased in various domains. Privacy challenges associated with linking sensitive information led to the development of privacy-preserving record linkage techniques. The multiple dynamic match-key encoding approach recently proposed by Randall et al. (IJPDS, 2019) is such a technique aimed at providing enough privacy for linkage applications while obtaining high linkage quality. However, the use of this encoding in large databases can reveal frequency information that can allow the re-identification of encoded values. OBJECTIVES: We propose a frequency-based attack to evaluate the privacy guarantees of multiple dynamic match-key encoding. We then present two recommendations that can be used in this match-key encoding approach to prevent such a privacy attack. METHODS: The proposed attack analyses the frequency distributions of individual match-keys in order to identify the attributes used for each match-key, where we assume the adversary has access to a plain-text database with similar characteristics as the encoded database. We employ a set of statistical correlation tests to compare the frequency distributions of match-key values between the encoded and plain-text databases. Once the attribute combinations used for match-keys are discovered, we then re-identify encoded sensitive values by utilising a frequency alignment method. Next, we propose two recommendations; one to alter the original frequency distributions and another to make the frequency distributions uniform. Both will help to prevent frequency-based attacks. RESULTS: We evaluate our privacy attack using two large real-world databases. The results show that in certain situations the attack can successfully re-identify a set of sensitive values encoded using the multiple dynamic match-key encoding approach. On the databases used in our experiments, the attack can re-identify plain-text values with a precision and recall of both up to 98%. Furthermore, we show that our proposed recommendations are able to make this attack harder to perform with only a small reduction in linkage quality. CONCLUSIONS: Our proposed privacy attack demonstrates the weaknesses of multiple match-key encoding that should be taken into consideration when linking databases that contain sensitive personal information. Our proposed recommendations ensure that the multiple dynamic match-key encoding approach can be used securely while retaining high linkage quality.

Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy.

During the development of hypertrophic cardiomyopathy, the heart returns to fetal energy metabolism where cells utilize more glucose instead of fatty acids as a source of energy. Metabolism of glucose can increase synthesis of the extracellular glycosaminoglycan hyaluronan, which has been shown to be involved in the development of cardiac hypertrophy and fibrosis. The aim of this study was to investigate hyaluronan metabolism in cardiac tissue from patients with hypertrophic cardiomyopathy in relation to cardiac growth. NMR and qRT-PCR analysis of human cardiac tissue from hypertrophic cardiomyopathy patients and healthy control hearts showed dysregulated glucose and hyaluronan metabolism in the patients. Gas phase electrophoresis revealed a higher amount of low molecular mass hyaluronan and larger cardiomyocytes in cardiac tissue from patients with hypertrophic cardiomyopathy. Histochemistry showed high concentrations of hyaluronan around individual cardiomyocytes in hearts from hypertrophic cardiomyopathy patients. Experimentally, we could also observe accumulation of low molecular mass hyaluronan in cardiac hypertrophy in a rat model. In conclusion, the development of hypertrophic cardiomyopathy with increased glucose metabolism affected both hyaluronan molecular mass and amount. The process of regulating cardiomyocyte size seems to involve fragmentation of hyaluronan.

Evaluation measure for group-based record linkage.

INTRODUCTION: The robustness of record linkage evaluation measures is of high importance since linkage techniques are assessed based on these. However, minimal research has been conducted to evaluate the suitability of existing evaluation measures in the context of linking groups of records. Linkage quality is generally evaluated based on traditional measures such as precision and recall. As we show, these traditional evaluation measures are not suitable for evaluating groups of linked records because they evaluate the quality of individual record pairs rather than the quality of records grouped into clusters. OBJECTIVES: We set out to highlight the shortcomings of traditional evaluation measures and then propose a novel method to evaluate clustering quality in the context of group-based record linkage. METHODS: The proposed linkage evaluation method assesses how well individual records have been allocated into predicted groups/clusters with respect to ground-truth data. We first identify the best representative predicted cluster for each ground-truth cluster and, based on the resulting mapping, each record in a ground-truth cluster is assigned to one of seven categories. These categories reflect how well the linkage technique assigned records into groups. RESULTS: We empirically evaluated our proposed method using real-world data and showed that it better reflects the quality of clusters generated by three group-based record linkage techniques. We also showed that traditional measures such as precision and recall can produce ambiguous results whereas our method does not. CONCLUSIONS: The proposed evaluation method provides unambiguous results regarding the assessed group-based record linkage approaches. The method comprises of seven categories which reflect how each record was predicted, providing more detailed information about the quality of the linkage result. This will help to make better-informed decisions about which linkage technique is best suited for a given linkage application.

The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling.

Pressure overload of the heart leads to cardiac remodeling that may progress into heart failure, a common, morbid and mortal condition. Increased mechanistic insight into remodeling is instrumental for development of novel heart failure treatment. Cardiac remodeling comprises cardiomyocyte hypertrophic growth, extracellular matrix alterations including fibrosis, and inflammation. Fibromodulin is a small leucine-rich proteoglycan that regulates collagen fibrillogenesis. Fibromodulin is expressed in the cardiac extracellular matrix, however its role in the heart remains largely unknown. We investigated fibromodulin levels in myocardial biopsies from heart failure patients and mice, subjected fibromodulin knock-out (FMOD-KO) mice to pressure overload by aortic banding, and overexpressed fibromodulin in cultured cardiomyocytes and cardiac fibroblasts using adenovirus. Fibromodulin was 3-10-fold upregulated in hearts of heart failure patients and mice. Both cardiomyocytes and cardiac fibroblasts expressed fibromodulin, and its expression was increased by pro-inflammatory stimuli. Without stress, FMOD-KO mice showed no cardiac phenotype. Upon aortic banding, left ventricles of FMOD-KO mice developed mildly exacerbated hypertrophic remodeling compared to wild-type mice, with increased cardiomyocyte size and altered infiltration of leukocytes. There were no differences in mortality, left ventricle dilatation, dysfunction or expression of heart failure markers. Although collagen amount and cross-linking were comparable in FMOD-KO and wild-type, overexpression of fibromodulin in cardiac fibroblasts in vitro decreased their migratory capacity and expression of fibrosis-associated molecules, i.e. the collagen-cross linking enzyme lysyl oxidase, transglutaminase 2 and periostin. In conclusion, despite a robust fibromodulin upregulation in clinical and experimental heart failure, FMOD-KO mice showed a relatively mild hypertrophic phenotype. In cultured cardiac fibroblasts, fibromodulin has anti-fibrotic effects.

Hypothermia elongates the contraction-relaxation cycle in explanted human failing heart decreasing the time for ventricular filling during diastole.

Targeted temperature management is part of the standardized treatment for patients in cardiac arrest. Hypothermia decreases cerebral oxygen consumption and induces bradycardia; thus, increasing the heart rate may be considered to maintain cardiac output. We hypothesized that increasing heart rate during hypothermia would impair diastolic function. Human left ventricular trabeculae obtained from explanted hearts of patients with terminal heart failure were stimulated at 0.5 Hz, and contraction-relaxation cycles were recorded. Maximal developed force (Fmax), maximal rate of development of force [(dF/d t)max], time to peak force (TPF), time to 80% relaxation (TR80), and relaxation time (RT = TR80 - TPF) were measured at 37, 33, 31, and 29°C. At these temperatures, stimulation frequency was increased from 0.5 to 1.0 and to 1.5 Hz. At 1.5 Hz, concentration-response curves for the ß-adrenergic receptor (ß-AR) agonist isoproterenol were performed. Fmax, TPF, and RT increased when temperature was lowered, whereas (dF/d t)max decreased. At all temperatures, increasing stimulation frequency increased Fmax and (dF/d t)max, whereas TPF and RT decreased. At 31 and 29°C, resting tension increased at 1.5 Hz, which was ameliorated by ß-AR stimulation. At all temperatures, maximal ß-AR stimulation increased Fmax, (dF/d t)max, and maximal systolic force, whereas resting tension decreased progressively with lowering temperature. ß-AR stimulation reduced TPF and RT to the same extent at all temperatures, despite the more elongated contraction-relaxation cycle at lower temperatures. Diastolic dysfunction during hypothermia results from an elongation of the contraction-relaxation cycle, which decreases the time for ventricular filling. Hypothermic bradycardia protects the heart from diastolic dysfunction and increasing the heart rate during hypothermia should be avoided. NEW & NOTEWORTHY Decreasing temperature increases the duration of the contraction-relaxation cycle in the human ventricular myocardium, significantly reducing the time for ventricular filling during diastole. During hypothermia, increasing heart rate further reduces the time for ventricular filling and in some situations increases resting tension further impairing diastolic function. Modest ß-adrenergic receptor stimulation can ameliorate these potentially detrimental changes during diastole while improving contractile force generation during targeted temperature management.

Increased Levels of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 in Ischemic Stroke and Transient Ischemic Attack.

BACKGROUND: Soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) has been shown to be increased in patients with acute ischemic stroke. Here, we evaluated plasma sLOX-1 levels and vascular carotid plaque LOX-1 (ie, OLR1) gene expression in patients with ischemic stroke and transient ischemic attack (TIA) with particular focus on their relation to time since symptom onset. METHODS AND RESULTS: Plasma sLOX-1 (n=232) and carotid plaque OLR1 gene expression (n=146) were evaluated in patients who were referred to evaluation for carotid endarterectomy, as well as in healthy control plasma (n=81). Patients were categorized according to presence of acute ischemic stroke or transient ischemic attack (n=35) ≤7 days, >7 days ≤3 months (n=90), >3 months (n=40), or no reported symptoms before study inclusion (n=67). Our major findings were the following: (1) Patients with carotid atherosclerosis had increased plasma sLOX-1 levels as compared with controls. (2) Plaque OLR1 mRNA levels were increased in carotid plaques (n=146) compared with nonatherosclerotic vessels (ie, common iliac arteries of organ donors, n=10). (3) There were no differences in sLOX plasma levels or OLR1 gene expression when analyzed according to the time since relevant cerebral ischemic symptoms. (4) Also patients with severe carotid atherosclerosis without any previous ischemic events had raised sLOX-1 levels. (5) Immunostaining showed colocalization between LOX-1 and macrophages within the carotid plaques. (6) Also patients with acute stroke (within 7 days) caused by atrial fibrillation (n=22) had comparable raised sLOX-1 levels. CONCLUSIONS: sLOX-1 levels are elevated in patients with ischemic stroke and transient ischemic attack independent of cause and time since the ischemic event.

Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation-contraction coupling, and inefficient Ca-induced Ca release remain major limitations. OBJECTIVE: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation-contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). METHODS AND RESULTS: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation-contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. CONCLUSIONS: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation-contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Increased Secondary/Primary Bile Acid Ratio in Chronic Heart Failure.

OBJECTIVE: Bile acids (BAs) are now recognized as signaling molecules and emerging evidence suggests that BAs affect cardiovascular function. The gut microbiota has recently been linked to the severity of heart failure (HF), and microbial metabolism has a major impact on BA homeostasis. We aimed to investigate the pattern of BAs, and particularly microbiota-transformed (secondary) BAs, in patients with chronic HF. METHODS AND RESULTS: This was a prospective, observational, single-center study including 142 patients with chronic HF and 20 age- and sex-matched healthy control subjects. We measured plasma levels of primary, secondary, and total BAs, and explored their associations with clinical characteristics and survival. Plasma levels of primary BAs were lower (P < .01) and the ratios of secondary to primary BAs higher (P < .001) in patients with HF compared with control subjects. Approximately 40% of patients in the upper tertile of the ratio of secondary to primary BAs died during 5.6 years of follow-up (unadjusted Cox regression: hazard ratio 1.93, 95% confidence interval 1.01-3.68, compared with the lower tertiles). However, this association was attenuated and no longer significant in multivariate analyses. CONCLUSIONS: Levels of primary BAs were reduced and specific secondary BAs increased in patients with chronic HF. This pattern was associated with reduced overall survival in univariate analysis, but not in multivariate analyses. Future studies should assess the regulation and potential role of BA metabolism in HF.

Wnt5a is associated with right ventricular dysfunction and adverse outcome in dilated cardiomyopathy.

The Wingless (Wnt) pathway has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). To explore the role of Wnt modulators Wnt5a and sFRP3 in DCM patients we analyzed the expression of Wnt5a and sFRP3 in plasma and myocardium of DCM patients and evaluated their effects on NFAT luciferase activity in neonatal mouse cardiomyocytes. Elevated circulating Wnt5a (n = 102) was associated with increased pulmonary artery pressures, decreased right ventricular function and adverse outcome, with a stronger association in more severely affected patients. A higher Wnt5a/sFRP3 ratio (n = 25) was found in the right ventricle vs. the left ventricle and was correlated with NFAT activation as well as pulmonary artery pressures. Wnt5a induced NFAT activation and sFRP3 release in cardiomyocytes in vitro, while sFRP3 antagonized Wnt5a. Wnt5a is associated with right ventricular dysfunction and adverse outcome in DCM patients and may promote the progression of DCM through NFAT signaling.

The Notch Ligands DLL1 and Periostin Are Associated with Symptom Severity and Diastolic Function in Dilated Cardiomyopathy.

In dilated cardiomyopathy (DCM), adverse myocardial remodeling is essential, potentially involving Notch signaling. We hypothesized that secreted Notch ligands would be dysregulated in DCM. We measured plasma levels of the canonical Delta-like Notch ligand 1 (DLL1) and non-canonical Notch ligands Delta-like 1 homologue (DLK1) and periostin (POSN) in 102 DCM patients and 32 matched controls. Myocardial mRNA and protein levels of DLL1, DLK1, and POSN were measured in 25 explanted hearts. Our main findings were: (i) Circulating levels of DLL1 and POSN were higher in patients with severe DCM and correlated with the degree of diastolic dysfunction and (ii) right ventricular tissue expressions of DLL1, DLK1, and POSN were oppositely associated with cardiac function indices, as high DLL1 and DLK1 expression corresponded to more preserved and high POSN expression to more deteriorated cardiac function. DLL1, DLK1, and POSN are dysregulated in end-stage DCM, possibly mediating different effects on cardiac function.

Limited Added Value of Circulating Inflammatory Biomarkers in Chronic Heart Failure.

OBJECTIVES: This study sought to evaluate whether a panel of biomarkers improved prognostication in patients with heart failure (HF) and reduced ejection fraction of ischemic origin using a systematized approach according to suggested requirements for validation of new biomarkers. BACKGROUND: Modeling combinations of multiple circulating markers could potentially identify patients with HF at particularly high risk and aid in the selection of individualized therapy. METHODS: From a panel of 20 inflammatory and extracellular matrix biomarkers, 2 different biomarker panels were created and added to the Seattle HF score and the prognostic model from the CORONA (Controlled Rosuvastatin Multinational Trial in Heart Failure) study (n = 1,497), which included conventional clinical characteristics and C-reactive protein and N-terminal pro-B-type natriuretic peptide. Interactions with statin treatment were also assessed. RESULTS: The two models-model 1 (endostatin, interleukin 8, soluble ST2, troponin T, galectin 3, and chemokine [C-C motif] ligand 21) and model 2 (troponin T, soluble ST2, galectin 3, pentraxin 3, and soluble tumor necrosis factor receptor 2)-significantly improved the CORONA and Seattle HF models but added only modestly to their Harrell's C statistic and net reclassification index. In addition, rosuvastatin had no effect on the levels of a wide range of inflammatory and extracellular matrix markers, but there was a tendency for patients with a lower level of biomarkers in the 2 panels to have a positive effect from statin treatment. CONCLUSIONS: In the specific HF patient population studied, a multimarker approach using the particular panel of biomarkers measured was of limited clinical value for identifying future risk of adverse outcomes.

Wnt5a is elevated in heart failure and affects cardiac fibroblast function.

Wnt signaling is dysregulated in heart failure (HF) and may promote cardiac hypertrophy, fibrosis, and inflammation. Blocking the Wnt ligand Wnt5a prevents HF in animal models. However, the role of Wnt5a in human HF and its functions in cardiac cells remain unclear. Here, we investigated Wnt5a regulation in HF patients and its effects on primary mouse and human cardiac fibroblasts. Serum Wnt5a was elevated in HF patients and associated with hemodynamic, neurohormonal, and clinical measures of disease severity. In failing human hearts, Wnt5a protein correlated with interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1. Wnt5a messenger RNA (mRNA) levels were markedly upregulated in failing myocardium and both mRNA and protein levels declined following left ventricular assist device therapy. In primary mouse and human cardiac fibroblasts, recombinant Wnt5a dose-dependently upregulated mRNA and protein release of IL-6 and TIMP-1. Wnt5a did not affect ß-catenin levels, but activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Importantly, inhibition of ERK1/2 activation attenuated Wnt5a-induced release of IL-6 and TIMP-1. In conclusion, our results show that Wnt5a is elevated in the serum and myocardium of HF patients and is associated with measures of progressive HF. Wnt5a induces IL-6 and TIMP-1 in cardiac fibroblasts, which might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression. KEY MESSAGES: • Wnt5a is elevated in serum and myocardium of HF patients and is associated with measures of progressive HF. • In cardiac fibroblasts, Wnt5a upregulates interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1 through the ERK pathway. • Wnt5a-mediated effects might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression.

The alternative complement pathway is dysregulated in patients with chronic heart failure.

The complement system, an important arm of the innate immune system, is activated in heart failure (HF). We hypothesized that HF patients are characterized by an imbalance of alternative amplification loop components; including properdin and complement factor D and the alternative pathway inhibitor factor H. These components and the activation product, terminal complement complex (TCC), were measured in plasma from 188 HF patients and 67 age- and sex- matched healthy controls by enzyme immunoassay. Our main findings were: (i) Compared to controls, patients with HF had significantly increased levels of factor D and TCC, and decreased levels of properdin, particularly patients with advanced clinical disorder (i.e., NYHA functional class IV), (ii) Levels of factor D and properdin in HF patients were correlated with measures of systemic inflammation (i.e., C-reactive protein), neurohormonal deterioration (i.e., Nt-proBNP), cardiac function, and deteriorated diastolic function, (iii) Low levels of factor H and properdin were associated with adverse outcome in univariate analysis and for factor H, this was also seen in an adjusted model. Our results indicate that dysregulation of circulating components of the alternative pathway explain the increased degree of complement activation and is related to disease severity in HF patients.

The Heparan Sulfate Proteoglycan Glypican-6 Is Upregulated in the Failing Heart, and Regulates Cardiomyocyte Growth through ERK1/2 Signaling.

Pressure overload is a frequent cause of heart failure. Heart failure affects millions of patients worldwide and is a major cause of morbidity and mortality. Cell surface proteoglycans are emerging as molecular players in cardiac remodeling, and increased knowledge about their regulation and function is needed for improved understanding of cardiac pathogenesis. Here we investigated glypicans (GPC1-6), a family of evolutionary conserved heparan sulfate proteoglycans anchored to the extracellular leaflet of the cell membrane, in experimental and clinical heart failure, and explored the function of glypican-6 in cardiac cells in vitro. In mice subjected to pressure overload by aortic banding (AB), we observed elevated glypican-6 levels during hypertrophic remodeling and dilated, end-stage heart failure. Consistently, glypican-6 mRNA was elevated in left ventricular myocardium from explanted hearts of patients with end-stage, dilated heart failure with reduced ejection fraction. Glypican-6 levels correlated negatively with left ventricular ejection fraction in patients, and positively with lung weight after AB in mice. Glypican-6 mRNA was expressed in both cardiac fibroblasts and cardiomyocytes, and the corresponding protein displayed different sizes in the two cell types due to tissue-specific glycanation. Importantly, adenoviral overexpression of glypican-6 in cultured cardiomyocytes increased protein synthesis and induced mRNA levels of the pro-hypertrophic signature gene ACTA1 and the hypertrophy and heart failure signature genes encoding natriuretic peptides, NPPA and NPPB. Overexpression of GPC6 induced ERK1/2 phosphorylation, and co-treatment with the ERK inhibitor U0126 attenuated the GPC6-induced increase in NPPA, NPPB and protein synthesis. In conclusion, our data suggests that glypican-6 plays a role in clinical and experimental heart failure progression by regulating cardiomyocyte growth through ERK signaling.

A Cross-Sectional Study of the Prevalence of Gastrointestinal Symptoms and Pathology in Patients With Common Variable Immunodeficiency.

OBJECTIVES: The objective of this study was to study the prevalence of gastrointestinal (GI) symptoms and histopathology in patients with common variable immunodeficiency (CVID) as well as linking the findings to GI infections and markers of systemic immune activation. METHODS: In this cross-sectional study, we addressed GI symptoms in 103 patients and GI histopathological findings in 53 patients who underwent upper and lower endoscopic examination. The most frequent histopathological findings were linked to GI symptoms, B-cell phenotype, and markers of systemic immune activation (soluble (s)CD14, sCD25, and sCD163). Microarray analysis compared "celiac-like disease" in CVID to celiac disease. Screening for selected bacterial and viral infections in fecal samples and gut mucosal biopsies was performed. RESULTS: The main findings of this study were as follows: most common GI symptoms were bloating (34%), pain (30%), and diarrhea (26%). The most frequent histopathological findings were increased intraepithelial lymphocytes in the descending part of the duodenum, i.e., "celiac-like disease" (46% of patients), decreased numbers of plasma cells in GI tract mucosa (62%), and lymphoid hyperplasia (38%), none of which were associated with GI symptoms. Reduced plasma cells in GI mucosa were associated with B-cell phenotypic characteristics of CVID, and increased serum levels of sCD14 (P=0.025), sCD25 (P=0.01), and sCD163 (P=0.04). Microarray analyses distinguished between CVID patients with "celiac-like disease" and celiac disease. Positive tests for bacterial and viral infections were scarce both in fecal samples and gut mucosal biopsies, including PCR test for norovirus in biopsy specimens (0 positive tests). CONCLUSIONS: In conclusion, GI pathology is common in CVID, but does not necessarily cause symptoms. However, reduced plasma cells in GI mucosa were linked to systemic immune activation, "celiac-like disease" in CVID and true celiac disease appear to be different disease entities, as assessed by gene expression, and infections (including norovirus) are rarely a cause of the CVID enteropathy.

Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA.

BACKGROUND: Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. METHODS: BER gene expression levels were analyzed in 162 carotid plaques, 8 disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls. RESULTS: Our major findings were: (i) The BER pathway showed a global increased transcriptional response in plaques as compared to control arteries, accompanied by increased expression of several BER proteins. (ii) Whereas nuclear DNA stability was maintained within carotid plaques, mtDNA integrity and copy number were decreased. (iii) Within carotid plaques, mRNA levels of several BER genes correlated with macrophage markers. (iv) In vitro, some of the BER genes were highly expressed in the anti-inflammatory and pro-resolving M2 macrophages, showing increased expression upon exposure to modified lipids. CONCLUSIONS: The increased transcriptional response of BER genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion.

High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis.

Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before.