Tumor necrosis factor as a pharmacological target

As indicated by its name, tumor necrosis factor (TNF), cloned in 1985, was originally described as a macrophage-derived endogenous mediator that can induce hemorrhagic necrosis of solid tumors and kill some tumor cell lines in vitro. Unfortunately, its promising use as an anticancer agent was biased by its toxicity, which was clear soon from the first clinical trials with TNF in cancer. Almost at the same time TNF was being developed as an anticancer drug, it became clear that TNF was identical to a mediator responsible for cachexia associated with sepsis, which was termed cachectin. This research led to the finding that TNF is, in fact, the main lethal mediator of sepsis and to the publication of a huge number of articles showing that TNF inhibits the toxic effects of bacterial endotoxins, which are now described as systemic inflammatory response. Although the clinical trials with anti-TNF in sepsis have not been successful thus far, undoubtedly as a result of the complexity of this clinical setting, these studies ultimately led to the identification of TNF as a key inflammatory mediator and to the development of anti-TNF molecules (soluble receptors and antibodies) for important diseases including rheumatoid arthritis and Crohn’s disease. On the other side, the mechanisms by which TNF and related molecules induce cell death have been studied in depth, and their knowledge might, in the future, suggest means of improve the therapeutic index of TNF in cancer.     

To hydrolyze or not to hydrolyze: the dilemma of platelet-activating factor acetylhydrolase

Mounting ambiguity persists around the functional role of the plasma form of platelet-activating factor acetylhydrolase (PAF-AH). Because PAF-AH hydrolyzes PAF and related oxidized phospholipids, it is widely accepted as an anti-inflammatory enzyme. On the other hand, its actions can also generate lysophosphatidylcholine (lysoPC), a component of bioactive atherogenic oxidized LDL, thus allowing the enzyme to have proinflammatory capabilities. Presence of a canonical lysoPC receptor has been seriously questioned for a multitude of reasons. Animal models of inflammation show that elevating PAF-AH levels is beneficial and not deleterious and overexpression of PAF receptor (PAF-R) also augments inflammatory responses. Further, many Asian populations have a catalytically inert PAF-AH that appears to be a severity factor in a range of inflammatory disorders. Correlation found with elevated levels of PAF-AH and CVDs has led to the design of a specific PAF-AH inhibitor, darapladib. However, in a recently concluded phase III STABILITY clinical trial, use of darapladib did not yield promising results. Presence of structurally related multiple ligands for PAF-R with varied potency, existence of multi-molecular forms of PAF-AH, broad substrate specificity of the enzyme and continuous PAF production by the so called bi-cycle of PAF makes PAF more enigmatic. This review seeks to address the above concerns.     

Telomere length and cardiovascular risk factors in a middle-aged population free of overt cardiovascular disease

Evidence assembled over the last decade shows that average telomere length (TL) acts as a biomarker for biological aging and cardiovascular disease (CVD) in particular. Although essential for a more profound understanding of the underlying mechanisms, little reference information is available on TL. We therefore sought to provide baseline TL information and assess the association of prevalent CVD risk factors with TL in subjects free of overt CVD within a small age range. We measured mean telomere restriction fragment length of peripheral blood leukocytes in a large, representative Asklepios study cohort of 2509 community-dwelling, Caucasian female and male volunteers aged approximately 35-55 years and free of overt CVD. We found a manifest age-dependent telomere attrition, at a significantly faster rate in men as compared to women. No significant associations were established with classical CVD risk factors such as cholesterol status and blood pressure, yet shorter TL was associated with increased levels of several inflammation and oxidative stress markers. Importantly, shorter telomere length was associated with an increasingly unhealthy lifestyle, particularly in men. All findings were age and gender adjusted where appropriate. With these cross-sectional results we show that TL of peripheral blood leukocytes primarily reflects the burden of increased oxidative stress and inflammation, whether or not determined by an increasingly unhealthy lifestyle, while the association with classical CVD risk factors is limited. This further clarifies the added value of TL as a biomarker for biological aging and might improve our understanding of how TL is associated with CVD.     

一种新型高脂血症易感大鼠的血脂水平与心血管并发症

目的观察一种新型高脂血症易感WSHc大鼠经高脂饲料诱导后血脂的动态变化和心血管并发症病理特点,为WSHc大鼠的应用提供参考。方法取7～8周龄雌性WSHc大鼠20只和Wistar大鼠10只,进行高脂饲喂,另取同周龄同性别的WSHc大鼠和Wistar大鼠各10只饲喂普通饲料,分别作为正常对照。观察高脂饲喂2、4、8、12、16周时的血脂动态变化,饲喂16周后采用心动超声检测大鼠心功能及左心结构,病理组织学观察心脏及主动脉病变。结果与正常对照组比,高脂饲喂2周后WSHc大鼠与Wistar大鼠血清总胆固醇(TC)与低密度脂蛋白胆固醇(LDL-c)均显著升高;高脂饲喂的WSHc大鼠TC、LDL-c水平为:(6.34±2.12)mmol/L、(2.56±0.94)mmol/L,而高脂饲喂的Wistar大鼠TC、LDL-c水平仅为(2.93±0.23)mmol/L,(0.63±0.12)mmol/L;在高脂饲喂期间,WSHc大鼠TC始终维持在高水平状态。饲喂16周后,WSHc大鼠心脏射血分数升高,左心室室壁增厚,并出现心肌纤维化,心肌细胞凋亡程度增高;主动脉内膜增厚,弹力纤维排列紊乱,形成早期动脉粥样硬化病变。而Wistar大鼠经高脂饲料诱导后,心功能及心血管病变不明显。结论 WSHc大鼠对外源性胆固醇敏感,高脂饲喂后易发高脂血症及心血管疾病,且TC、LDL-c水平与临床高脂血症更接近,相较于普通大鼠,更适用于高脂血症及相关心血管并发症的实验研究。     

Growth differentiation factor 15 in cardiovascular diseases: from bench to bedside

Context: Growth differentiation factor 15 (GDF-15) is a novel cytokine showing close association with cardiovascular diseases. The biological mechanism and clinical use of GDF-15 in cardiovascular diseases have been well demonstrated. We review recent investigations from both basic research and clinical trials into the biological role of GDF-15.

Methods: The data were obtained mainly from MedLine via PubMed and from our own investigations.

Results: Laboratory investigations revealed that GDF-15 has biphasic effects on cellular survival by several signaling pathways. GDF-15 participates in several cardiovascular pathological processes such as cardiac remodeling, ischemia/reperfusion injury and atherosclerotic plaque formation. As well, GDF-15 was found a prognostic biomarker of heart failure and acute coronary syndrome. The evidence for diagnostic or therapeutic utility is poor.

Conclusion: GDF-15 has great potential as a biomarker in cardiovascular diseases, especially for prognosis, and is seen as a myocardial protective cytokine, but the exact mechanism of GDF-15 in cardiovascular diseases remains unknown.     

The impact of obesity to antioxidant defense parameters in adolescents with increased cardiovascular risk

BackgroundThe goal of this study was to assess the oxidative stress status through the values of antioxidant defense parameters: superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and total antioxidant status (TAS), as well as cardiovascular risk factors (total cholesterol, LDL-cholesterol, VLDL-cholesterol, non-HDL-cholesterol and triglycerides), anthropometric parameters (Body mass index-BMI, waist circumference-WC, hipp circumferemce-HC, waist-to-hipp ratio-WHR and inflammatory markers (high sensitive C-reactive protein) in a group of obese adolescents.MethodsA total of 238 students of both sexes, age of 22.32 ± 1.85 yr. were included in the study. According to the values of BMI lower and higher than 25 kg/m2 and WC higher and lower than 94 cm (males)/80 cm (females) the tested group of students was divided into 2 subgroups: Group 1 (increased risk for CVD) and Group 2 (lower risk for CVD).ResultsSignificantly reduced SOD and GPx with increased GR, TAS, inflammatory and lipoprotein parameters were obtained in Group 1 compared to Group 2. Significant positive association of hsCRP (OR:1.41; 95%CI 1.08-1.83; P=0.007), TAS (OR:827.2; 95%CI 19.27-35498; P=0.007) and GR (OR:1.13; 95%CI 1.05-1.21; P=0.002) and negative association of GPx (OR:0.97; 95%CI 0.94-1.003; P=0.043) and HDL-cholesterol (OR:0.41; 95%CI 0.176-0.963; P=0.0014) with cardiovascular risk factors were found in obese students. According to the ROC analysis GR>44.8 U/L, TAS>1.35 mmol/L, hsCRP>1.71 mg/L and HDL-cholesterol <1.13 mmol/L have sufficient predictive ability for cardiovascular disease in obese students.ConclusionsSignificant association of antioxidant defense parameters with anthropometric, lipid and inflammatory markers in obese students with increased cardiovascular risk suggest that screening of these parameters is necessary and highly recommended.     

Tissue factor expression on monocytes induced by anti-phospholipid antibodies as a strong risk factor for thromboembolic complications in SLE patients

Our aim was to clarify the role of anti-phospholipid antibodies in the pathogenesis of monocyte tissue factor (TF) expression and thromboembolic complications (TE) in patients with SLE. We examined cell surface expression of TF on monocytes in 93 SLE patients. Monocyte TF expression was significantly higher in SLE patients who had TE than in other SLE patients, and confirmed that the high expression of monocyte TF was a strong risk factor for TE. Furthermore, the presence of anti-cardiolipin/β2-glycoprotein I antibodies (anti-CL/β2-GPI) was strongly associated with the high expression of monocyte TF. We therefore studied the in vitro effect of IgG anti-CL/β2-GPI on lipopolysaccharide (LPS)-induced expression of TF on monocytes in healthy peripheral blood and found that purified IgG containing anti-CL/β2-GPI significantly enhanced LPS-induced monocyte TF expression. These results suggest that anti-CL/β2-GPI cause persistently high TF expression on monocyte, which may contribute to the risk of thromboembolic events in SLE patients.     

Antagonism by ethanol of endotoxin-induced tissue factor activation in relation to the depressed endotoxin binding to monocyte-like U937 cells

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)-induced tissue factor (TF)-activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP-1 cells. The present study shows a strong correlation (r=0·92; p<0·01) between TF-activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)-LPS or -anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC-LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti-CD14 mAb (UCHM-1) inhibited LPS binding in a dose-dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC-UCHM-1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS-induced TF-activation. © 1997 John Wiley & Sons, Ltd.     

Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite

Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93 > Arg-165 > or = Lys-169 > Lys-236 > Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.     

Targeting FGF21 in cardiovascular and metabolic diseases: from mechanism to medicine

Cardiovascular and metabolic disease (CVMD) is becoming increasingly prevalent in developed and developing countries with high morbidity and mortality. In recent years, fibroblast growth factor 21 (FGF21) has attracted intensive research interest due to its purported role as a potential biomarker and critical player in CVMDs, including atherosclerosis, coronary artery disease, myocardial infarction, hypoxia/reoxygenation injury, heart failure, type 2 diabetes, obesity, and nonalcoholic steatohepatitis. This review summarizes the recent developments in investigating the role of FGF21 in CVMDs and explores the mechanism whereby FGF21 regulates the development of CVMDs. Novel molecular targets and related pathways of FGF21 (adenosine 5''-monophosphate-activated protein kinase, silent information regulator 1, autophagy-related molecules, and gut microbiota-related molecules) are highlighted in this review. Considering the poor pharmacokinetics and biophysical properties of native FGF21, the development of new generations of FGF21-based drugs has tremendous therapeutic potential. Related preclinical and clinical studies are also summarized in this review to foster clinical translation. Thus, our review provides a timely and insightful overview of the physiology, biomarker potential, molecular targets, and therapeutic potential of FGF21 in CVMDs.     

Attention‐deficit/hyperactivity disorder as a risk factor for cardiovascular diseases: a nationwide population‐based cohort study

Accumulating evidence suggests a higher risk for cardiovascular diseases among individuals with mental disorders, but very little is known about the risk for overall and specific groups of cardiovascular diseases in people with attention‐deficit/hyperactivity disorder (ADHD). To fill this knowledge gap, we investigated the prospective associations between ADHD and a wide range of cardiovascular diseases in adults. In a nationwide population‐based cohort study, we identified 5,389,519 adults born between 1941 and 1983, without pre‐existing cardiovascular diseases, from Swedish registers. The study period was from January 1, 2001 to December 31, 2013. Incident cardiovascular disease events were identified according to ICD codes. Hazard ratios (HR) with 95% confidence intervals (CI) were calculated using Cox proportional hazards regression model, with ADHD as a time‐varying exposure. After an average 11.80 years of follow‐up, 38.05% of individuals with ADHD versus 23.57% of those without ADHD had at least one diagnosis of cardiovascular disease (p<0.0001). ADHD was significantly associated with increased risk of any cardiovascular disease (HR=2.05, 95% CI: 1.98‐2.13) after adjusting for sex and year of birth. Further adjustments for education level, birth country, type 2 diabetes mellitus, obesity, dyslipidemia, sleep problems and heavy smoking attenuated the association, which however remained significant (HR=1.84, 95% CI: 1.77‐1.91). Further adjustment for psychiatric comorbidities attenuated but could not fully explain the association (HR=1.65, 95% CI: 1.59‐1.71). The strongest associations were found for cardiac arrest (HR=2.28, 95% CI: 1.81‐2.87), hemorrhagic stroke (HR=2.16, 95% CI: 1.68‐2.77), and peripheral vascular disease/arteriosclerosis (HR=2.05, 95% CI: 1.76‐2.38). Stronger associations were observed in males and younger adults, while comparable associations were found among individuals with or without psychotropic medications and family history of cardiovascular diseases. These data suggest that ADHD is an independent risk factor for a wide range of cardiovascular diseases. They highlight the importance of carefully monitoring cardiovascular health and developing age‐appropriate and individualized strategies to reduce the cardiovascular risk in individuals with ADHD.     

Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N-terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+-mediated allosteric down-regulation of FVIIa's activity.     

Dynamic hyperinflation is associated with a poor cardiovascular response to exercise in COPD patients

Background

Pulmonary hyperinflation has the potential for significant adverse effects on cardiovascular function in COPD. The aim of this study was to investigate the relationship between dynamic hyperinflation and cardiovascular response to maximal exercise in COPD patients.

Methods

We studied 48 patients (16F; age 68 yrs ± 8; BMI 26 ± 4) with COPD. All patients performed spirometry, plethysmography, lung diffusion capacity for carbon monoxide (TLco) measurement, and symptom-limited cardiopulmonary exercise test (CPET). The end-expiratory lung volume (EELV) was evaluated during the CPET. Cardiovascular response was assessed by change during exercise in oxygen pulse (ΔO₂Pulse) and double product, i.e. the product of systolic blood pressure and heart rate (DP reserve), and by the oxygen uptake efficiency slope (OUES), i.e. the relation between oxygen uptake and ventilation.

Results

Patients with a peak exercise EELV (%TLC) ≥ 75% had a significantly lower resting FEV₁/VC, FEF₅₀/FIF₅₀ ratio and IC/TLC ratio, when compared to patients with a peak exercise EELV (%TLC) < 75%. Dynamic hyperinflation was strictly associated to a poor cardiovascular response to exercise: EELV (%TLC) showed a negative correlation with ΔO₂Pulse (r = - 0.476, p = 0.001), OUES (r = - 0.452, p = 0.001) and DP reserve (r = - 0.425, p = 0.004). Furthermore, according to the ROC curve method, ΔO₂Pulse and DP reserve cut-off points which maximized sensitivity and specificity, with respect to a EELV (% TLC) value ≥ 75% as a threshold value, were ≤ 5.5 mL/bpm (0.640 sensitivity and 0.696 specificity) and ≤ 10,000 Hg · bpm (0.720 sensitivity and 0.783 specificity), respectively.

Conclusion

The present study shows that COPD patients with dynamic hyperinflation have a poor cardiovascular response to exercise. This finding supports the view that in COPD patients, dynamic hyperinflation may affect exercise performance not only by affecting ventilation, but also cardiac function.     

The length of the linker between the epidermal growth factor‐like domains in factor VIIa is critical for a productive interaction with tissue factor

Formation of the factor VIIa (FVIIa)‐tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure‐based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)‐like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ‐carboxyglutamic acid (Gla)‐containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly‐Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF‐mediated stabilization of the active conformation of FVIIa and abrogated TF‐induced activity enhancement. In addition, FVIIa variants with short linkers associated 80‐fold slower with soluble TF (sTF) as compared with wild‐type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca²⁺‐free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)‐FVIIa reaching the level of Gla‐domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo‐FVIIa activity.     

Population dynamics of aquatic bacteria in relation to environment change as measured by factor analysis

In order to determine the relationship changes in river water bacteria populations and environmental changes, a multivariate statistical analysis, factor analysis was performed on two datasets: one the bacterial test responses, the other certain physicochemical parameters of the water. When sampling bacteria from natural environments, the process is highly selective and the question of the representative nature of the population arises. If a relationship between the environment and the predominant bacterial population can be established, there is an increased level of confidence in the ecological significance of the observed population.Bacterial isolates from river water were examined for positive and negative responses to 223 physiological and nutritional tests. The dichotomous database was factor analyzed. Each factor was interpreted according to its major biological attributes. A parallel dataset on 19 physical and chemical parameters was also analysed for factors. The two datasets were correlated and a relationship was demonstrated between the physiological attributes of the bacterial population and the environmental parameters was also analyzed for factors. The two datasets were correlated and a relationship was bacteria was related to ion concentration and specific conductance. Subtile changes in nutrient and oxygen levels were shown to relate to shifts from nitrate reduction to nutrification. The analytical method demonstrates the possibility of expanding the measurement of the parameters controlling bacterial populations by multivariate statistics without loss of the essential variance.     

Genetic analysis of factor V Leiden in a family with history of thrombosis and venous leg ulcers

Inherited resistance to activated protein C has been recognized as a major risk factor for thrombosis. The factor V Leiden mutation, which is detectable by molecular DNA techniques, is responsible for 95% of cases of activated protein C resistance. In our study one patient with venous leg ulcers from a family with a history of thrombosis showed factor V Leiden mutation. Genotypic analysis demonstrated that the patient was homozygous for factor V Leiden. All family members of the index subject showed the same abnormalities. Two were homozygous and 3 were heterozygous for factor V Leiden mutation. The polymerase chain reaction was used to amplify exon 10 of the factor V gene, followed by enzymatic digestion with MnlI for mutation detection. Patients with a family history of thrombosis and factor V Leiden have an increased risk of venous leg ulcers. Screening for factor V Leiden may be indicated in patients with venous leg ulcers and their family members.