Identification of Serum Interleukin-22 as Novel Biomarker in Pulmonary Hypertension: A Translational Study.

Growing evidence suggests the crucial involvement of inflammation in the pathogenesis of pulmonary hypertension (PH). The current study analyzed the expression of interleukin (IL)-17a and IL-22 as potential biomarkers for PH in a preclinical rat model of PH as well as the serum levels in a PH patient collective. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in 10 Sprague Dawley rats (PH) and compared to 6 sham-treated controls (CON) as well as 10 monocrotalin-induced, macitentan-treated rats (PH_MAC). Lung and cardiac tissues were subjected to histological and immunohistochemical analysis for the ILs, and their serum levels were quantified using ELISA. Serum IL levels were also measured in a PH patient cohort. IL-22 expression was significantly increased in the lungs of the PH and PH_MAC groups (p = 0.002), whereas increased IL17a expression was demonstrated only in the lungs and RV of the PH (p < 0.05) but not the PH_MAC group (p = n.s.). The PH group showed elevated serum concentrations for IL-22 (p = 0.04) and IL-17a (p = 0.008). Compared to the PH group, the PH_MAC group demonstrated a decrease in IL-22 (p = 0.021) but not IL17a (p = n.s.). In the PH patient collective (n = 92), increased serum levels of IL-22 but not IL-17a could be shown (p < 0.0001). This elevation remained significant across the different etiological groups (p < 0.05). Correlation analysis revealed multiple significant relations between IL-22 and various clinical, laboratory, functional and hemodynamic parameters. IL-22 could serve as a promising inflammatory biomarker of PH with potential value for initial diagnosis, functional classification or even prognosis estimation. Its validation in larger patients' cohorts regarding outcome and survival data, as well as the probability of promising therapeutic target structures, remains the object of further studies.

Expression of Inflammatory Genes in Murine Lungs in a Model of Experimental Pulmonary Hypertension: Effects of an Antibody-Based Targeted Delivery of Interleukin-9.

BACKGROUND: Pathogenesis of pulmonary hypertension (PH) is a multifactorial process driven by inflammation and pulmonary vascular remodeling. To target these two aspects of PH, we recently tested a novel treatment: Interleukin-9 (IL9) fused to F8, an antibody that binds to the extra-domain A of fibronectin (EDA+ Fn). As EDA+ Fn is not found in healthy adult tissue but is expressed during PH, IL9 is delivered specifically to the tissue affected by PH. We found that F8IL9 reduced pulmonary vascular remodeling and attenuated PH compared with sham-treated mice. PURPOSE: To evaluate possible F8IL9 effects on PH-associated inflammatory processes, we analysed the expression of genes involved in pulmonary immune responses. METHODS: We applied the monocrotaline (MCT) model of PH in mice (n = 44). Animals were divided into five experimental groups: sham-induced animals without PH (control, n = 4), MCT-induced PH without treatment (PH, n = 8), dual endothelin receptor antagonist treatment (dual ERA, n = 8), F8IL9 treatment (n = 12, 2 formats with n = 6 each), or with KSFIL9 treatment (KSFIL9, n = 12, 2 formats with n = 6 each, KSF: control antibody with irrelevant antigen specificity). After 28 days, a RT-PCR gene expression analysis of inflammatory response (84 genes) was performed in the lung. RESULTS: Compared with the controls, 19 genes exhibited relevant (+2.5-fold) upregulation in the PH group without treatment. Gene expression levels in F8IL9-treated lung tissue were reduced compared to the PH group without treatment. This was the case especially for CCL20, CXCL5, C-reactive protein, pentraxin related (CRPPR), and Kininogen-1 (KNG1). CONCLUSION: In accordance with the hypothesis stated above, F8IL9 treatment diminished the upregulation of some genes associated with inflammation in a PH animal model. Therefore, we hypothesize that IL9-based immunocytokine treatment will likely modulate various inflammatory pathways.

Targeted Interleukin-9 delivery in pulmonary hypertension: Comparison of immunocytokine formats and effector cell study.

AIMS: Pulmonary hypertension (PH) is accompanied by pulmonary vascular remodelling. By targeted delivery of Interleukin-9 (IL9) via the immunocytokine F8IL9, beneficial effects could be demonstrated in a mouse model of PH. This study aimed to compare two immunocytokine formats (single-chain Fv and full IgG) and to identify potential target cells of IL9. METHODS: The Monocrotaline mouse model of PH (PH, n = 12) was chosen to evaluate the treatment effects of F8IL9F8 (n = 12) and F8IgGIL9 (n = 6) compared with sham-induced animals (control, n = 10), the dual endothelin receptor antagonist Macitentan (MAC, n = 12) or IL9-based immunocytokines with irrelevant antigen specificity (KSFIL9KSF, n = 12; KSFIgGIL9 n = 6). Besides comparative validation of treatment effects, the study was focused on the detection and quantification of mast cells (MCs) and regulatory T cells (Tregs). RESULTS: There was a significantly elevated systolic right ventricular pressure (104 ± 36 vs. 45 ± 17 mmHg) and an impairment of right ventricular echocardiographic parameters (RVbasal: 2.52 ± 0.25 vs. 1.94 ± 0.13 mm) in untreated PH compared with controls (p < 0.05). Only the groups treated with F8IL9, irrespective of the format, showed consistent beneficial effects (p < 0.05). Moreover, F8IL9F8 but not F8IgGIL9 treatment significantly reduced lung tissue damage compared with untreated PH mice (p < 0.05). There was a significant increase in Tregs in F8IL9-treated compared with control animals, the untreated PH and the MAC group (p < 0.05). CONCLUSIONS: Beneficial treatment effects of targeted IL9 delivery in a preclinical model of PH could be convincingly validated. IL9-mediated recruitment of Tregs into lung tissue might play a crucial role in the induction of anti-inflammatory and anti-proliferative mechanisms potentially contributing to a novel disease-modifying concept.

Prediction of one- and two-year mortality after transcatheter aortic valve implantation: proposal of a fast sum-score system integrating a novel biomarker of cardiac extracellular matrix accumulation and fibrosis.

BACKGROUND: Prediction of long-term mortality in patients with severe symptomatic aortic valve stenosis undergoing transcatheter aortic valve implantation (TAVI) is still challenging but of great impact with respect to the selection of treatment strategy. Whereas most of the established scores address perioperative risk and/or short-term mortality, the aim of our current study was the integrative investigation of a multitude of patients' characteristics including novel biomarkers of cardiovascular remodeling with respect to their value for the prediction of long-term mortality. METHODS: In a first subset of patients (n = 122, identification group) a wide range of baseline characteristics were assigned to three clusters with 4 to 10 items each (classical clinical parameters; risk assessment scores; novel biomarkers of cardiovascular remodeling) and tested with respect to their predictive value for one-year mortality. Thereby, a sum-score system (Jena Mortality Score, JMS) was defined and tested in a larger collective of TAVI patients (n = 295, validation group) with respect to one- and two-year mortality prediction. RESULTS: In the identification cohort, binary logistic regression analysis, with one-year mortality as dependent variable and the items per cluster as cofounders, revealed atrial fibrillation (Afib; odds ratio [OR] 7.583, 95% confidence interval [95% CI]: 2.051-28.040, p = 0.002), clinical frailty scale (CFS; OR 2.258, 95% CI: 1.262-4.039, p = 0.006) and Tissue-Inhibitor of Metalloproeinase-1 (TIMP-1; OR 1.006, 95% CI: 1.001-1.011, p = 0.019) as independent predictors of one-year mortality. These 3 parameters were integrated into a simplified sum-score as follows: presence of Afib (no = 0, yes = 1); dichotomized CFS (1 to 4 = 0; 5 to 9 = 1); TIMP-1 range (cut-off value 187.2 ng/mL; below = 0, above = 1). The resulting sum-score (JMS) ranged from 0 to 3. By binary logistic regression analysis in the validation cohort with one- and two-year mortality as dependent variable and Society of Thoracic Surgeons (STS) score (STS), staging of extra-valvular cardiac damage (stage), presence of high gradient aortic stenosis (HGAS), EQ visual analogue scale score (EQ-VAS) and JMS as cofounders, besides STS score, only JMS could be proven to serve as independent predictor of both, one-year (OR 1.684, 95% CI: 1.094-2.592, p = 0.018) and two-year (OR 1.711, 95% CI: 1.136-2.576, p = 0.010) mortality. After dichotomization of patients into a low-risk and a high-risk group according to JMS, Kaplan-Meier survival analysis displayed a significant survival benefit for the low-risk group after one and two years (p < 0.001). CONCLUSION: JMS, including TIMP-1 as a novel biomarker of cardiac extracellular matrix accumulation and fibrosis, could serve as a novel simple tool to assess long-term mortality risk after TAVI and might thereby contribute to a more precise stratification of individual risk.

Host-pathogen interactions of clinical S. aureus isolates to induce infective endocarditis.

To evaluate potential pathomechanisms in the induction of infective endocarditis (IE), 34 Staphylococcus aureus (S. aureus) isolates, collected from patients with S. aureus endocarditis and from healthy individuals were investigated both in vitro and in vivo. S. aureus isolates were tested in vitro for their cytotoxicity, invasion and the association with platelets. Virulence factor expression profiles and cellular response were additionally investigated and tested for correlation with the ability of S. aureus to induce vegetations on the aortic valves in vivo. In an animal model of IE valvular conspicuity was assessed by in vivo magnetic resonance imaging at 9.4 T, histology and enrichment gene expression analysis. All S. aureus isolates tested in vivo caused a reliable infection and inflammation of the aortic valves, but could not be differentiated and categorized according to the measured in vitro virulence profiles and cytotoxicity. Results from in vitro assays did not correlate with the severity of IE. However, the isolates differed substantially in the activation and inhibition of pathways connected to the extracellular matrix and inflammatory response. Thus, comprehensive approaches of host-pathogen interactions and corresponding immune pathways are needed for the evaluation of the pathogenic capacity of bacteria. An improved understanding of the interaction between virulence factors and immune response in S. aureus infective endocarditis would offer novel possibilities for the development of therapeutic strategies and specific diagnostic imaging markers.

Serum Liberation of Fetal Fibronectin Variants in Patients with Pulmonary Hypertension: ED-A+ Fn as Promising Novel Biomarker of Pulmonary Vascular and Right Ventricular Myocardial Remodeling.

BACKGROUND AND AIMS: Pulmonary Hypertension (PH) represents an aetiologically and clinically heterogeneous disorder accompanied by a severely impaired prognosis. Key steps of PH pathogenesis are vascular and right ventricular myocardial remodelling entailing the re-occurrence of fetal variants of the cell adhesion modulating protein fibronectin (Fn) being virtually absent in healthy adult tissues. These variants are liberated into circulation and are therefore qualified as excellent novel serum biomarkers. Moreover, these molecules might serve as promising therapeutic targets. The current study was aimed at quantifying the serum levels of two functionally important fetal Fn variants (ED-A+ and ED-B+ Fn) in patients suffering from PH due to different aetiologies compared to healthy controls. METHODS: Serum levels of ED-A+ and ED-B+ Fn were quantified using novel ELISA protocols established and validated in our group in 80 PH patients and 40 controls. Results were analysed with respect to clinical, laboratory, echocardiographic and functional parameters. RESULTS: Serum levels of ED-A+ Fn (p = 0.001) but not ED-B+ Fn (p = 0.722) were significantly increased in PH patients compared to healthy controls. Thus, the following analyses were performed only for ED-A+ Fn. When dividing PH patients into different aetiological groups according to current ESC guidelines, the increase in ED-A+ Fn in PH patients compared to controls remained significant for group 1 (p = 0.032), 2 (p = 0.007) and 3 (p = 0.001) but not for group 4 (p = 0.156). Correlation analysis revealed a significant relation between ED-A+ Fn and brain natriuretic peptide (BNP) (r = 0.310; p = 0.002), six minutes' walk test (r = -0.275; p = 0.02) and systolic pulmonary artery pressure (PAPsys) (r = 0.364; p < 0.001). By logistic regression analysis (backward elimination WALD) including a variety of potentially relevant patients' characteristics, only chronic kidney disease (CKD) (OR: 8.866; CI: 1.779-44.187; p = 0.008), C reactive protein (CRP) (OR: 1.194; CI: 1.011-1.410; p = 0.037) and ED-A+ Fn (OR: 1.045; CI: 1.011-1.080; p = 0.009) could be identified as independent predictors of the presence of PH. CONCLUSIONS: Against the background of our results, ED-A+ Fn could serve as a promising novel biomarker of PH with potential value for initial diagnosis and aetiological differentiation. Moreover, it might contribute to more precise risk stratification of PH patients. Beyond that, the future role of ED-A+ Fn as a therapeutic target has to be evaluated in further studies.

Therapeutic Evaluation of Antibody-Based Targeted Delivery of Interleukin 9 in Experimental Pulmonary Hypertension.

BACKGROUND AND AIMS: Pulmonary hypertension (PH) is a heterogeneous disorder associated with poor prognosis. For the majority of patients, only limited therapeutic options are available. Thus, there is great interest to develop novel treatment strategies focusing on pulmonary vascular and right ventricular remodeling. Interleukin 9 (IL9) is a pleiotropic cytokine with pro- and anti-inflammatory functions. The aim of this study was to evaluate the therapeutic activity of F8IL9F8 consisting of IL9 fused to the F8 antibody, specific to the alternatively-spliced EDA domain of fibronectin, which is abundantly expressed in pulmonary vasculature and right ventricular myocardium in PH. METHODS: The efficacy of F8IL9F8 in attenuating PH progression in the monocrotaline mouse model was evaluated in comparison to an endothelin receptor antagonist (ERA) or an IL9 based immunocytokine with irrelevant antibody specificity (KSFIL9KSF). Treatment effects were assessed by right heart catheterization, echocardiography as well as histological and immunohistochemical tissue analyses. RESULTS: Compared to controls, systolic right ventricular pressure (RVPsys) was significantly elevated and a variety of right ventricular echocardiographic parameters were significantly impaired in all MCT-induced PH groups except for the F8IL9F8 group. Both, F8IL9F8 and ERA treatments lead to a significant reduction in RVPsys and an improvement of echocardiographic parameters when compared to the MCT group not observable for the KSFIL9KSF group. Only F8IL9F8 significantly reduced lung tissue damage and displayed a significant decrease of leukocyte and macrophage accumulation in the lungs and right ventricles. CONCLUSIONS: Our study provides first pre-clinical evidence for the use of F8IL9F8 as a new therapeutic agent for PH in terms of a disease-modifying concept addressing cardiovascular remodeling.

Pulmonary Arteriovenous Pressure Gradient and Time-Averaged Mean Velocity of Small Pulmonary Arteries Can Serve as Sensitive Biomarkers in the Diagnosis of Pulmonary Arterial Hypertension: A Preclinical Study by 4D-Flow MRI.

(1) Background: Pulmonary arterial hypertension (PAH) is a serious condition that is associated with many cardiopulmonary diseases. Invasive right heart catheterization (RHC) is currently the only method for the definitive diagnosis and follow-up of PAH. In this study, we sought a non-invasive hemodynamic biomarker for the diagnosis of PAH. (2) Methods: We applied prospectively respiratory and cardiac gated 4D-flow MRI at a 9.4T preclinical scanner on three different groups of Sprague Dawley rats: baseline (n = 11), moderate PAH (n = 8), and severe PAH (n = 8). The pressure gradients as well as the velocity values were analyzed from 4D-flow data and correlated with lung histology. (3) Results: The pressure gradient between the pulmonary artery and vein on the unilateral side as well as the time-averaged mean velocity values of the small pulmonary arteries were capable of distinguishing not only between baseline and severe PAH, but also between the moderate and severe stages of the disease. (4) Conclusions: The current preclinical study suggests the pulmonary arteriovenous pressure gradient and the time-averaged mean velocity as potential biomarkers to diagnose PAH.

Serum Biomarkers of Cardiovascular Remodelling Reflect Extra-Valvular Cardiac Damage in Patients with Severe Aortic Stenosis.

In patients with aortic stenosis (AS), a novel staging classification of extra-valvular left and right heart damage with prognostic relevance was introduced in 2017. The aim of the study was to evaluate the biomarkers of cardiovascular tissue remodelling in relation to this novel staging classification. Patients were categorized according to the novel staging classification into stages 0 to 4. The levels of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinases 1 (TIMP-1), B and C domain containing tenascin-C (B+ Tn-C, C+ Tn-C), the ED-A and ED-B domain containing fibronectin (ED-A+ Fn, ED-B+ Fn), endothelin 1 (ET-1) and neutrophil gelatinase-associated lipocalin (NGAL) were determined in serum by ELISA. There were significantly decreased serum levels of MMP-9 and increased levels of B+ Tn-C and C+ Tn-C when comparing stages 0 and 1 with stage 2, with no further dynamics in stages 3 and 4. In contrast, for TIMP-1, C+ Tn-C, ED-A+ Fn, ET-1 and NGAL, significantly increased serum levels could be detected in stages 3 and 4 compared to both stages 0 and 1 and stage 2. ED-A+ Fn and ET-1 could be identified as independent predictors of the presence of stage 3 and/or 4. To the best of our knowledge, this is the first study identifying novel serum biomarkers differentially reflecting the patterns of left and right heart extra-valvular damage in patients suffering from AS. Our findings might indicate a more precise initial diagnosis and risk stratification.

Cellular inflammation in pulmonary hypertension: Detailed analysis of lung and right ventricular tissue, circulating immune cells and effects of a dual endothelin receptor antagonist.

Growing evidence suggests that inflammation is crucially involved in the pathogenesis of pulmonary hypertension (PH) and consecutive right heart failure. The present study analyzed the inflammatory response in lung and right ventricle in a rat model of PH and evaluated the effects of the dual endothelin receptor antagonist (ERA) Macitentan. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in Sprague-Dawley rats (PH, n = 10) and compared to healthy controls (CON, n = 10) as well as monocrotalin-induced, macitentan-treated rats (THER, n = 10). Detection of Dendritic cells (DCs), regulatory T cells (Tregs) and others as well as RT-PCR based inflammatory gene expression analysis were performed. Circulating DCs and Tregs were quantified by flow cytometry in the rat model and in PH patients (n = 70) compared to controls (n = 52). Inflammatory cells were increased in lung and right ventricular tissue, whereas DCs and Tregs were decreased in blood. Expression of 17 genes in the lung and 20 genes in the right ventricle were relevantly (>2.0 fold) regulated in the PH group. These effects were, at least in part, attenuated in response to Macitentan treatment. In humans as well as rats, immune cells showed significant correlations to clinical, echocardiographic, and haemodynamic parameters. PH is accompanied by a distinct inflammatory response in lung and right but not left ventricular tissue attenuated by Macitentan. Correlations of circulating DCs as well as tissue resident immune cells with parameters reflecting right ventricular function raise the idea of both, promising biomarkers and novel treatment strategies.

Cystatin A suppresses tumor cell growth through inhibiting epithelial to mesenchymal transition in human lung cancer.

Cystatin A (CSTA), belonging to type 1 cystatin super-family, is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by cathepsins B, H and L. CSTA acts as a tumor suppressor in esophageal cancer, however, its role in lung cancer has not yet been elucidated. Here we found that CSTA was down-regulated in all lung cancer cell lines compared to normal lung epithelial cells. CSTA was restored in most lung cancer cell lines after treatment with demethylation agent 5-aza-2-deoxycytidine and deacetylation agent Trichostatin. Bisulfite sequencing revealed that CSTA was partially methylated in the promoter and exon 1. In primary lung tumors, squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (p<0.00001), and higher expression of CSTA was significantly associated with lower tumor grade (p<0.01). CSTA stable transfection reduced the activity of cathepsin B and inhibited the ability of colony formation, migration and invasion, and enhanced gemcitabine-induced apoptosis. CSTA overexpression resulted in reduced activity of ERK, p-38, and AKT. Additionally, CSTA overexpression led to a mesenchymal to epithelial transition (MET) and prevented the TGF-ß1-induced epithelial to mesenchymal transition (EMT) through inhibiting the ERK/MAPK pathway. In conclusion, our date indicate 1) epigenetic regulation is associated with CSTA gene silencing; 2) CSTA exerts tumor suppressive function through inhibiting MAPK and AKT pathways; 3) Overexpression of CSTA leads to MET and prevents TGF-ß1-induced EMT by modulating the MAPK pathway; 4) CSTA may be a potential biomarker for lung SCC and tumor differentiation.

Increased Serum Levels of Fetal Tenascin-C Variants in Patients with Pulmonary Hypertension: Novel Biomarkers Reflecting Vascular Remodeling and Right Ventricular Dysfunction?

Pulmonary vascular remodeling is a pathophysiological feature that common to all classes of pulmonary hypertension (PH) and right ventricular dysfunction, which is the major prognosis-limiting factor. Vascular, as well as cardiac tissue remodeling are associated with a re-expression of fetal variants of cellular adhesion proteins, including tenascin-C (Tn-C). We analyzed circulating levels of the fetal Tn-C splicing variants B⁺ and C⁺ Tn-C in serum of PH patients to evaluate their potential as novel biomarkers reflecting vascular remodeling and right ventricular dysfunction. Serum concentrations of B⁺ and C⁺ Tn-C were determined in 80 PH patients and were compared to 40 healthy controls by enzyme-linked immunosorbent assay. Clinical, laboratory, echocardiographic, and functional data were correlated with Tn-C levels. Serum concentrations of both Tn-C variants were significantly elevated in patients with PH (p < 0.05). Significant correlations could be observed between Tn-C and echocardiographic parameters, including systolic pulmonary artery pressure (B⁺ Tn-C: r = 0.31, p < 0.001, C⁺ Tn-C: r = 0.26, p = 0.006) and right atrial area (B⁺ Tn-C: r = 0.46, p < 0.001, C⁺ Tn-C: r = 0.49, p < 0.001), and laboratory values like BNP (B⁺ Tn-C: r = 0.45, p < 0.001, C⁺ Tn-C: r = 0.42, p < 0.001). An inverse correlation was observed between Tn-C variants and 6-minute walk distance as a functional parameter (B⁺ Tn-C: r = -0.54, p < 0.001, C⁺ Tn-C: r = -0.43, p < 0.001). In a multivariate analysis, B⁺ Tn-C, but not C⁺ Tn-C, was found to be an independent predictor of pulmonary hypertension. Both fetal Tn-C variants may represent novel biomarkers that are capable of estimating both pulmonary vascular remodeling and right ventricular load. The potential beneficial impact of Tn-C variants for risk stratification in patients with PH needs further investigation.

Arginase Inhibition Reverses Monocrotaline-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous disorder associated with a poor prognosis. Thus, the development of novel treatment strategies is of great interest. The enzyme arginase (Arg) is emerging as important player in PH development. The aim of the current study was to determine the expression of ArgI and ArgII as well as the effects of Arg inhibition in a rat model of PH. PH was induced in 35 Sprague-Dawley rats by monocrotaline (MCT, 60 mg/kg as single-dose). There were three experimental groups: sham-treated controls (control group, n = 11), MCT-induced PH (MCT group, n = 11) and MCT-induced PH treated with the Arg inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA; MCT/NorNoha group, n = 13). ArgI and ArgII expression was determined by immunohistochemistry and Western blot. Right ventricular systolic pressure (RVPsys) was measured and lung tissue remodeling was determined. Induction of PH resulted in an increase in RVPsys (81 ± 16 mmHg) compared to the control group (41 ± 15 mmHg, p = 0.002) accompanied by a significant elevation of histological sum-score (8.2 ± 2.4 in the MCT compared to 1.6 ± 1.6 in the control group, p < 0.001). Both, ArgI and ArgII were relevantly expressed in lung tissue and there was a significant increase in the MCT compared to the control group (p < 0.01). Arg inhibition resulted in a significant reduction of RVPsys to 52 ± 19 mmHg (p = 0.006) and histological sum-score to 5.8 ± 1.4 compared to the MCT group (p = 0.022). PH leads to increased expression of Arg. Arg inhibition leads to reduction of RVPsys and diminished lung tissue remodeling and therefore represents a potential treatment strategy in PH.

Lung tissue remodelling in MCT-induced pulmonary hypertension: a proposal for a novel scoring system and changes in extracellular matrix and fibrosis associated gene expression.

Pulmonary hypertension (PH) is associated with vasoconstriction and remodelling. We studied lung tissue remodelling in a rat model of PH with special focus on histology and extracellular matrix (ECM) remodelling. After induction of PH by monocrotaline, lung tissue was analysed histologically, by gene expression analysis and immunofluorescence labelling of ED-A domain containing fibronectin (ED-A+ Fn), B domain containing tenascin-C (B+ Tn-C) as well as alpha-smooth muscle actin (α-SMA). Serum concentrations of ED-A+ Fn were determined by ELISA. Systolic right ventricular pressure (RVPsys) values were significantly elevated in PH (n = 18; 75 ± 26.4 mmHg) compared to controls (n = 10; 29 ± 19.3 mmHg; p = 0.015). The histological sum-score was significantly increased in PH (8.0 ± 2.2) compared to controls (2.5 ± 1.6; p < 0.001). Gene expression analysis revealed relevant induction of several key genes of extracellular matrix remodelling. Increased protein deposition of ED-A+ Fn but not of B+ Tn-C and α-SMA in lung tissue was found in PH (2.88 ± 3.19 area%) compared to controls (1.32 ± 0.16 area%; p = 0.030). Serum levels of ED-A+ Fn were significantly higher in PH (p = 0.007) positively correlating with RVPsys (r = 0.618, p = 0.019). We here present a novel histological scoring system to assess lung tissue remodelling in PH. Gene expression analysis revealed induction of candidate genes involved in collagen matrix turnover, fibrosis and vascular remodelling. The stable increased tissue deposition of ED-A+ Fn in PH as well as its dynamics in serum suggests a role as a promising novel biomarker and potential therapeutic target.

Detection of Soluble ED-A(+) Fibronectin and Evaluation as Novel Serum Biomarker for Cardiac Tissue Remodeling.

Background and Aims. Fibronectin containing the extra domain A (ED-A(+) Fn) was proven to serve as a valuable biomarker for cardiac remodeling. The study was aimed at establishing an ELISA to determine ED-A(+) Fn in serum of heart failure patients. Methods. ED-A(+) Fn was quantified in serum samples from 114 heart failure patients due to ischemic (ICM, n = 44) and dilated (DCM, n = 39) cardiomyopathy as well as hypertensive heart disease (HHD, n = 31) compared to healthy controls (n = 12). Results. In comparison to healthy volunteers, heart failure patients showed significantly increased levels of ED-A(+) Fn (p < 0.001). In particular in ICM patients there were significant associations between ED-A(+) Fn serum levels and clinical parameters, for example, increased levels with rising NYHA class (p = 0.013), a negative correlation with left ventricular ejection fraction (p = 0.026, r: -0.353), a positive correlation with left atrial diameter (p = 0.008, r: 0.431), and a strong positive correlation with systolic pulmonary artery pressure (p = 0.002, r: 0.485). In multivariate analysis, ED-A(+) Fn was identified as an independent predictor of an ischemic heart failure etiology. Conclusions. The current study could clearly show that ED-A(+) Fn is a promising biomarker in cardiovascular diseases, especially in heart failure patients due to an ICM. We presented a valid ELISA method, which could be applied for further studies investigating the value of ED-A(+) Fn.

Targeted delivery of interleukin-10 to chronic cardiac allograft rejection using a human antibody specific to the extra domain A of fibronectin.

BACKGROUND AND AIMS: Management of chronic rejection is challenging since there are not sufficient preventive or therapeutic strategies. The rejection process leads to overexpression of ED-A(+) fibronectin (ED-A(+) Fn). The human antibody F8, specific to ED-A(+) Fn, may serve as a vehicle for targeted delivery of bioactive payloads, e.g. interleukin 10 (IL-10). The aim of this study was to investigate the therapeutic effects of the fusion protein F8-interleukin-10 (F8-IL10) in the process of chronic rejection development. METHODS: A heterotopic rat heart transplantation model was used to induce chronic rejection. For therapeutic interventions, the immunocytokines F8-humanIL10 (DEKAVIL), F8-ratIL10 as well as KSF-humanIL10 (irrelevant antigen-specificity) were used. Treatment was performed weekly for 10 weeks starting at day 7 after transplantation (1mg/animal). RESULTS: In the cardiac allografts, treatment with F8-huIL10 or F8-ratIL10 was associated with increased heart weights, a higher grade of chronic rejection, increased CIF, higher protein expression levels of alpha-smooth muscle actin (α-SMA), an augmented infiltration with inflammatory cells (CD4+, CD8+ and CD68+ cells) and higher serum levels of brain natriuretic peptide (BNP) compared to the control groups. CONCLUSIONS: All observed treatment effects are transplantation-specific since the F8 antibody is specific to ED-A(+) Fn that is not expressed in healthy hearts. A clear targeting effect of F8-huIL10 as well as F8-ratIL10 could be proven. Against that background, a further study is needed to address the question, if F8-IL10 treatment is capable to reduce CAV and CIF starting at a time point when chronic rejection has fully developed (therapeutic approach).

Serum levels of tenascin-C variants in congestive heart failure patients: comparative analysis of ischemic, dilated, and hypertensive cardiomyopathy.

BACKGROUND: Tissue remodelling in ischemic cardiomyopathy (ICM), dilated cardiomyopathy (DCM), and hypertensive heart disease (HHD) is accompanied by the re-occurrence of fetal tenascin-C (Tn-C) variants. The study was aimed to comparatively analyze the serum levels of Tn-C containing the FNIIIB (B+ Tn-C) or FNIIIC (C+ Tn-C) domain in heart failure patients due to ICM, DCM, and HHD. METHODS: 119 male patients with congestive heart failure (45 with ICM, 43 with DCM, 31 with HHD) were included. Measurement of serum levels of B+ and C+ Tn-C was performed using Enzyme Linked Immunosorbent Assay (ELISA). Results were correlated to clinical, laboratory, echocardiographic, and spiroergometric parameters. RESULTS: Analysis of Tn-C concentrations according to heart failure etiology revealed no significant differences. There was an association of C+ Tn-C serum levels to enlargement of the left atrium in DCM (p < 0.01) and the left ventricle in HHD (p < 0.05). In patients with ICM, C+ Tn-C showed a strong negative correlation to the stress test performance (p = 0.002, R2: -0.691). Most strikingly, there was a strong correlation between BNP and B+ Tn-C (p = 0.038, R2: 0.466) as well as C+ Tn-C (p = 0.001, R2: 0.814) in DCM patients. CONCLUSIONS: The present study highlights the impact of Tn-C variants as biomarkers reflecting the extent of cardiac remodeling in heart failure patients. Furthermore, B+ Tn-C can be suggested as an additional tool to estimate ICM performance in patients. Especially in combination with BNP, analysis of Tn-C might pave the way for a more precise evaluation of heart failure patients.

De novo expression of fetal ED-A(+) fibronectin and B (+) tenascin-C splicing variants in human cardiac allografts: potential impact for targeted therapy of rejection.

Management of acute and especially chronic rejection after human cardiac transplantation is still challenging. Chronic rejection, represented by allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) is known to cause severe long-term complications. Rejection associated tissue-remodelling entails the reoccurrence of fetal variants of Fibronectin (Fn) and Tenascin-C (Tn-C), which are virtually absent in adult human organs. In a rat model, an extensive re-expression could be demonstrated for ED-A(+) Fn with spatial association to CAV and CIF. Thus, it is of great interest to investigate the cardiac tissue expression and distribution in human samples. From 48 heart transplanted patients, 64 tissue specimens derived from right ventricular biopsies were available. Histopathological analysis was performed according to the International Society for Heart and Lung Transplantation (ISHLT) guidelines for the detection of acute rejection. By immunohistochemistry, protein expression of ED-A(+) Fn, B(+) Tn-C, alpha-smooth muscle actin, CD31 and CD45 was assessed and analysed semiquantitatively. Co-localisation studies were performed by means of immunofluorescence double labelling. Histopathological analysis of the 64 samples revealed different ISHLT grades (0R in 36 cases, 1R in 20 cases and 2R in 8 cases). There was a distinct and quantitatively relevant re-occurrence of ED-A(+) Fn and B(+) Tn-C in most samples. Semi-quantitative evaluation did not show any correlation to the acute rejection grade for all markers. Interestingly, significant correlations to the extent of inflammation could be shown for ED-A(+) Fn (r = 0.442, p = 0.000) and B(+) Tn-C (r = 0.408, p = 0.001) as well as between both proteins (r = 0.663, p = 0.000). A spatial association of ED-A(+) Fn and B(+) Tn-C to CAV and CIF could be demonstrated. A relevant re-occurrence of ED-A(+) Fn and B(+) Tn-C following human heart transplantation could be demonstrated with spatial association to signs of rejection and a significant correlation to tissue inflammation. These data might contribute to the identification of novel biomarkers reflecting the rejection process and to the development of promising strategies to image, prevent or treat cardiac rejection.

Matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, B⁺ tenascin-C and ED-A⁺ fibronectin in dilated cardiomyopathy: potential impact on disease progression and patients' prognosis.

BACKGROUND: Dilated cardiomyopathy (DCM) is associated with heart failure and increased mortality and there is no reliable biomarker to estimate patients' prognosis. During cardiac remodeling, an extensive reorganization of the extracellular matrix occurs. The study was aimed to investigate matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1) and fetal tenascin-C (B(+) Tn-C) and fibronectin (ED-A(+) Fn) variants known to be involved in that process. METHODS AND RESULTS: In 187 patients with DCM, levels of MMP-9, TIMP-1 and B(+) Tn-C in serum as well as B(+) Tn-C and ED-A(+) Fn in tissue were quantified and subjected to univariate analysis. For all serum markers, concentrations above a calculated threshold were associated with decreased survival (MMP-9: p = 0.008, TIMP-1: p = 0.001, B(+) Tn-C: p < 0.001) and a significantly higher risk to die or undergo transplantation. In tissue, a reexpression of B(+) Tn-C and ED-A(+) Fn could be shown. Protein deposition levels of ≥4.5% for B(+) Tn-C and ≥2.1% for ED-A(+) Fn were associated with a significantly decreased survival (p = 0.001 for B(+) Tn-C, p = 0.031 for ED-A(+) Fn) and an increased risk to die or undergo transplantation. In a multivariate analysis, TIMP-1 is the superior parameter to predict transplantation free survival (p = 0.027). CONCLUSIONS: Serum levels of MMP-9, TIMP-1 and B(+) Tn-C and tissue levels of B(+) Tn-C and ED-A(+) Fn are promising markers for risk assessment. The reoccurrence of ED-A(+) Fn and the availability of a human antibody usable as a vehicle for targeted drug delivery might be the basis for novel therapeutic strategies.

Selective imaging of chronic cardiac rejection using a human antibody specific to the alternatively spliced EDA domain of fibronectin.

BACKGROUND: Chronic cardiac rejection is intimately associated with cardiac allograft vasculopathy and fibrosis, both causing severe complications that cannot be reversed. Thus, there is an urgent need for early diagnosis and for development of therapeutic agents. Chronic rejection is accompanied by the dramatic upregulation of EDA(+) fibronectin (EDA(+) Fn), which is virtually undetectable in the normal healthy adult. METHODS: In this study, we evaluated the potential of the monoclonal antibody F8, specific to that molecule, to selectively accumulate in chronically rejected allografts. RESULTS: A syngeneic immunocompetent heterotopic rat heart transplantation model was used to induce chronic rejection within 70 days. The F8 antibody or an antibody of irrelevant specificity, labeled with the dye DY-682, was administered and near-infrared fluorescence (NIRF) imaging was performed. Targeting performance was assessed by macroscopic organ imaging and fluorescence microscopy. A selective accumulation of the F8 antibody (but not of the negative control antibody) was observed by NIRF imaging in cardiac allografts. The antibody localized to diseased blood vessels as well as to fibrotic regions, where the cognate antigen is abundantly expressed. CONCLUSIONS: This is the first example of antibody-mediated imaging of chronic cardiac rejection. The findings pave the way to immuno-positron emission tomography (immuno-PET) imaging of this clinical condition in patients using the human F8 antibody labeled with a suitable radionuclide (e.g., iodine-124). Furthermore, it would be conceivable to use the F8 antibody as a delivery vehicle to assess experimentally whether a bioactive payload (e.g., drug or cytokine) may be able to reduce disease progression.