RT-PCR assay to detect FGFR3::TACC3 fusions in formalin-fixed, paraffin-embedded glioblastoma samples.

Background: One targeted treatment option for isocitrate dehydrogenase (IDH)-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next-generation sequencing (NGS) is not routinely performed, and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using reverse transcription polymerase chain reaction (RT-PCR) on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin-embedded (FFPE) tissue is available. Aim: To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples. Methods: Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 (n = 13) were subjected to FGFR3::TACC3 RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS (n = 6), FGFR3 Fluorescence In Situ Hybridization (n = 6), and RNA sequencing (n = 5). Results: Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an FGFR3::TACC3 fusion in 7 samples, covering 3 different fusion variants. For 5 of these FF was available, and the presence of the fusion was confirmed through RT-PCR on FF. With RNA sequencing, 1 additional sample was found to harbor an FGFR3::TACC3 fusion (variant not covered by current RT-PCR for FFPE). The frequency of FGFR3::TACC3 fusion in this cohort was 9/353 (2.5%). Conclusions: RT-PCR for FGFR3::TACC3 fusions can successfully be performed on FFPE material, with a specificity of 100% and (due to limited primer sets) a sensitivity of 83.3%. This assay allows for the identification of potential targeted treatment options when only formalin-fixed tissue is available.

Histological assessment of the sclerotic graft-versus-host response in the humanized RAG2-/-γc-/- mouse model.

Graft-versus-host disease (GVHD) remains a frequently occurring and difficult-to-treat complication in human allogeneic stem cell transplantation. Murine transplantation models are often used to study and understand the complex pathogenesis of GVHD and to explore new treatment strategies. Although GVHD kinetics may differ in murine and human models, adequate models are essential for identification of the crucial factors responsible for the major pathology in GVHD. We present a detailed description of the specific histological features of a graft-versus-host-induced fibrotic response in xenogeneic RAG2(-/-)γc(-/-) mice after total body irradiation and injection with human peripheral blood mononuclear cells. We describe the full morphological features of this reaction, including a detailed analysis of the specific tissue infiltration patterns of the human peripheral blood mononuclear cells. Our data show the development of fibrosis, predominantly near blood vessels, and reveal different cell populations and specific cell migration patterns in the affected organs. The combination of immunohistochemical cell characterization and mRNA expression analysis of both human (donor)- and murine (host)-derived cytokines reveals an interaction between host tissues and donor-derived cells in an entangled cytokine profile, in which both donor- and host-derived cytokines contribute to the formation of fibrosis.

Consensus molecular subtype 4 (CMS4)-targeted therapy in primary colon cancer: A proof-of-concept study.

Background: Mesenchymal Consensus Molecular Subtype 4 (CMS4) colon cancer is associated with poor prognosis and therapy resistance. In this proof-of-concept study, we assessed whether a rationally chosen drug could mitigate the distinguishing molecular features of primary CMS4 colon cancer. Methods: In the ImPACCT trial, informed consent was obtained for molecular subtyping at initial diagnosis of colon cancer using a validated RT-qPCR CMS4-test on three biopsies per tumor (Phase-1, n=69 patients), and for neoadjuvant CMS4-targeting therapy with imatinib (Phase-2, n=5). Pre- and post-treatment tumor biopsies were analyzed by RNA-sequencing and immunohistochemistry. Imatinib-induced gene expression changes were associated with molecular subtypes and survival in an independent cohort of 3232 primary colon cancer. Results: The CMS4-test classified 52/172 biopsies as CMS4 (30%). Five patients consented to imatinib treatment prior to surgery, yielding 15 pre- and 15 post-treatment samples for molecular analysis. Imatinib treatment caused significant suppression of mesenchymal genes and upregulation of genes encoding epithelial junctions. The gene expression changes induced by imatinib were associated with improved survival and a shift from CMS4 to CMS2. Conclusion: Imatinib may have value as a CMS-switching drug in primary colon cancer and induces a gene expression program that is associated with improved survival.

Accurate detection of circulating tumor DNA using nanopore consensus sequencing.

Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ~60×, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in clinical workflows.

Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy.

OBJECTIVE: To investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity. METHODS: We performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1-4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood. RESULTS: We identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing. CONCLUSIONS: PLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.

Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells.

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2-/- γc-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-ß. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response.

The Interleukin-33/ST2 Pathway Is Expressed in the Failing Human Heart and Associated with Pro-fibrotic Remodeling of the Myocardium.

The interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) pathway is a potential pathophysiological mediator of cardiac fibrosis. Soluble ST2 (sST2) is one of the main isoforms of ST2 with strong prognostic value in cardiac disease. The exact role of sST2 in cardiac fibrosis is unknown. The aim of this study was (1) to investigate myocardial expression of the IL-33/ST2 pathway in relation to myocardial fibrosis in end-stage heart failure patients and (2) to study whether plasma sST2 is associated with histologically determined cardiac fibrosis. In 38 patients undergoing left ventricular assist device implantation, mRNA expression of sST2, total ST2, and IL-33 was measured in cardiac tissue obtained during the implantation. In the same tissue, histological fibrosis was digitally quantified and mRNA expression of pro-fibrotic signaling molecules, connective tissue growth factor (CTGF) and transforming growth factor beta 1 (TGFß1), was measured. In addition, plasma levels of sST2 were determined. Expression levels of IL-33/ST2 pathway factors in myocardial tissue were significantly associated with cardiac fibrosis and the expression levels of CTGF and TGFß1. Plasma levels of sST2 did not correlate with tissue expression of ST2, the amount of fibrosis or myocardial expression of pro-fibrotic signaling proteins. The interleukin-33/ST2 pathway is expressed in the failing human heart and its expression is associated with cardiac fibrosis and pro-fibrotic signaling proteins, suggesting a role in pro-fibrotic myocardial remodeling. Soluble ST2 levels in the circulation did not correlate with the amount of cardiac fibrosis or myocardial ST2 expression, however. Therefore, other pathophysiological processes such as inflammation might also substantially affect sST2 plasma levels.

The chemokine and chemokine receptor profile of infiltrating cells in the wall of arteries with cardiac allograft vasculopathy is indicative of a memory T-helper 1 response.

BACKGROUND: Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development. METHODS AND RESULTS: We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell-alpha chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx-CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon-gamma was highly expressed in HTx+CAV compared with HTx-CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T-helper 1 (TH1) C+CR, were more abundant than in the HTx-CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups. CONCLUSIONS: In coronary arteries with CAV, most T cells are CD4+ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.

Type IV collagen degradation in the myocardial basement membrane after unloading of the failing heart by a left ventricular assist device.

After left ventricular assist device (LVAD) support in patients with end-stage cardiomyopathy, cardiomyocytes decrease in size. We hypothesized that during this process, known as reverse remodeling, the basement membrane (BM), which is closely connected to, and forms the interface between the cardiomyocytes and the extracellular matrix, will be severely affected. Therefore, the changes in the myocardial BM in patients with end-stage heart failure before and after LVAD support were studied. The role of MMP-2 in this process was also investigated. Transmission electron microscopy showed that the BM thickness decreased post-LVAD compared to pre-LVAD. Immunohistochemistry indicated a reduced immunoreactivity for type IV collagen in the BM after LVAD support. Quantitative PCR showed a similar mRNA expression for type IV collagen pre- and post-LVAD. MMP-2 mRNA almost doubled post-LVAD (P<0.01). In addition, active MMP-2 protein as identified by gelatin zymography and confirmed by Western blot analysis was detected after LVAD support and in controls, but not before LVAD support. Active MMP was localized in the BM of the cardiomyocyte, as detected by type IV collagen in situ zymography. Furthermore, in situ hybridization/immunohistochemical double staining showed that MMP-2 mRNA was expressed in cardiomyocytes, macrophages, T-cells and endothelial cells. Taken together, these findings show reduced type IV collagen content in the BM of cardiomyocytes after LVAD support. This reduction is at least in part the result of increased MMP-2 activity and not due to reduced synthesis of type IV collagen.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future.

Myocardial fibrosis and pro-fibrotic markers in end-stage heart failure patients during continuous-flow left ventricular assist device support.

OBJECTIVES: During support with a left ventricular assist device (LVAD), partial reverse remodelling takes place in which fibrosis plays an important role. In this study, we analysed the histological changes and expression of fibrotic markers in patients with advanced heart failure (HF) during continuous-flow LVAD (cf-LVAD) support. METHODS: In 25 patients, myocardial tissue at the time of LVAD implantation (pre-LVAD) was compared with tissue from the explanted left ventricle (post-LVAD). Interstitial fibrosis and cardiomyocyte size were analysed pre- and post-LVAD. Plasma was obtained from all patients before and during LVAD support. Plasma levels, cardiac mRNA and protein expression of brain natriuretic peptide (BNP), galectin-3 (Gal-3), connective tissue growth factor (CTGF), osteopontin (OPN) and transforming growth factor ß-1 were determined. RESULTS: Fibrosis increased during cf-LVAD unloading (P < 0.05). Cardiomyocytes elongated (P < 0.05), whereas cross-sectional area did not change. BNP, Gal-3, CTGF and OPN were significantly elevated pre-LVAD in comparison with controls. BNP decreased significantly after 1 month of cf-LVAD support (P < 0.001) to near-normal levels. Pro-fibrotic markers remained elevated in comparison with controls. CONCLUSIONS: cf-LVAD support is associated with lengthening of cardiomyocytes, without alterations in diameter size. Remarkably, myocardial fibrosis increased as well as circulating pro-fibrotic markers. Whether the morphological changes are a direct effect of reduced pulsatility during cf-LVAD support or due to HF progression requires further investigation.

MicroRNA Expression in Myocardial Tissue and Plasma of Patients with End-Stage Heart Failure during LVAD Support: Comparison of Continuous and Pulsatile Devices.

AIM: Pulsatile flow left ventricular assist devices (pf-LVADs) are being replaced by continuous flow LVADs (cf-LVADs) in patients with end-stage heart failure (HF). MicroRNAs (miRs) play an important role in the onset and progression of HF. Our aim was to analyze cardiac miR expression patterns associated with each type of device, to analyze differences in the regulation of the induced cardiac changes. METHODS AND RESULTS: Twenty-six miRs were selected (based on micro-array data and literature studies) and validated in myocardial tissue before and after pf- (n = 17) and cf-LVAD (n = 17) support. Of these, 5 miRs displayed a similar expression pattern among the devices (miR-129*, miR-146a, miR-155, miR-221, miR-222), whereas others only changed significantly during pf-LVAD (miR-let-7i, miR-21, miR-378, miR-378*) or cf-LVAD support (miR-137). In addition, 4 miRs were investigated in plasma of cf-LVAD supported patients (n = 18) and healthy controls (n = 10). Circulating miR-21 decreased at 1, 3, and 6 months after LVAD implantation. MiR-146a, miR-221 and miR-222 showed a fluctuating time pattern post-LVAD. CONCLUSION: Our data show a different miR expression pattern after LVAD support, suggesting that differentially expressed miRs are partially responsible for the cardiac morphological and functional changes observed after support. However, the miR expression patterns do not seem to significantly differ between pf- and cf-LVAD implying that most cardiac changes or clinical outcomes specific to each device do not relate to differences in miR expression levels.

Acute cardiac transplant rejection is associated with low frequencies of interleukin-4 producing helper T-lymphocytes rather than with interleukin-4 promoter or splice variants.

Interleukin-4 (IL-4) is a cytokine of the Th2 subtype. It is suggested that Th2 cytokines are involved in induction of tolerance towards the graft after organ transplantation. Therefore, we studied the association between the frequencies of IL-4 producing helper T lymphocytes (IL-4 HTL) and acute rejection in a panel of 31 cardiac transplant patients. It was also investigated whether these frequencies were influenced by: (1) a single nucleotide polymorphism (SNP) at position -590 in the promoter region of the IL-4 gene, which influences the production level of IL-4; and (2) the expression of an IL-4 splice variant (IL-4delta2), which inhibits the IL-4 receptor. Frequencies of IL-4 HTL were determined by limiting dilution analysis. Genotyping for the SNP was carried out by sequencing. The ratio of wild type versus IL-4delta2 mRNA was determined by quantitative RT-PCR of mRNA isolated from stimulated MNC of cardiac transplant patients. Frequencies of IL-4 HTL were significantly higher in patients who did not suffer from acute cardiac transplant rejection, than in patients that suffered from at least one rejection episode requiring treatment in the first year after heart transplantation. The genotype of the promoter SNP and the ratio between wild type/splice variant IL-4 mRNA did not influence the measured frequencies of IL-4 HTL or the presence of transplant rejection itself.

Cardiomyocyte death in patients with end-stage heart failure before and after support with a left ventricular assist device: low incidence of apoptosis despite ubiquitous mediators.

BACKGROUND: Left ventricular assist device (LVAD) implantation in patients with end-stage heart failure results in impressive hemodynamic improvement. The effects on myocardial apoptosis and its mediators are unknown. METHODS: Myocardial biopsies from 17 patients at the time of LVAD implantation and after explantation, at the time of heart transplantation (HTx), were examined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction and with antibodies against Fas ligand (FasL), Fas, tumor necrosis factor (TNF)-alpha receptor 1 (TNF-R1), TNF-alpha receptor 2 (TNF-R2), TNF-alpha, TNF-alpha-converting enzyme (TACE), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) (PAR), caspase-3 and FLICE inhibitory protein (FLIP). RESULTS: Apoptosis incidence was low: 0.8% (range 0% to 3%) positive cardiomyocytes nuclei before support, and 0.1% (range 0% to 0.6%) after support (p < 0.01). This was accompanied by low expression of caspase-3 and high expression of the DNA repair enzyme, PARP. Its product, PAR, increased after support. Mediators and receptors inducing apoptosis as well as FLIP were widely present before and after support. CONCLUSIONS: Despite the abundant presence of mediators and receptors inducing apoptosis, the incidence of apoptosis itself was low before and after mechanical support. The abundant expression of FLIP may suggest an important role for this protein in the inhibition of cardiomyocyte death.

Post-transcriptional regulation of α-1-antichymotrypsin by microRNA-137 in chronic heart failure and mechanical support.

BACKGROUND: Better understanding of the molecular mechanisms of remodeling has become a major objective of heart failure (HF) research to stop or reverse its progression. Left ventricular assist devices (LVADs) are being used in patients with HF, leading to partial reverse remodeling. In the present study, proteomics identified significant changes in α-1-antichymotrypsin (ACT) levels during LVAD support. Moreover, the potential role of ACT in reverse remodeling was studied in detail. METHODS AND RESULTS: Expression of ACT mRNA (quantitative-polymerase chain reaction) decreased significantly in post-LVAD myocardial tissue compared with pre-LVAD tissue (n=15; P<0.01). Immunohistochemistry revealed that ACT expression and localization changed during LVAD support. Circulating ACT levels were elevated in HF patients (n=18) as compared with healthy controls (n=6; P=0.001) and normalized by 6 months of LVAD support. Because increasing evidence implicates that microRNAs (miRs) are involved in myocardial disease processes, we also investigated whether ACT is post-transcriptionally regulated by miRs. Bioinformatics analysis pointed miR-137 as a potential regulator of ACT. The miR-137 expression is inversely correlated with ACT mRNA in myocardial tissue. Luciferase activity assays confirmed ACT as a direct target for miR-137, and in situ hybridization indicated that ACT and miR-137 were mainly localized in cardiomyocytes and stromal cells. CONCLUSIONS: High ACT plasma levels in HF normalized during LVAD support, which coincides with decreased ACT mRNA in heart tissue, whereas miR-137 levels increased. MiR-137 directly targeted ACT, thereby indicating that ACT and miR-137 play a role in the pathophysiology of HF and reverse remodeling during mechanical support.

Integrin expression during reverse remodeling in the myocardium of heart failure patients.

BACKGROUND: The main anchoring proteins of myocardial cells with each other and with the extracellular matrix are integrins present in the membranes of myocardial cells. These integrins are important for maintaining the architecture of the myocardial tissue and the mechanotransduction in the heart. Heart failure leads to various alterations in the myocardium, such as changes in morphology, and in expression of mRNAs, miRNAs, and proteins. Left ventricular assist device (LVAD) support in heart failure patients has been described to induce reverse remodeling of the myocardium and thus to (some degree of) reversal of the aforementioned alterations. In this study, we evaluated whether changes in expression of integrins α-1, -3, -5, -6, -7, -10, -11 and ß-1, -3, -5 and -6 play a role during reverse remodeling. METHODS: Three-step immunoperoxidase staining procedures were applied on frozen heart tissue sections to locate the various integrins tested. Integrin mRNA expression was established by standard Q-PCR procedures. RESULTS: It was shown that mRNA expression of several integrins changes significantly during LVAD support, however without subsequent changes in immunohistochemical detectable quantities. Various integrins showed different locations within the myocardium. CONCLUSION: LVAD-induced reversed remodeling did not result in significant integrin protein expression, although changes in integrin mRNA expression suggested an adaptation to unloading.

Circulating growth differentiation factor-15 correlates with myocardial fibrosis in patients with non-ischaemic dilated cardiomyopathy and decreases rapidly after left ventricular assist device support.

AIMS: Growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine and is emerging as a biomarker of cardiac remodelling. Left ventricular assist devices (LVADs) provide unloading of the left ventricle, resulting in partial reverse remodelling. Our aim was to study GDF-15 in patients with a non-ischaemic dilated cardiomyopathy (DCM) during LVAD support. METHODS AND RESULTS: We analysed circulating GDF-15 in 30 patients before and 1, 3, and 6 months after LVAD implantation and before heart transplantation or explantation. In addition, mRNA and protein expression of GDF-15 were evaluated in myocardial tissue obtained prior to and after LVAD support. Circulating GDF-15 was significantly higher before LVAD implantation as compared with healthy controls (P < 0.001). After 1 month of mechanical support, GDF-15 levels were significantly decreased compared with pre-implantation levels (P < 0.001) and remained stable thereafter. Circulating GDF-15 was significantly correlated with kidney function and the severity of myocardial fibrosis. Interestingly, GDF-15 mRNA and protein expression in the myocardium were hardly detectable. CONCLUSIONS: High circulating levels of GDF-15 in patients with end-stage non-ischaemic DCM correlate with myocardial fibrosis and kidney function and decline strongly after 1 month of mechanical unloading, remaining stable thereafter. However, cardiac mRNA and protein expression of GDF-15 are very low, suggesting that the heart is not an important source of GDF-15 production in these patients.

Intimal fibrosis in human cardiac allograft vasculopathy.

Human Cardiac Allograft Vasculopathy (CAV) is one of the major complications for patients after heart transplantation. It is characterized by a concentric luminal narrowing due to (neo) intimal expansion in the coronary arteries of donor hearts after heart transplantation. In this process fibrosis plays an important role. Aim of this study is to analyze the factors and cells involved in this fibrotic process. Coronary arteries from five heart transplantation patients and three controls were obtained at autopsy. Quantitative real-time PCR was performed on mRNA obtained from various arterial layers isolated by laser micro dissection. Positive gene expression was confirmed by immunohistochemistry and/or in situ hybridisation. The strongest mRNA expression of fibrotic factors (predominantly pro-fibrotic) was found in the neo-intima. Especially, connective tissue growth factor expression was higher in the CAV vessels than in the controls. The lymphocyte activity of interferon gamma was only detected in CAV vessels. Furthermore as shown by in situ hybridisation, the lymphocytes producing interferon gamma also expressed transforming growth factor beta. Anti-fibrotic factors, such as bone morphogenic protein 4, were only expressed in CD3(-)/CD68(-) stromal cells. Macrophages present in the CAV and control vessels showed to be of the M2 type and did not produce any fibrotic factor(s). In conclusion, T-cells producing both interferon gamma and transforming growth factor beta, may play an important role in the fibrotic process in CAV vessels by upregulation of connective tissue growth factor production.

Osteopontin: a potential biomarker for heart failure and reverse remodeling after left ventricular assist device support.

BACKGROUND: Left ventricular assist device (LVAD) support in end-stage heart failure (HF) leads to recovery of the patient's condition, size reduction of cardiomyocytes, and also volume reduction and change in the composition of the extracellular matrix (ECM). Myocardial expression of ECM osteopontin (OPN) protein increases with the severity of HF. We analyzed whether OPN messenger RNA expression in heart tissue and/or OPN protein in plasma are associated with reverse remodeling during LVAD support. METHODS: Plasma and heart tissue specimens of 22 end-stage HF patients before and after LVAD implantation and subsequent heart transplantation (HTx) were used to determine the concentrations of OPN protein (EIA) and OPN messenger RNA (mRNA) by quantitative polymerase chain reaction. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to locate OPN protein and mRNA. RESULTS: The high OPN protein levels in plasma of HF patients did not differ significantly before and after LVAD support in ischemic heart disease (IHD) (pre-LVAD 167 ± 32 ng/ml; post-LVAD 165 ± 28 ng/ml) and in dilated cardiomyopathy (DCM) (pre-LVAD 99 ± 12 ng/ml; post-LVAD (142 ± 6 ng/ml). The OPN plasma levels after HTx decreased to control levels (IHD, 48 ± 6; DCM, 40 ± 5; control, 31 ± 3 ng/ml). In contrast, expression of OPN mRNA in heart biopsy specimens decreased significantly after LVAD support (the relative quantity decreased > 90% in IHD and 50% in DCM). ISH and IHC revealed that OPN was present in cardiomyocytes and in the ECM. CONCLUSIONS: Levels of OPN mRNA in the myocardium of HF patients showed a significant decrease after LVAD support but OPN protein expression did not. LVAD support only induced a decrease of OPN plasma levels in individual patients, whereas OPN plasma levels reduced significantly in all patients after HTx.