[Digitalization in Migraine Therapy]. / Digitalisierung in der Migränetherapie.

Digitalization is a commonly used keyword in medicine and also in the area of migraine and its therapy. However, digitalization should not be an end in itself, but should improve the treatment of patients and make the work of practitioners easier. This article summarizes the use of e-health applications for migraine medicine, today and in the future.

Persistent effectiveness of CGRP antibody therapy in migraine and comorbid medication overuse or medication overuse headache - a retrospective real-world analysis.

BACKGROUND: Management of patients with migraine who have concomitant medication overuse (MO) or medication overuse headache (MOH) is a major problem in clinical practice. Detoxification of acute analgesics before or during initiation of prophylactic therapy has long been recommended although this concept has recently been questioned. Additionally, relapse after detoxification is a common problem. This real-world study analyses the initial and sustained effectiveness of prophylactic migraine therapy with CGRP (receptor) antibodies without prior detoxification in patients with comorbid MO or MOH for up to one year. METHODS: A retrospective real-world analysis was performed on 291 patients (episodic migraine (EM) with MO (EM-MO; n = 35), EM without MO (EM-noMO; n = 77), chronic migraine (CM) with MOH (CM-MOH; n = 109), CM without MOH (CM-noMOH; n = 70). All patients began treatment with either erenumab (n = 173), fremanezumab (n = 70) or galcanezumab (n = 48) without prior detoxification. Data were available for up to 12 months of treatment. Responder rates for monthly headache days (MHD), monthly migraine days (MMD) and monthly acute medication intake (AMD) were analysed. RESULTS: All groups showed a significant reduction in MHD, MMD and AMD at the last observed time point compared to baseline. In patients with CM and MOH, 60.6% (66/109) no longer fulfilled the definition of MO or MOH and a further 13.8% (15/109) had only EM-MO. In the EM cohort, 89% (31/35) of MO patients lost their MO during therapy. MHD and AMD 30% responder rates were comparable for CM-MOH and CM-noMOH (MHD: CM-MOH: 56.0% vs. CM-noMOH: 41.4%, p = 0.058, AMD: CM-MOH: 66.1% vs. CM-noMOH: 52.9%, p = 0.077). MMD responder rate did not differ significantly (after Bonferroni adjustment) (CM-MOH: 62.4% vs. CM-noMOH: 47.1%, p = 0.045, α = 0.017). After successful initiation of therapy, 15.4% of the initial CM-MOH patients relapsed and met the criterion for CM-MOH at the end of follow-up. There were no antibody specific differences in response to therapy. CONCLUSIONS: Our data confirms the effectiveness of CGRP antibody treatment in migraine patients with additional MOH or MO in a real-world setting. Low relapse rates after initial successful therapy support an early start of CGRP antibody treatment in patients with MOH or MO. TRIAL REGISTRATION: No registration, retrospective analysis.

Effectiveness and tolerability of eptinezumab in treating patients with migraine resistant to conventional preventive medications and CGRP (receptor) antibodies: a multicentre retrospective real-world analysis from Germany.

BACKGROUND: Eptinezumab is a monoclonal antibody that targets calcitonin gene-related peptide (CGRP mAb) and is used for migraine prophylaxis. Efficacy data are mainly from clinical trials, real-world data are hardly available yet. Reimbursement policy in Germany leads to eptinezumab mainly being used in patients having failed pre-treatment with other CGRP mAb. To date, it is unclear whether eptinezumab is efficacious and well tolerated in this population and how the treatment response differs from patients who are naive to CGRP mAbs. METHODS: We analysed clinical routine data of 79 patients (episodic migraine (EM): n = 19; chronic migraine (CM): n = 60) from four different centres in Germany. All patients were treated with eptinezumab (100mg). Differences in monthly headache (MHD), migraine (MMD) and acute medication days (AMD) after three months were analysed. The correlation of response with the number of CGRP mAb failures was evaluated. Significance level has been corrected (alpha = 0.017). RESULTS: After three months MHD, MMD and AMD were significantly reduced. In EM, the median reduction for MHD was 4.0 days (IQR: -6.5 to -1.0; p = 0.001), for MMD 3.0 days (IQR: -5.5 to -1.5; p < 0.001) and for AMD 2.0 days (IQR: -5.0 to -0.5; p = 0.006). In CM, median reduction of MHD was 4 days (IQR: -8.0 to 0.0; p < 0.001), 3.0 days (IQR: -6.0 to-1.0; p < 0.001) for MMD and 1.0 day (IQR: -5.0 to 0.0; p < 0.001) for AMD. All patients were resistant to conventional preventive therapies and most to CGRP mAbs. Fourteen patients had never received a CGRP mAb and 65 patients had received at least one mAb without sufficient effectiveness and/or intolerability (one: n = 20, two: n = 28, three: n = 17). There was a significant association between the number of prior therapies and the 30% MHD responder rate (none: 78.6%, one: 45.0%, two: 32.1%, three: 23.5%, p = 0.010). Regarding tolerability, 10.4% (8/77) reported mild side effects. CONCLUSIONS: The effectiveness of eptinezumab is significantly reduced in patients who have not previously responded to other CGRP mAbs. However, limitations such as the retrospective nature of the analysis, the small sample size and the short treatment period with only the lower dose of eptinezumab must be considered when interpreting the results.

Matrix metalloproteinases in coronary artery disease and myocardial infarction.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Most cardiovascular deaths are caused by ischaemic heart diseases such as myocardial infarction (MI). Hereby atherosclerosis in the coronary arteries often precedes disease manifestation. Since tissue remodelling plays an important role in the development and progression of atherosclerosis as well as in outcome after MI, regulation of matrix metalloproteinases (MMPs) as the major ECM-degrading enzymes with diverse other functions is crucial. Here, we provide an overview of the expression profiles of MMPs in coronary artery and left ventricular tissue using publicly available data from whole tissue to single-cell resolution. To approach an association between MMP expression and the development and outcome of CVDs, we further review studies investigating polymorphisms in MMP genes since polymorphisms are known to have an impact on gene expression. This review therefore aims to shed light on the role of MMPs in atherosclerosis and MI by summarizing current knowledge from publically available datasets, human studies, and analyses of polymorphisms up to preclinical and clinical trials of pharmacological MMP inhibition.

An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

Hypnic Headache - What do we know in 2022?

BACKGROUND: Hypnic Headache, also known as "alarm clock headache", is a rare primary headache disorder. It is characterized by frequently recurring headache attacks, which only develop during sleep, especially nighttime sleep. OBJECTIVE: This article gives a narrative review on the current knowledge about Hypnic Headache with a focus on secondary Hypnic Headache. METHODS: Based on literature research, using Pubmed and Google Scholar, latest case reports, studies, and systematic reviews about Hypnic Headache were analyzed and summarized focusing on therapeutic options and causes of secondary Hypnic Headache. CONCLUSION: Hypnic Headache mainly occurs in elderly patients. However, younger patients and children may also suffer from Hypnic Headache. Many different causes of secondary Hypnic Headache are described in the literature and ought to be ruled out before diagnosing primary Hypnic Headache. The pathophysiology of primary Hypnic Headache remains unclear, but a dysfunction of the hypothalamus seems to play a key role.

cAMP Imaging at Ryanodine Receptors Reveals ß2-Adrenoceptor Driven Arrhythmias.

[Figure: see text].

Myeloid differentiation factor-2 activates monocytes in patients with dilated cardiomyopathy.

Plasma levels of myeloid differentiation factor-2 (MD-2), a co-receptor of toll-like-receptor 4 (TLR4), independently predict mortality in patients with dilated cardiomyopathy (DCM). We tested whether monocyte activation by MD-2 contributes to immune activation and inflammatory status in DCM patients. We found increased MD-2 plasma levels in 25 patients with recent-onset DCM (1250 ± 80.7 ng/ml) compared to 25 age- and gender-matched healthy controls (793.4 ± 52.0 ng/ml; p < 0.001). Monocytes isolated from DCM patients showed a higher expression (141.7 ± 12.4%; p = 0.006 vs. controls) of the MD-2 encoding gene, LY96 and an increased NF-κB-activation. Further, the TLR4-activator lipopolysaccharide (LPS) caused a higher increase in interleukin (IL)-6 in monocytes from DCM patients compared to controls (mean fluorescence intensity: 938.7 ± 151.0 vs. 466.9 ± 51.1; p = 0.005). MD-2 increased IL-6 secretion in a TLR4/NF-κB-dependent manner in monocyte-like THP-1-cells as demonstrated by TLR4-siRNA and NF-κB-inhibition. Since endothelial cells (ECs) are responsible for recruiting monocytes to the site of inflammation, ECs were treated with MD-2 leading to an activation of Akt and increased secretion of monocyte-chemoattractant-protein-1 (MCP-1). Activation of ECs by MD-2 was accompanied by an increased expression of the adhesion molecules CD54, CD106 and CD62E, resulting in an increased monocyte recruitment, which was attenuated by CD54 inhibition. In addition, in murine WT but not LY96-KO bone marrow-derived macrophages LPS increased the amount of CD54 and CD49d/CD29. MD-2 facilitates a pro-inflammatory status of monocytes and EC-mediated monocyte recruitment via TLR4/NF-κB. Elevated MD-2 plasma levels are possibly involved in monocyte-related inflammation-promoting disease progression in DCM. Our results suggest that MD-2 contributes to increasing monocytic inflammatory activity and triggers the recruitment of monocytes to ECs in DCM.

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells.

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL-2, TNF and interferon-γ and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells.

G Protein-Coupled Receptor 15 Expression Is Associated with Myocardial Infarction.

Beyond the influence of lifestyle-related risk factors for myocardial infarction (MI), the mechanisms of genetic predispositions for MI remain unclear. We sought to identify and characterize differentially expressed genes in early-onset MI in a translational approach. In an observational case−control study, transcriptomes from 112 early-onset MI individuals showed upregulated G protein-coupled receptor 15 (GPR15) expression in peripheral blood mononuclear cells compared to controls (fold change = 1.4, p = 1.87 × 10−7). GPR15 expression correlated with intima-media thickness (ß = 0.8498, p = 0.111), C-reactive protein (ß = 0.2238, p = 0.0052), ejection fraction (ß = −0.9991, p = 0.0281) and smoking (ß = 0.7259, p = 2.79 × 10−10). The relation between smoking and MI was diminished after the inclusion of GPR15 expression as mediator in mediation analysis (from 1.27 (p = 1.9 × 10−5) to 0.46 (p = 0.21)). The DNA methylation of two GPR15 sites was 1%/5% lower in early-onset MI individuals versus controls (p = 2.37 × 10−6/p = 0.0123), with site CpG3.98251219 significantly predicting risk for incident MI (hazard ratio = 0.992, p = 0.0177). The nucleotide polymorphism rs2230344 (C/T) within GPR15 was associated with early-onset MI (odds ratio = 3.61, p = 0.044). Experimental validation showed 6.3-fold increased Gpr15 expression in an ischemic mouse model (p < 0.05) and 4-fold increased Gpr15 expression in cardiomyocytes under ischemic stress (p < 0.001). After the induction of MI, Gpr15gfp/gfp mice showed lower survival (p = 0.042) and deregulated gene expression for response to hypoxia and signaling pathways. Using a translational approach, our data provide evidence that GPR15 is linked to cardiovascular diseases, mediating the adverse effects of smoking.

Precursor proadrenomedullin influences cardiomyocyte survival and local inflammation related to myocardial infarction.

Increased adrenomedullin (ADM) levels are associated with various cardiac diseases such as myocardial infarction (MI). ADM is cleaved off from the full-length precursor protein proadrenomedullin (ProADM) during its posttranslational processing. To date, no biological effect of ProADM is reported, while ADM infusion leads to antiapoptotic effects and improved cardiac function. Using an MI mouse model, we found an induction of ProADM gene as well as protein expression during the early phase of MI. This was accompanied by apoptosis and increasing inflammation, which substantially influence the post-MI remodeling processes. Simulating ischemia in vitro, we demonstrate that ProADM expression was increased in cardiomyocytes and cardiac fibroblasts. Subsequently, we treated ischemic cardiomyocytes with either ProADM or ADM and found that both proteins increased survival. This effect was diminishable by blocking the ADM1 receptor. To investigate whether ProADM and ADM play a role in the regulation of cardiac inflammation, we analyzed chemokine expression after treatment of cells with both proteins. While ProADM induced an expression of proinflammatory cytokines, thus promoting inflammation, ADM reduced chemokine expression. On leukocytes, both proteins repressed chemokine expression, revealing antiinflammatory effects. However, ProADM but not ADM dampened concurrent activation of leukocytes. Our data show that the full-length precursor ProADM is biologically active by reducing apoptosis to a similar extent as ADM. We further assume that ProADM induces local inflammation in affected cardiac tissue but attenuates exaggerated inflammation, whereas ADM has low impact. Our data suggest that both proteins are beneficial during MI by influencing apoptosis and inflammation.

Cytokine-Mediated Alterations of Human Cardiac Fibroblast's Secretome.

Fibroblasts contribute to approximately 20% of the non-cardiomyocytic cells in the heart. They play important roles in the myocardial adaption to stretch, inflammation, and other pathophysiological conditions. Fibroblasts are a major source of extracellular matrix (ECM) proteins whose production is regulated by cytokines, such as TNF-α or TGF-ß. The resulting myocardial fibrosis is a hallmark of pathological remodeling in dilated cardiomyopathy (DCM). Therefore, in the present study, the secretome and corresponding transcriptome of human cardiac fibroblasts from patients with DCM was investigated under normal conditions and after TNF-α or TGF-ß stimulation. Secreted proteins were quantified via mass spectrometry and expression of genes coding for secreted proteins was analyzed via Affymetrix Transcriptome Profiling. Thus, we provide comprehensive proteome and transcriptome data on the human cardiac fibroblast's secretome. In the secretome of quiescent fibroblasts, 58% of the protein amount belonged to the ECM fraction. Interestingly, cytokines were responsible for 5% of the total protein amount in the secretome and up to 10% in the corresponding transcriptome. Furthermore, cytokine gene expression and secretion were upregulated upon TNF-α stimulation, while collagen secretion levels were elevated after TGF-ß treatment. These results suggest that myocardial fibroblasts contribute to pro-fibrotic and to inflammatory processes in response to extracellular stimuli.

Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice.

The tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b/PEX5R) is an interaction partner and auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are key for rhythm generation in the brain and in the heart. Since TRIP8b is expressed in central neurons but not in cardiomyocytes, the TRIP8b-HCN interaction has been studied intensely in the brain, but is deemed irrelevant in the cardiac conduction system. Still, to date, TRIP8b has not been studied in the intrinsic cardiac nervous system (ICNS), a neuronal network located within epicardial fat pads. In vitro electrophysiological studies revealed that TRIP8b-deficient mouse hearts exhibit increased atrial refractory and atrioventricular nodal refractory periods, compared to hearts of wild-type littermates. Meanwhile, heart rate, sino-nodal recovery time, and ventricular refractory period did not differ between genotypes. Trip8b mRNA was detected in the ICNS by quantitative polymerase chain reaction. RNAscope in situ hybridization confirmed Trip8b localization in neuronal somata and nerve fibers. Additionally, we found a very low amount of mRNAs in the sinus node and atrioventricular node, most likely attributable to the delicate fibers innervating the conduction system. In contrast, TRIP8b protein was not detectable. Our data suggest that TRIP8b in the ICNS may play a role in the modulation of atrial electrophysiology beyond HCN-mediated sino-nodal control of the heart.

The role of fibroblast - Cardiomyocyte interaction for atrial dysfunction in HFpEF and hypertensive heart disease.

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

Inflammation and fibrosis in murine models of heart failure.

Heart failure is a consequence of various cardiovascular diseases and associated with poor prognosis. Despite progress in the treatment of heart failure in the past decades, prevalence and hospitalisation rates are still increasing. Heart failure is typically associated with cardiac remodelling. Here, inflammation and fibrosis are thought to play crucial roles. During cardiac inflammation, immune cells invade the cardiac tissue and modulate tissue-damaging responses. Cardiac fibrosis, however, is characterised by an increased amount and a disrupted composition of extracellular matrix proteins. As evidence exists that cardiac inflammation and fibrosis are potentially reversible in experimental and clinical set ups, they are interesting targets for innovative heart failure treatments. In this context, animal models are important as they mimic clinical conditions of heart failure patients. The advantages of mice in this respect are short generation times and genetic modifications. As numerous murine models of heart failure exist, the selection of a proper disease model for a distinct research question is demanding. To facilitate this selection, this review aims to provide an overview about the current understanding of the pathogenesis of cardiac inflammation and fibrosis in six frequently used murine models of heart failure. Hence, it compares the models of myocardial infarction with or without reperfusion, transverse aortic constriction, chronic subjection to angiotensin II or deoxycorticosterone acetate, and coxsackievirus B3-induced viral myocarditis in this context. It furthermore provides information about the clinical relevance and the limitations of each model, and, if applicable, about the recent advancements in their methodological proceedings.

Heart non-specific effector CD4+ T cells protect from postinflammatory fibrosis and cardiac dysfunction in experimental autoimmune myocarditis.

Heart-specific CD4+ T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4+ T cells in the progression of the disease. Heart non-specific CD4+ T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4+ T cells expressed exclusively effector (Teff) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific Teff infiltrated the heart shortly after injection, heart non-specific Teff effectively accumulated during myocarditis and became the major heart-infiltrating CD4+ T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4+ T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific Teff and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific Teff. Adoptive transfer of heart non-specific Teff in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human Teff stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific Teff cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.

Pancreatic polypeptide is recognized by two hydrophobic domains of the human Y4 receptor binding pocket.

Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr(2.64) interacts with Tyr(27) of hPP through hydrophobic contacts. Asn(7.32) is affected by modifications on position Arg(33) of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe(7.35) is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6-TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.

Single-target RNA interference for the blockade of multiple interacting proinflammatory and profibrotic pathways in cardiac fibroblasts.

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.

Cardiac fibroblasts support cardiac inflammation in heart failure.

Cardiac remodeling and inflammation are hallmarks of cardiac failure and correlate with outcome in patients. However, the basis for the development of both remains unclear. We have previously reported that cardiac inflammation triggers transdifferentiation of fibroblasts to myofibroblasts and therefore increase accumulation of cardiac collagen, one key pathology in cardiac remodeling. Hence, identifying key pathways for inflammation would be beneficial for patients suffering from heart failure also. Besides their well-characterized function in matrix regulation, we here investigate the role of fibroblasts in the inflammatory process. We address for the first time the role of fibroblasts as inflammatory supporter cells in heart failure. Using endomyocardial biopsies from patients with heart failure and dilated cardiomyopathy, we created a primary human cardiac fibroblast cell culture system. We found that mechanical stretch mimicking cardiac dilation in heart failure induces activation of fibroblasts and not only stimulates production of extracellular matrix but more interestingly up-regulates chemokine production and triggers typical inflammatory pathways in vitro. Moreover, the cell culture supernatant of stretched fibroblasts activates inflammatory cells and induces further recruitment of monocytes by allowing transendothelial migration into the cardiac tissue. Our findings reveal that cardiac fibroblasts provide pro-inflammatory mediators and may act as sentinel cells activated by mechanical stress. Those cells are able to recruit inflammatory cells into the cardiac tissue, a process known to aggravate outcome of patients. This might be important in different forms of heart failure and therefore may be one general mechanism specific for fibroblasts.