Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human ...cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV-cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter
miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application.
Purpose
Mesenchymal stromal cells (MSC) are an attractive tool for treatment of diabetic cardiomyopathy. Syndecan‐2/CD362 has been identified as a functional marker for MSC isolation. Imaging mass ...spectrometry (IMS) allows for the characterization of therapeutic responses in the left ventricle. This study aims to investigate whether IMS can assess the therapeutic effect of CD362+‐selected MSC on early onset experimental diabetic cardiomyopathy.
Experimental Design
1 × 106 wild type (WT), CD362−, or CD362+ MSC are intravenously injected into db/db mice. Four weeks later, mice are hemodynamically characterized and subsequently sacrificed for IMS combined with bottom‐up mass spectrometry, and isoform and phosphorylation analyses of cardiac titin.
Results
Overall alterations of the cardiac proteome signatures, especially titin, are observed in db/db compared to control mice. Interestingly, only CD362+ MSC can overcome the reduced titin intensity distribution and shifts the isoform ratio toward the more compliant N2BA form. In contrast, WT and CD362− MSCs improve all‐titin phosphorylation and protein kinase G activity, which is reflected in an improvement in diastolic performance.
Conclusions and Clinical Relevance
IMS enables the characterization of differences in titin intensity distribution following MSC application. However, further analysis of titin phosphorylation is needed to allow for the assessment of the therapeutic efficacy of MSC.
This study investigated the spontaneous clinical course of patients with endomyocardial biopsy (EMB)-proven lymphocytic myocarditis and cardiac human herpesvirus 6 (HHV6) DNA presence, and the ...effectiveness of steroid-based intervention in HHV6-positive patients.
756 heart failure (HF) patients underwent an EMB procedure to determine the underlying cause of unexplained HF. Low levels of HHV6 DNA, detectable by nested PCR only, were found in 10.4% of the cases (
= 79) of which 62% (
= 49) showed myocardial inflammation. The spontaneous course of patients with EMB-proven HHV6 DNA-associated lymphocytic myocarditis (
= 26) showed significant improvements in the left ventricular ejection fraction (LVEF) and clinical symptoms, respectively, in 15/26 (60%) patients, 3-12 months after disease onset. EMB mRNA expression of components of the NLRP3 inflammasome pathway and protein analysis of cardiac remodeling markers, analyzed by real-time PCR and MALDI mass spectrometry, respectively, did not differ between HHV6-positive and -negative patients. In another cohort of patients with ongoing symptoms related to lymphocytic myocarditis associated with cardiac levels of HHV6-DNA copy numbers <500 copies/µg cardiac DNA, quantified by real-time PCR, the efficacy and safety of steroid-based immunosuppression for six months was investigated. Steroid-based immunosuppression improved the LVEF (≥5%) in 8/10 patients and reduced cardiac inflammation in 7/10 patients, without an increase in cardiac HHV6 DNA levels in follow-up EMBs.
Low HHV6 DNA levels are frequently detected in the myocardium, independent of inflammation. In patients with lymphocytic myocarditis with low levels of HHV6 DNA, the spontaneous clinical improvement is nearby 60%. In selected symptomatic patients with cardiac HHV6 DNA copy numbers less than 500 copies/µg cardiac DNA and without signs of an active systemic HHV6 infection, steroid-based therapy was found to be effective and safe. This finding needs to be further confirmed in large, randomized trials.
Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is ...associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.
Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet ...sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features.
Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1β) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFβ. Further investigations identified CD14
cells as major recipient cell subset of CardAP-EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14
cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3
T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3
T cells were co-cultured with EV-primed CD14
cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells.
CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14
immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.
Background Myocarditis is an important cause of acute and chronic heart failure. Men with myocarditis have worse recovery and an increased need for transplantation compared with women, but the reason ...for the sex difference remains unclear. Elevated sera soluble (s) ST2 predicts mortality from acute and chronic heart failure, but has not been studied in myocarditis patients. Methods and Results Adults with a diagnosis of clinically suspected myocarditis (n=303, 78% male) were identified according to the 2013 European Society of Cardiology position statement. Sera sST2 levels were examined by ELISA in humans and mice and correlated with heart function according to sex and age. Sera sST2 levels were higher in healthy men ( P=8×10
) and men with myocarditis ( P=0.004) compared with women. sST2 levels were elevated in patients with myocarditis and New York Heart Association class III - IV heart failure ( P=0.002), predominantly in men ( P=0.0003). Sera sST2 levels were associated with New York Heart Association class in men with myocarditis who were ≤50 years old ( r=0.231, P=0.0006), but not in women ( r=0.172, P=0.57). Sera sST2 levels were also significantly higher in male mice with myocarditis ( P=0.005) where levels were associated with cardiac inflammation. Gonadectomy with hormone replacement showed that testosterone ( P<0.001), but not estradiol ( P=0.32), increased sera sST2 levels in male mice with myocarditis. Conclusions We show in a well-characterized subset of heart failure patients with clinically suspected and biopsy-confirmed myocarditis that elevated sera sST2 is associated with an increased risk of heart failure based on New York Heart Association class in men ≤50 years old.
Our aim was to investigate the effect of nitric oxide (NO)-independent activation of soluble guanylyl cyclase (sGC) on cardiomyocyte function in a hypertensive animal model with diastolic dysfunction ...and in biopsies from human heart failure with preserved ejection fraction (HFpEF).
Dahl salt-sensitive (DSS) rats and control rats were fed a high-salt diet for 10 weeks and then acutely treated
with the sGC activator BAY 58-2667 (cinaciguat) for 30 min. Single skinned cardiomyocyte passive stiffness (F
) was determined in rats and human myocardium biopsies before and after acute treatment. Titin phosphorylation, activation of the NO/sGC/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade, as well as hypertrophic pathways including NO/sGC/cGMP/PKG, PKA, calcium-calmodulin kinase II (CaMKII), extracellular signal-regulated kinase 2 (ERK2), and PKC were assessed. In addition, we explored the contribution of pro-inflammatory cytokines and oxidative stress levels to the modulation of cardiomyocyte function. Immunohistochemistry and electron microscopy were used to assess the translocation of sGC and connexin 43 proteins in the rat model before and after treatment.
High cardiomyocyte F
was found in rats and human myocardial biopsies compared to control groups, which was attributed to hypophosphorylation of total titin and to deranged site-specific phosphorylation of elastic titin regions. This was accompanied by lower levels of PKG and PKA activity, along with dysregulation of hypertrophic pathway markers such as CaMKII, PKC, and ERK2. Furthermore, DSS rats and human myocardium biopsies showed higher pro-inflammatory cytokines and oxidative stress compared to controls. DSS animals benefited from treatment with the sGC activator, as F
, titin phosphorylation, PKG and the hypertrophic pathway kinases, pro-inflammatory cytokines, and oxidative stress markers all significantly improved to the level observed in controls. Immunohistochemistry and electron microscopy revealed a translocation of sGC protein toward the intercalated disc and t-tubuli following treatment in both control and DSS samples. This translocation was confirmed by staining for the gap junction protein connexin 43 at the intercalated disk. DSS rats showed a disrupted connexin 43 pattern, and sGC activator was able to partially reduce disruption and increase expression of connexin 43. In human HFpEF biopsies, the high F
, reduced titin phosphorylation, dysregulation of the NO-sGC-cGMP-PKG pathway and PKA activity level, and activity of kinases involved in hypertrophic pathways CaMKII, PKC, and ERK2 were all significantly improved by sGC treatment and accompanied by a reduction in pro-inflammatory cytokines and oxidative stress markers.
Our data show that sGC activator improves cardiomyocyte function, reduces inflammation and oxidative stress, improves sGC-PKG signaling, and normalizes hypertrophic kinases, indicating that it is a potential treatment option for HFpEF patients and perhaps also for cases with increased hypertrophic signaling.
Aim
The acute phase of a coxsackievirus 3 (CVB3)‐induced myocarditis involves direct toxic cardiac effects and the systemic activation of the immune system, including the cardiosplenic axis. ...Consequently, the nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing‐3 (NLRP3) inflammasome pathway is activated, which plays a role in disease pathogenesis and progression. The anti‐inflammatory drug colchicine exerts its effects, in part, via reducing NLRP3 activity, and has been shown to improve several cardiac diseases, including acute coronary syndrome and pericarditis. The aim of the present study was to evaluate the potential of colchicine to improve experimental CVB3‐induced myocarditis.
Methods and results
C57BL6/j mice were intraperitoneally injected with 1 × 105 plaque forming units of CVB3. After 24 h, mice were treated with colchicine (5 μmol/kg body weight) or phosphate‐buffered saline (PBS) via oral gavage (p.o.). Seven days post infection, cardiac function was haemodynamically characterized via conductance catheter measurements. Blood, the left ventricle (LV) and spleen were harvested for subsequent analyses. In vitro experiments on LV‐derived fibroblasts (FB) and HL‐1 cells were performed to further evaluate the anti‐(fibro)inflammatory and anti‐apoptotic effects of colchicine via gene expression analysis, Sirius Red assay, and flow cytometry. CVB3 + colchicine mice displayed improved LV function compared with CVB3 + PBS mice, paralleled by a 4.7‐fold (P < 0.01) and 1.7‐fold (P < 0.001) reduction in LV CVB3 gene expression and cardiac troponin‐I levels in the serum, respectively. Evaluation of components of the NLRP3 inflammasome revealed an increased percentage of apoptosis‐associated speck‐like protein containing a CARD domain (ASC)‐expressing, caspase‐1‐expressing, and interleukin‐1β‐expressing cells in the myocardium and in the spleen of CVB3 + PBS vs. control mice, which was reduced in CVB3 + colchicine compared with CVB3 + PBS mice. This was accompanied by 1.4‐fold (P < 0.0001), 1.7‐fold (P < 0.0001), and 1.7‐fold (P < 0.0001) lower numbers of cardiac dendritic cells, natural killer cells, and macrophages, respectively, in CVB3 + colchicine compared with CVB3 + PBS mice. A 1.9‐fold (P < 0.05) and 4.6‐fold (P < 0.001) reduced cardiac gene expression of the fibrotic markers, Col1a1 and lysyl oxidase, respectively, was detected in CVB3 + colchicine mice compared with CVB3 + PBS animals, and reflected by a 2.2‐fold (P < 0.05) decreased Collagen I/III protein ratio. Colchicine further reduced Col3a1 mRNA and collagen protein expression in CVB3‐infected FB and lowered apoptosis and viral progeny release in CVB3‐infected HL‐1 cells. In both CVB3 FB and HL‐1 cells, colchicine down‐regulated the NLRP3 inflammasome‐related components ASC, caspase‐1, and IL‐1β.
Conclusions
Colchicine improves LV function in CVB3‐induced myocarditis, involving a decrease in cardiac and splenic NLRP3 inflammasome activity, without exacerbation of CVB3 load.
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