[Inherited heart diseases and storage diseases with cardiac involvement]. / Genetische Herzerkrankungen und Speichererkrankungen mit kardialer Beteiligung.

Rare diseases mostly have a genetic cause. Many rare cardiovascular diseases also have a genetic cause. For target-oriented cardiogenetic diagnostics, expert knowledge in human genetics as well as in clinical cardiology is needed. In recent years, the genetic cause of a number of heart diseases have been, at least in part, elucidated. Especially, certain arrhythmias and cardiomyopathy forms have a monogenetic cause. An early genetic diagnosis means that patients can be treated more effectively. Rare storage diseases also usually have a genetic cause and can manifest themselves in the heart; prominent examples are Fabry disease and amyloidosis. As patients with Fabry disease or amyloidosis suffer from a diverse and variable symptomatology, the correct diagnosis is often difficult.

Genetic determinants of heart failure: facts and numbers.

The relevance of gene mutations leading to heart diseases and hence heart failure has become evident. The risk for and the course of heart failure depends on genomic variants and mutations underlying the so-called genetic predisposition. Genetic contribution to heart failure is highly heterogenous and complex. For any patient with a likely inherited heart failure syndrome, genetic counselling is recommended and important. In the last few years, novel sequencing technologies (named next-generation sequencing - NGS) have dramatically improved the availability of molecular testing, the efficiency of genetic analyses, and moreover reduced the cost for genetic testing. Due to this development, genetic testing has become increasingly accessible and NGS-based sequencing is now applied in clinical routine diagnostics. One of the most common reasons of heart failure are cardiomyopathies such as the dilated or the hypertrophic cardiomyopathy. Nearly 100 disease-associated genes have been identified for cardiomyopathies. The knowledge of a pathogenic mutation can be used for genetic counselling, risk and prognosis determination, therapy guidance and hence for a more effective treatment. Besides, family cascade screening for a known familial, pathogenic mutation can lead to an early diagnosis in affected individuals. At that timepoint, a preventative intervention could be used to avoid or delay disease onset or delay disease progression. Understanding the cellular basis of genetic heart failure syndromes in more detail may provide new insights into the molecular biology of physiological and impaired cardiac (cell) function. As our understanding of the molecular and genetic pathophysiology of heart failure will increase, this might help to identify novel therapeutic targets and may lead to the development of new and specific treatment options in patients with heart failure.

Predictors of High Post-Procedural Gradients after Catheter-Based Aortic Valve Implantation Using Direct Flow Medical Bioprostheses.

BACKGROUND: The Direct Flow Medical (DFM) valve is a new non-metallic and repositionable bioprosthesis used for transcatheter aortic valve implantation (TAVI). The study aim was to investigate procedural and post-implant valve data in patients receiving differently sized DFM bioprostheses. METHODS: Procedural, echocardiographic and computed tomography findings of 28 patients receiving either a 25, 27 or 29 mm DFM bioprosthesis were analyzed. RESULTS: Implantation of a 29 mm bioprosthesis was associated with longer procedure (p <0.05) and radiation (p <0.05) times, and a higher dose-area product (p <0.01) compared to the 25 mm valve. A high mean post-interventional aortic gradient indicating a suboptimal result was found in 44% patients receiving a 29 mm bioprosthesis, whereas none of the patients with a 25 or 27 mm valve had a high gradient (p <0.05). Aortic valve calcification was greatest in the 29 mm group and correlated with a higher dose-area product (p <0.01). CONCLUSIONS: DFM bioprosthesis size significantly influences the TAVI procedure and post-implant valve function. Valve calcification and use of the 29 mm DFM bioprosthesis per se possibly predict a more complicated procedure. Therefore, annulus size and valve calcification severity should be taken into consideration when deciding which bioprosthesis type might be best suited for individual patients.

CCL5 deficiency reduces neointima formation following arterial injury and thrombosis in apolipoprotein E-deficient mice.

Activated platelets secrete different chemokines, among others CCL5, thereby triggering inflammatory cell recruitment into the vessel wall. Here, we investigated how CCL5 deficiency influences vascular remodeling processes. Experiments were performed in apolipoprotein E and CCL5 double deficient (ApoE(-/-)×CCL5(-/-)) mice, using ApoE(-/-)×CCL5(+/+) mice as controls. The ferric chloride model was applied to induce thrombosis at the site of carotid artery injury within minutes and the formation of a smooth muscle cell-rich neointima within 3weeks. In both groups, vascular injury resulted in thrombus formation. CCL5 deficiency did not alter thrombus resolution examined at day 7. Analysis at 21days revealed that CCL5 absence was associated with a significant reduction in the neointima area (p<0.05), neointima-to-media ratio (p<0.05) and lumen stenosis (p<0.05) compared to ApoE(-/-)×CCL5(+/+) mice. Immunohistochemical analysis of CCL5 receptors showed decreased CCR5 positive staining in ApoE(-/-)×CCL5(-/-) mice (p<0.01), whereas the amount of CCR1 (p=0.053) and Mac2-positive macrophages (p<0.05) was increased. The amount of SMA-positive smooth muscle cells was lower in ApoE(-/-) mice lacking CCL5 (p<0.05). Positive staining for Krüppel-like factor 4 (KLF4), an atheroprotective transcription factor, was increased in the neointima of ApoE(-/-)×CCL5(-/-) mice (p<0.05) and found to co-localize with smooth muscle cells. In summary, CCL5 deficiency resulted in reduced neointima formation after carotid artery injury and thrombosis. Hemodynamic and histochemical analyses suggested that this was not due to differences in thrombus formation or resolution. Possibly, the atheroprotective effect of CCL5 deficiency is mediated by KLF4 upregulation in smooth muscle cells.

Circulating Endothelial Cells Expressing the Angiogenic Transcription Factor Krüppel-Like Factor 4 are Decreased in Patients with Coronary Artery Disease.

OBJECTIVE: The zinc finger transcription factor KLF4 is known to control diverse EC functions. METHODS: The functional role of KLF4 for angiogenesis and its association with CAD was examined in HUVECs and human CECs. RESULTS: In two different angiogenesis assays, siRNA-mediated KLF4 downregulation impaired HUVEC sprouting and network formation. Conversely, KLF4 overexpression increased HUVEC sprouting and network formation. Similar findings were observed after incubation of HUVECs with CdM from KLF4 cDNA-transfected cells, suggesting a role of paracrine factors for mediating angiogenic KLF4 effects. In this regard, VEGF expression was increased in KLF4-overexpressing HUVECs, whereas its expression was reduced in HUVECs transfected with KLF4 siRNA. To examine the relevance of our in vitro findings for human endothelial dysfunction, we analyzed the expression of KLF4 in CECs of patients with stable CAD. Flow cytometry analyses revealed decreased numbers of KLF4-positive CECs in peripheral blood from CAD patients compared to healthy controls. CONCLUSIONS: Our findings suggest that KLF4 may represent a potential biomarker for EC dysfunction. In the future, (therapeutic) modulation of KLF4 may be useful in regulating EC function during vascular disease processes.

In vitro and in vivo effects of human monocytes and their subsets on new vessel formation.

OBJECTIVE: Human monocytes can be divided into CD16(-) monocytes and CD16(+) monocytes. Studies in mice suggested differential effects of monocyte subsets during new vessel formation. METHODS: The functional role of human monocyte subsets in neovascularization processes was investigated. For in vivo experiments, nude mice underwent unilateral hindlimb ischemia surgery before being injected with either total monocytes, CD16(-) monocytes or CD16(+) monocytes isolated from healthy individuals. RESULTS: In vitro, cytokine array analysis demonstrated that monocytes release numerous angiogenic cytokines, some of which were differentially expressed in monocyte subsets. Sprout length was enhanced in EC spheroids being cultured in conditioned medium obtained from total monocytes and, to a lesser extent, also in supernatants of CD16(-) monocytes. Laser Doppler perfusion imaging up to day 28 after surgery revealed a trend toward improved revascularization in mice treated with monocytes, but no significant differences between monocyte subsets. Histological analyses four weeks after surgery showed an increased arteriole size in mice having received CD16(+) monocytes, whereas the number of capillaries did not significantly differ between groups. CONCLUSIONS: Our findings suggest additive and differential effects of monocyte subsets during neovascularization processes, possibly due to an altered secretion of angiogenic factors and their paracrine capacity to stimulate new vessel formation.

Stage-dependent detection of CD14+ and CD16+ cells in the human heart after myocardial infarction.

Monocytes are critically involved in cardiovascular wound healing processes. Human monocytes can be classified into two subsets based on the expression of CD14 and CD16. Here, we examined the temporal and spatial distribution of CD14⁺ and CD16⁺ cells after myocardial infarction (MI) in human heart and spleen tissue and correlated it with markers of cardiac repair. Heart samples obtained at autopsy were histologically classified into acute (AMI; n = 11), subacute (SAMI; n = 10) and old (OMI; n = 16) MI, or control myocardium (CONTR; n = 8). Histochemical analyses revealed marked fibrosis in OMI (p < 0.001 vs. CONTR). The adhesion molecule CD56 was also strongly expressed in OMI (p < 0.01 vs. CONTR) and found to correlate with fibrosis (p < 0.001). The number of capillaries was reduced in OMI (p < 0.01 vs. CONTR; p < 0.05 vs. AMI), whereas the hypoxia indicator carbonic anhydrase IX was predominantly expressed in AMI (p < 0.01 vs. OMI and CONTR) and SAMI (p < 0.05 vs. OMI and CONTR). The monocyte chemoattractrant osteopontin was also more highly expressed in hearts of SAMI patients (p < 0.01 vs. CONTR). Numbers of CD14⁺ monocytes were found to correlate with CD16⁺ cells (p < 0.05) and inversely with fibrosis (p < 0.05). Regarding a MI-associated release of monocytes from spleen reservoirs, a non-significant reduction of splenic CD14⁺ and CD16⁺ cells was detected in subjects with AMI. In conclusion, disease stage-specific alterations in CD14⁺ and CD16 cells in human heart may contribute to cardiac repair processes following MI.

Leptin-dependent and leptin-independent paracrine effects of perivascular adipose tissue on neointima formation.

OBJECTIVE: Clinical and experimental evidence suggests that periadventitial adipose tissue may modulate vascular lesion formation. The aim of this study was to determine the role of perivascular leptin expression on neointima formation and to differentiate it from local inflammation and systemically elevated leptin levels. APPROACH AND RESULTS: Increased neointima formation after carotid artery injury was observed in hyperleptinemic, diet-induced obese wild-type mice, but not in leptin-deficient ob/ob mice. High-fat diet was associated with increased leptin expression in visceral adipose tissue (VAT) as well as in perivascular adipose tissue. Perivascular leptin overexpression achieved by adenoviral vectors enhanced intimal cell proliferation and neointima formation in wild-type mice, but not in leptin receptor-deficient mice. Perivascular transplantation of VAT from high-fat diet-induced obese wild-type mice around the carotid artery of immunodeficient mice also promoted neointima formation, without affecting body weight or systemic leptin levels, and this effect was absent, if VAT from ob/ob mice was used. On the contrary, perivascular transplantation of VAT from ob/ob mice fed high-fat diet, characterized by marked immune cell accumulation, promoted neointimal hyperplasia also in the absence of leptin. In vitro, recombinant leptin and VAT-conditioned medium increased human arterial smooth muscle cell proliferation in a (partly) leptin-dependent manner. CONCLUSIONS: Our findings suggest that locally elevated leptin levels may promote neointima formation, independent of obesity and systemic hyperleptinemia, but also underline the importance of perivascular inflammation in mediating the increased cardiovascular risk in obesity.

VEGF-A-induced chemotaxis of CD16+ monocytes is decreased secondary to lower VEGFR-1 expression.

BACKGROUND: Monocyte recruitment into the vessel wall is a crucial initial step in vascular repair, arteriogenesis and atherogenesis. Two distinct human monocyte subpopulations can be classified according to their CD14/16 surface expression, namely CD14++CD16-monocytes (CD16-mo) and CD14+CD16+ monocytes (CD16+mo). We investigated different functional properties of the two monocyte subsets. METHODS: CD16-/CD16+mo were isolated from human blood by an immunological selection. We assessed monocyte chemokinesis, chemotaxis, adhesion and Vascular-Endothelial Growth Factor (VEGF) receptor expression. Furthermore, generation of reactive oxygen species (ROS) as well as expression of antioxidant enzymes was investigated. RESULTS: Chemokinesis of CD16+mo was decreased compared to CD16-mo (p<0.01). Likewise, adhesion capacity of CD16+mo was weaker (p<0.05). CD16+mo chemotaxis towards the angiogenic ligands vascular endothelial growth factor-A (VEGF-A) and placenta growth factor-1 (PlGF-1) was reduced compared to CD16-mo. VEGFR-1 is the receptor for VEGF-A and PlGF-1 on monocytes. VEGFR-1 protein expression was lower in CD16+mo than in CD16-mo (p<0.05). The impaired VEGF-A- and PlGF-1-induced CD16+mo chemotaxis might therefore be attributed to the reduced VEGFR-1 expression. CD16+mo exhibited less spontaneous ROS production than CD16-mo. Additionally, the antioxidant enzyme manganese superoxide dismutase was expressed at higher levels in CD16+mo (p<0.05); this might partly explain the higher oxidative resistance of CD16+mo. CONCLUSION: These novel functional differences between CD16-mo and CD16+mo may predict different functional roles of both monocyte subsets in vascular repair, arteriogenesis and atherogenesis.

Strenuous physical exercise adversely affects monocyte chemotaxis.

Physical exercise is important for proper cardiovascular function and disease prevention, but it may influence the immune system. We evaluated the effect of strenuous exercise on monocyte chemotaxis. Monocytes were isolated from blood of 13 young, healthy, sedentary individuals participating in a three-week training program which consisted of repeated exercise bouts. Monocyte chemotaxis and serological biomarkers were investigated at baseline, after three weeks training and after four weeks recovery. Chemotaxis towards vascular endothelial growth factor-A (VEGF-A) and transforming growth factor-ß1 (TGF-ß1) was completely inhibited immediately after training (p<0.01), and remained so after four weeks recovery. Likewise, monocyte chemoattractant protein-1 (MCP-1)-induced migration declined after training (p<0.01) and improved only partially during the recovery period. MCP-1 serum levels were significantly reduced after four weeks recovery compared to baseline (p<0.01). Total blood antioxidant capacity was enhanced at this time point (p<0.01). Monocyte chemokinesis, TGF-ß1 and nitric oxide serum levels remained unchanged during the study. Strenuous three-week training consisting of repeated exercise bouts in healthy, sedentary individuals reduces monocyte chemotaxis. It remains to be established, whether this is a sound adaptation to increased stimuli or an untoward reaction to overtraining. Nevertheless, the effect remains for several weeks with no exercise.

Chemotaxis analysis of circulating monocytes in patients with a recent acute coronary syndrome.

BACKGROUND: Monocytes/macrophages are crucially involved in the process of atherogenesis. Presence of an acute coronary syndrome (ACS) is associated with macrophage activation. We investigated whether ligand-induced monocyte chemotaxis can serve as biomarker in recent ACS and discriminate ACS from stable coronary artery disease (CAD). METHODS: In a prospective study, the migratory response of monocytes towards the chemotactic ligands vascular endothelial growth factor-A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1) in patients with recent ACS (n=29) (median time period since cardiovascular event, 11 days) and stable CAD patients (n=41) was analysed. Furthermore, blood levels of C-reactive protein (CRP), VEGF-A and soluble vascular endothelial growth factor receptor-1 (sVEGRF-1) were determined. RESULTS: Unexpectedly, VEGF-A-induced monocyte chemotaxis did not differ between ACS and CAD. The same was true for the chemotactic response of monocytes towards MCP-1. In addition, we could not find any difference in VEGF-A and sVEGFR-1 levels between recent ACS and stable CAD. CRP was significantly enhanced in the ACS group, but did not correlate with the VEGF-A- and MCP-1-induced chemotaxis. CONCLUSIONS: Recent ACS is not associated with enhanced monocyte chemotaxis towards VEGF-A and MCP-1. Therefore, VEGF-A- and MCP-1-induced monocyte chemotaxis as a potential novel biomarker remains unaffected by recent ACS.

Monocyte function is severely impaired by the fluorochrome calcein acetomethylester.

For rapid chemotaxis quantification, cell prelabelling is often performed with the fluorochrome calcein acetomethylester (calcein AM). We investigated whether calcein AM-prelabelling is reliable for monocyte migration analysis. Human monocytes were either preexposed to calcein AM or unlabelled. Monocyte migration towards the potent chemoattractants transforming growth factor-beta1 (TGF-beta1) and N-formyl-Methionin-Leucin-Phenylalanin (fMLP) was assessed using a 48-well micro-chemotaxis chamber. For quantification, cells were visualized by light microscopy and counted. Surprisingly, random migration of calcein AM-prelabelled cells was significantly impaired compared to the unlabelled control. Accordingly, monocyte chemotaxis towards either TGF-beta1 or fMLP dramatically declined. Adherence of calcein AM-labelled monocytes on plastic was also significantly decreased compared to control cells. As adhesion is regarded as an essential component of monocyte migration, the reduced migration observed in calcein AM-labelled monocytes might be explained by a fluorochrome-induced adhesion defect. Therefore, use of the fluorochrome calcein AM cannot be recommended for functional testing of monocytes.

The BTB-Kelch protein KLEIP controls endothelial migration and sprouting angiogenesis.

Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1alpha. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF- and basic fibroblast growth factor-induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF- or basic fibroblast growth factor-mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases.