VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Platelets regulate ischemia-induced revascularization and angiogenesis by secretion of growth factor-modulating factors.

In ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization. We assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in patients with coronary artery disease as well as those with peripheral artery disease and used genetic mouse models to characterize its significance for ischemia and growth factor-driven revascularization. The presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model. Ischemia-driven angiogenesis was significantly improved in C5aR1-/- mice but not in C5-/- mice, suggesting a specific role of C5aR1. Experiments using the supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. Using C5aR1-/-CXCL4-/- mice, we observed no additional effect in the revascularization response, underscoring a strong dependence of CXCL4 secretion on the C5a-C5aR1-axis. We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by the release of antiangiogenic CXCL4.

Improved mid-term stability of MR reduction with an increased number of clips after percutaneous mitral valve repair in functional MR.

Background: Percutaneous mitral valve repair (PMVR) has evolved to be a standard procedure in suitable patients with mitral regurgitation (MR) not accessible for open surgery. Here, we analyzed the influence of the number and positioning of the clips implanted during the procedure on MR reduction analyzing also sub-collectives of functional and degenerative MR (DMR). Results: We included 410 patients with severe MR undergoing PMVR using the MitraClip® System. MR and reduction of MR were analyzed by TEE at the beginning and at the end of the PMVR procedure. To specify the clip localization, we sub-divided segment 2 into 3 sub-segments using the segmental classification of the mitral valve. Results: We found an enhanced reduction of MR predominantly in DMR patients who received more than one clip. Implantation of only one clip led to a higher MR reduction in patients with functional MR (FMR) in comparison to patients with DMR. No significant differences concerning pressure gradients could be observed in degenerative MR patients regardless of the number of clips implanted. A deterioration of half a grade of the achieved MR reduction was observed 6 months post-PMVR independent of the number of implanted clips with a better stability in FMR patients, who got 3 clips compared to patients with only one clip. Conclusions: In patients with FMR, after 6 months the reduction of MR was more stable with an increased number of implanted clips, which suggests that this specific patient collective may benefit from a higher number of clips.

Protocol to isolate and analyze mouse bone marrow derived dendritic cells (BMDC).

Different types of immune cells are involved in atherogenesis and may act atheroprotective or atheroprogressive. Here, we describe an in vitro approach to analyze CD11c+ cells and CD11c+-derived ApoE in atherosclerosis. The major steps include harvesting mouse bone marrow, plating cells in culture dishes, treating them with differentiation factors, and collecting cells after removal of undesirable populations. This protocol can be adapted for CD11c+ cells in different contexts, thus, serving as models for different diseases and to analyze cell-specific molecules. For complete details on the use and execution of this protocol, please refer to Sauter et al. (2021).

Platelets and the Role of P2X Receptors in Nociception, Pain, Neuronal Toxicity and Thromboinflammation.

P2X receptors belong to a family of cation channel proteins, which respond to extracellular adenosine 5'-triphosphate (ATP). These receptors have gained increasing attention in basic and translational research, as they are central to a variety of important pathophysiological processes such as the modulation of cardiovascular physiology, mediation of nociception, platelet and macrophage activation, or neuronal-glial integration. While P2X1 receptor activation is long known to drive platelet aggregation, P2X7 receptor antagonists have recently been reported to inhibit platelet activation. Considering the role of both P2X receptors and platelet-mediated inflammation in neuronal diseases such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, and stroke, targeting purinergic receptors may provide a valuable novel therapeutic approach in these diseases. Therefore, the present review illuminates the role of platelets and purinergic signaling in these neurological conditions to evaluate potential translational implications.

The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1-/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

Platelets and Immune Responses During Thromboinflammation.

Besides mediating hemostatic functions, platelets are increasingly recognized as important players of inflammation. Data from experiments in mice and men revealed various intersection points between thrombosis, hemostasis, and inflammation, which are addressed and discussed in this review in detail. One such example is the intrinsic coagulation cascade that is initiated after platelet activation thereby further propagating and re-enforcing wound healing or thrombus formation but also contributing to the pathophysiology of severe diseases. FXII of the intrinsic pathway connects platelet activation with the coagulation cascade during immune reactions. It can activate the contact system thereby either creating an inflammatory state or accelerating inflammation. Recent insights into platelet biology could show that platelets are equipped with complement receptors. Platelets are important for tissue remodeling after injury has been inflicted to the endothelial barrier and to the subendothelial tissue. Thus, platelets are increasingly recognized as more than just cells relevant for bleeding arrest. Future insights into platelet biology are to be expected. This research will potentially offer novel opportunities for therapeutic intervention in diseases featuring platelet abundance.

Elevated Mitral Valve Pressure Gradient Is Predictive of Long-Term Outcome After Percutaneous Edge-to-Edge Mitral Valve Repair in Patients With Degenerative Mitral Regurgitation ( MR ), But Not in Functional MR.

Background This study analyzed the effects on long-term outcome of residual mitral regurgitation ( MR ) and mean mitral valve pressure gradient ( MVPG ) after percutaneous edge-to-edge mitral valve repair using the MitraClip system. Methods and Results Two hundred fifty-five patients who underwent percutaneous edge-to-edge mitral valve repair were analyzed. Kaplan-Meier and Cox regression analyses were performed to evaluate the impact of residual MR and MVPG on clinical outcome. A combined clinical end point (all-cause mortality, MV surgery, redo procedure, implantation of a left ventricular assist device) was used. After percutaneous edge-to-edge mitral valve repair, mean MVPG increased from 1.6±1.0 to 3.1±1.5 mm Hg ( P<0.001). Reduction of MR severity to ≤2+ postintervention was achieved in 98.4% of all patients. In the overall patient cohort, residual MR was predictive of the combined end point while elevated MVPG >4.4 mm Hg was not according to Kaplan-Meier and Cox regression analyses. We then analyzed the cohort with degenerative and that with functional MR separately to account for these different entities. In the cohort with degenerative MR , elevated MVPG was associated with increased occurrence of the primary end point, whereas this was not observed in the cohort with functional MR . Conclusions MVPG >4.4 mm Hg after MitraClip implantation was predictive of clinical outcome in the patient cohort with degenerative MR . In the patient cohort with functional MR , MVPG >4.4 mm Hg was not associated with increased clinical events.

The Mutual Relation of Platelet Activation and Innate Immunity.

Platelets are known to be central regulators of haemostasis, inflammation and immune response. Formed by megakaryocytes in the bone marrow and the lungs, platelets express a broad range of adhesion receptors and release cytokines and platelet microparticles which enable them to interact with both immune cells and pathogens. In bacterial and viral infections, thrombophilia and thrombocytopenia are commonly seen symptoms, indicating the close relationship between haemostasis and immune defence. Indeed, platelets contribute both directly and via immune mediation to pathogen clearance. In sterile inflammation, a pathogen-free process which is often triggered by cell necrosis and autoimmune reactions, platelets are also of central importance. Recently, platelet inflammasome has been extensively studied in this context. Both sterile inflammation and infection are affected by the interactions of platelets and innate immunity, notably the complement system. Although the general elements of this interplay have been known for long, more and more insights into disease-specific mechanisms could be gained recently. This review gives an outline of the current findings in the field of platelet-immune cell interactions and points out possible implications for clinical therapy.

Myocardial expression of the anaphylatoxin receptor C3aR is associated with cardiac inflammation and prognosis in patients with non-ischaemic heart failure.

AIM: The aim of this study is to analyse the prognostic value of complement anaphylatoxin receptors in patients with non-ischaemic cardiomyopathy undergoing endomyocardial biopsy. METHODS AND RESULTS: In 102 patients (72.5% male patients, median age 54 years) with non-ischaemic cardiomyopathy, myocardial expression of C3aR was assessed among other parameters. The primary study endpoint was a composite of death, heart transplantation, heart failure-related re-hospitalization, and deterioration of left ventricular ejection fraction within a mean follow-up of 11.9 months. The number of cells, which stained positive for C3aR, was significantly increased in patients with inflammatory compared with non-inflammatory cardiomyopathy (1.75 ± 0.31 cells in inflammatory cardiomyopathy vs. 0.94 ± 0.26 in non-inflammatory cardiomyopathy, P = 0.049). Subsequently, positive expression for C3aR was judged based on a semi-quantitative scoring system. Significantly, more patients with positive MHCII and CD68 expression showed an increased number of C3aR-positive cells. C3aR expression based on this score was more pronounced in patients with human herpesvirus 6 viral genome detection. Kaplan-Meier curves illustrate that the C3aR-negative group reached the primary endpoint significantly more often (mean follow-up 11.9 months, log rank 5.963, P = 0.015). Lack of C3aR expression was a strong independent predictor for the primary endpoint in Cox regression analysis [hazard ratio 0.46 (0.26-0.82, P = 0.009)]. CONCLUSIONS: C3aR-positive cells are found more often in patients with inflammatory cardiomyopathy. The relevance of C3aR-positive cells in patients with non-ischaemic cardiomyopathy should be further evaluated as potential predictors or modulators of adverse cardiac remodelling, the substrate of progressive heart failure.

3D echocardiography derived right ventricular function is associated with right ventricular failure and mid-term survival after left ventricular assist device implantation.

BACKGROUND: Right heart failure remains a major cause of morbidity and mortality after left ventricular assist device (LVAD) implantation. Multiple 2D echocardiography derived parameters are associated with right ventricular failure (RV failure), but none of them has been proven to be a reliable predictor to date. We hypothesized that novel 3D-echocardiography (3DE) based parameters are associated with RV failure and predict long term outcome in patients undergoing LVAD implantation. METHODS: This single-center study retrospectively enrolled 26 patients undergoing continuous-flow LVAD implantation. RV failure was defined as prolonged inotropic support for >14 days after LVAD implantation or consecutive implantation of a right ventricular assist device. Based on transesophageal 3DE datasets acquired prior to surgery right ventricular size, ejection fraction and longitudinal strains were calculated. RESULTS: The overall RV failure rate was 19.2%. Patients suffering from RV failure had a significantly impaired 3D-right ventricular ejection fraction (3D-RVEF; 28 ±â€¯2% vs. 19 ±â€¯3%, p = 0.0145) and 3D derived RV free wall longitudinal strain (3D-RV-fws; -13.2 ±â€¯0.97% vs. -6.4 ±â€¯1.98%; p = 0.0056) when compared to patients without RV failure. ROC analysis for 3D-RV-fws (AUC 0.914) and 3D-RVEF (AUC 0.876) showed high discriminative capabilities in regard to detection of RV failure. Kaplan-Meier analysis showed an improved long-term survival of patients with a 3D-RV-fws <-11.9%. CONCLUSIONS: 3D-echocardiography derived RV ejection fraction and RV free wall strain are associated with right ventricular failure and long term outcome in patients undergoing LVAD implantation. These parameters have the potential to be future predictors for right heart failure in LVAD surgery.

Improved mitral valve coaptation and reduced mitral valve annular size after percutaneous mitral valve repair (PMVR) using the MitraClip system.

Aims: Improved mitral valve leaflet coaptation with consecutive reduction of mitral regurgitation (MR) is a central goal of percutaneous mitral valve repair (PMVR) with the MitraClip® system. As influences of PMVR on mitral valve geometry have been suggested before, we examined the effect of the procedure on mitral annular size in relation to procedural outcome. Methods and results: Geometry of the mitral valve annulus was evaluated in 183 patients undergoing PMVR using echocardiography before and after the procedure and at follow-up. Mitral valve annular anterior-posterior (ap) diameter decreased from 34.0 ± 4.3 to 31.3 ± 4.9 mm (P < 0.001), and medio-lateral (ml) diameter from 33.2 ± 4.8 to 32.4 ± 4.9 mm (P < 0.001). Accordingly, we observed an increase in MV leaflet coaptation after PMVR. The reduction of mitral valve ap diameter showed a significant inverse correlation with residual MR. Importantly, the reduction of mitral valve ap diameter persisted at follow-up (31.3 ± 4.9 mm post PMVR, 28.4 ± 5.3 mm at follow-up). Conclusion: This study demonstrates mechanical approximation of both mitral valve annulus edges with improved mitral valve annular coaptation by PMVR using the MitraClip® system, which correlates with residual MR in patients with MR.

Comparison of Deep Sedation With General Anesthesia in Patients Undergoing Percutaneous Mitral Valve Repair.

BACKGROUND: Percutaneous edge-to-edge mitral valve repair (PMVR) has become an established treatment option for mitral regurgitation in patients not eligible for surgical repair. Currently, most procedures are performed under general anesthesia (GA). An increasing number of centers, however, are performing the procedure under deep sedation (DS). Here, we compared patients undergoing PMVR with GA or DS. METHODS AND RESULTS: A total of 271 consecutive patients underwent PMVR at our institution between May 2014 and December 2016. Seventy-two procedures were performed under GA and 199 procedures under DS. We observed that in the DS group, doses of propofol (743±228 mg for GA versus 369±230 mg for DS, P<0.001) and norepinephrine (1.1±1.6 mg for GA versus 0.2±0.3 mg for DS, P<0.001) were significantly lower. Procedure time, fluoroscopy time, and dose area product were significantly higher in the GA group. There was no significant difference between GA and DS with respect to overall bleeding complications, postinterventional pneumonia (4% for GA versus 5% for DS), or C-reactive protein levels (361±351 nmol/L for GA versus 278±239 nmol/L for DS). Significantly fewer patients with DS needed a postinterventional stay in the intensive care unit (96% for GA versus 19% for DS, P<0.001). Importantly, there was no significant difference between DS and GA regarding intrahospital or 6-month mortality. CONCLUSIONS: DS for PMVR is safe and feasible. No disadvantages with respect to procedural outcome or complications in comparison to GA were observed. Applying DS may simplify the PMVR procedure.

Immediate increase of cardiac output after percutaneous mitral valve repair (PMVR) determined by echocardiographic and invasive parameters: Patzelt: Increase of cardiac output after PMVR.

BACKGROUND: Successful percutaneous mitral valve repair (PMVR) in patients with severe mitral regurgitation (MR) causes changes in hemodynamics. Echocardiographic calculation of cardiac output (CO) has not been evaluated in the setting of PMVR, so far. Here we evaluated hemodynamics before and after PMVR with the MitraClip system using pulmonary artery catheterization, transthoracic (TTE) and transesophageal (TEE) echocardiography. METHODS: 101 patients with severe MR not eligible for conventional surgery underwent PMVR. Hemodynamic parameters were determined during and after the intervention. We evaluated changes in CO and pulmonary artery systolic pressure before and after PMVR. CO was determined with invasive parameters using the Fick method (COi) and by a combination of TTE and TEE (COe). RESULTS: All patients had successful clip implantation, which was associated with increased COi (from 4.6±1.4l/min to 5.4±1.6l/min, p<0.001). Furthermore, pulmonary artery systolic pressure (PASP) showed a significant decrease after PMVR (47.6±16.1 before, 44.7±15.5mmHg after, p=0.01). In accordance with invasive measurements, COe increased significantly (COe from 4.3±1.7l/min to 4.8±1.7l/min, p=0.003). Comparing both methods to calculate CO, we observed good agreement between COi and COe using Bland Altman plots. CONCLUSIONS: CO increased significantly after PMVR as determined by echocardiography based and invasive calculation of hemodynamics during PMVR. COe shows good agreement with COi before and after the intervention and, thus, represents a potential non-invasive method to determine CO in patients with MR not accessible by conventional surgery.

Effects of Mechanical Ventilation on Heart Geometry and Mitral Valve Leaflet Coaptation During Percutaneous Edge-to-Edge Mitral Valve Repair.

OBJECTIVES: This study sought to evaluate a ventilation maneuver to facilitate percutaneous edge-to-edge mitral valve repair (PMVR) and its effects on heart geometry. BACKGROUND: In patients with challenging anatomy, the application of PMVR is limited, potentially resulting in insufficient reduction of mitral regurgitation (MR) or clip detachment. Under general anesthesia, however, ventilation maneuvers can be used to facilitate PMVR. METHODS: A total of 50 consecutive patients undergoing PMVR were included. During mechanical ventilation, different levels of positive end-expiratory pressure (PEEP) were applied, and parameters of heart geometry were assessed using transesophageal echocardiography. RESULTS: We found that increased PEEP results in elevated central venous pressure. Specifically, central venous pressure increased from 14.0 ± 6.5 mm Hg (PEEP 3 mm Hg) to 19.3 ± 5.9 mm Hg (PEEP 20 mm Hg; p < 0.001). As a consequence, the reduced pre-load resulted in reduction of the left ventricular end-systolic diameter from 43.8 ± 10.7 mm (PEEP 3 mm Hg) to 39.9 ± 11.0 mm (PEEP 20 mm Hg; p < 0.001), mitral valve annulus anterior-posterior diameter from 32.4 ± 4.3 mm (PEEP 3 mm Hg) to 30.5 ± 4.4 mm (PEEP 20 mm Hg; p < 0.001), and the medio-lateral diameter from 35.4 ± 4.2 mm to 34.1 ± 3.9 mm (p = 0.002). In parallel, we observed a significant increase in leaflet coaptation length from 3.0 ± 0.8 mm (PEEP 3 mm Hg) to 5.4 ± 1.1 mm (PEEP 20 mm Hg; p < 0.001). The increase in coaptation length was more pronounced in MR with functional or mixed genesis. Importantly, a coaptation length >4.9 mm at PEEP of 10 mm Hg resulted in a significant reduction of PMVR procedure time (152 ± 49 min to 116 ± 26 min; p = 0.05). CONCLUSIONS: In this study, we describe a novel ventilation maneuver improving mitral valve coaptation length during the PMVR procedure, which facilitates clip positioning. Our observations could help to improve PMVR therapy and could make nonsurgical candidates accessible to PMVR therapy, particularly in challenging cases with functional MR.

Platelets induce apoptosis via membrane-bound FasL.

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL(△m/△m)) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre(+) FasL(fl/fl) mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.