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.

Modification of the GRACE Risk Score for Risk Prediction in Patients With Acute Coronary Syndromes.

Importance: The Global Registry of Acute Coronary Events (GRACE) risk score, a guideline-recommended risk stratification tool for patients presenting with acute coronary syndromes (ACS), does not consider the extent of myocardial injury. Objective: To assess the incremental predictive value of a modified GRACE score incorporating high-sensitivity cardiac troponin (hs-cTn) T at presentation, a surrogate of the extent of myocardial injury. Design, Setting, and Participants: This retrospectively designed longitudinal cohort study examined 3 independent cohorts of 9803 patients with ACS enrolled from September 2009 to December 2017; 2 ACS derivation cohorts (Heidelberg ACS cohort and Newcastle STEMI cohort) and an ACS validation cohort (SPUM-ACS study). The Heidelberg ACS cohort included 2535 and the SPUM-ACS study 4288 consecutive patients presenting with a working diagnosis of ACS. The Newcastle STEMI cohort included 2980 consecutive patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention. Data were analyzed from March to June 2023. Exposures: In-hospital, 30-day, and 1-year mortality risk estimates derived from an updated risk score that incorporates continuous hs-cTn T at presentation (modified GRACE). Main Outcomes and Measures: The predictive value of continuous hs-cTn T and modified GRACE risk score compared with the original GRACE risk score. Study end points were all-cause mortality during hospitalization and at 30 days and 1 year after the index event. Results: Of 9450 included patients, 7313 (77.4%) were male, and the mean (SD) age at presentation was 64.2 (12.6) years. Using continuous rather than binary hs-cTn T conferred improved discrimination and reclassification compared with the original GRACE score (in-hospital mortality: area under the receiver operating characteristic curve [AUC], 0.835 vs 0.741; continuous net reclassification improvement [NRI], 0.208; 30-day mortality: AUC, 0.828 vs 0.740; NRI, 0.312; 1-year mortality: AUC, 0.785 vs 0.778; NRI, 0.078) in the derivation cohort. These findings were confirmed in the validation cohort. In the pooled population of 9450 patients, modified GRACE risk score showed superior performance compared with the original GRACE risk score in terms of reclassification and discrimination for in-hospital mortality end point (AUC, 0.878 vs 0.780; NRI, 0.097), 30-day mortality end point (AUC, 0.858 vs 0.771; NRI, 0.08), and 1-year mortality end point (AUC, 0.813 vs 0.797; NRI, 0.056). Conclusions and Relevance: In this study, using continuous rather than binary hs-cTn T at presentation, a proxy of the extent of myocardial injury, in the GRACE risk score improved the mortality risk prediction in patients with ACS.

Effector T cell chemokine IP-10 predicts cardiac recovery and clinical outcomes post-myocardial infarction.

Background and aims: Preclinical data suggest that activation of the adaptive immune system is critical for myocardial repair processes in acute myocardial infarction. The aim of the present study was to determine the clinical value of baseline effector T cell chemokine IP-10 blood levels in the acute phase of ST-segment elevation myocardial infarction (STEMI) for the prediction of the left ventricular function changes and cardiovascular outcomes after STEMI. Methods: Serum IP-10 levels were retrospectively quantified in two independent cohorts of STEMI patients undergoing primary percutaneous coronary intervention. Results: We report a biphasic response of the effector T cell trafficking chemokine IP-10 characterized by an initial increase of its serum levels in the acute phase of STEMI followed by a rapid reduction at 90min post reperfusion. Patients at the highest IP-10 tertile presented also with more CD4 effector memory T cells (CD4 TEM cells), but not other T cell subtypes, in blood. In the Newcastle cohort (n=47), patients in the highest IP-10 tertile or CD4 TEM cells at admission exhibited an improved cardiac systolic function 12 weeks after STEMI compared to patients in the lowest IP-10 tertile. In the Heidelberg cohort (n=331), STEMI patients were followed for a median of 540 days for major adverse cardiovascular events (MACE). Patients presenting with higher serum IP-10 levels at admission had a lower risk for MACE after adjustment for traditional risk factors, CRP and high-sensitivity troponin-T levels (highest vs. rest quarters: HR [95% CI]=0.420 [0.218-0.808]). Conclusion: Increased serum levels of IP-10 in the acute phase of STEMI predict a better recovery in cardiac systolic function and less adverse events in patients after STEMI.

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.

Novel Aspects Targeting Platelets in Atherosclerotic Cardiovascular Disease-A Translational Perspective.

Platelets are important cellular targets in cardiovascular disease. Based on insights from basic science, translational approaches and clinical studies, a distinguished anti-platelet drug treatment regimen for cardiovascular patients could be established. Furthermore, platelets are increasingly considered as cells mediating effects "beyond thrombosis", including vascular inflammation, tissue remodeling and healing of vascular and tissue lesions. This review has its focus on the functions and interactions of platelets with potential translational and clinical relevance. The role of platelets for the development of atherosclerosis and therapeutic modalities for primary and secondary prevention of atherosclerotic disease are addressed. Furthermore, novel therapeutic options for inhibiting platelet function and the use of platelets in regenerative medicine are considered.

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).

Protocol to visualize CD11c+ cells in atherosclerosis using LacZ reporter mice.

Here, we describe an in vivo approach to visualize CD11c+ cells in atherosclerosis. In particular, we use a protocol for X-Gal staining of immune cells within atherosclerotic plaques, which can be used as an alternative to analyze plaque composition and cell-specific molecules in atherogenesis. LacZ knockin mice have to be bred to mice carrying the CD11ccre recombinase-both brought onto an ApoE-/- background-to be able to visualize this cell type of interest in the plaques by X-Gal staining. With this approach, different immune cells in atherogenesis can be examined. For complete details on the use and execution of this protocol, please refer to Sauter et al. (2021).

Platelet depletion does not alter long-term functional outcome after cerebral ischaemia in mice.

Platelets are key mediators of thrombus formation and inflammation during the acute phase of ischaemic stroke. Particularly, the platelet glycoprotein (GP) receptors GPIbα and GPVI have been shown to mediate platelet adhesion and activation in the ischaemic brain. GPIbα and GPVI blockade could reduce infarct volumes and improve functional outcome in mouse models of acute ischaemic stroke, without concomitantly increasing intracerebral haemorrhage. However, the functional role of platelets during long-term stroke recovery has not been elucidated so far. Thus, we here examined the impact of platelet depletion on post-stroke recovery after transient middle cerebral artery occlusion (tMCAO) in adult male mice. Platelet depleting antibodies or isotype control were applied from day 3-28 after tMCAO in mice matched for infarct size. Long-term functional recovery was assessed over the course of 28 days by behavioural testing encompassing motor and sensorimotorical functions, as well as anxiety-like or spontaneous behaviour. Whole brain flow cytometry and light sheet fluorescent microscopy were used to identify resident and infiltrated immune cell types, and to determine the effects of platelet depletion on the cerebral vascular architecture, respectively. We found that delayed platelet depletion does not improve long-term functional outcome in the tMCAO stroke model. Immune cell abundance, the extent of thrombosis and the organisation of the cerebral vasculature were also comparable between platelet-depleted and control mice. Our study demonstrates that, despite their critical role in the acute stroke setting, platelets appear to contribute only marginally to tissue reorganisation and functional recovery at later stroke stages.

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.

ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

Platelets, Thromboinflammation and Neurovascular Disease.

The brain and spinal cord are immune-privileged organs, but in the disease state protection mechanisms such as the blood brain barrier (BBB) are ineffective or overcome by pathological processes. In neuroinflammatory diseases, microglia cells and other resident immune cells contribute to local vascular inflammation and potentially a systemic inflammatory response taking place in parallel. Microglia cells interact with other cells impacting on the integrity of the BBB and propagate the inflammatory response through the release of inflammatory signals. Here, we discuss the activation and response mechanisms of innate and adaptive immune processes in response to neuroinflammation. Furthermore, the clinical importance of neuroinflammatory mediators and a potential translational relevance of involved mechanisms are addressed also with focus on non-classical immune cells including microglia cells or platelets. As illustrative examples, novel agents such as Anfibatide or Revacept, which result in reduced recruitment and activation of platelets, a subsequently blunted activation of the coagulation cascade and further inflammatory process, demonstrating that mechanisms of neuroinflammation and thrombosis are interconnected and should be further subject to in depth clinical and basic research.

Activated Platelets Upregulate ß2 Integrin Mac-1 (CD11b/CD18) on Dendritic Cells, Which Mediates Heterotypic Cell-Cell Interaction.

Recent evidence suggests interaction of platelets with dendritic cells (DCs), while the molecular mechanisms mediating this heterotypic cell cross-talk are largely unknown. We evaluated the role of integrin Mac-1 (αMß2, CD11b/CD18) on DCs as a counterreceptor for platelet glycoprotein (GP) Ibα. In a dynamic coincubation model, we observed interaction of human platelets with monocyte-derived DCs, but also that platelet activation induced a sharp increase in heterotypic cell binding. Inhibition of CD11b or GPIbα led to significant reduction of DC adhesion to platelets in vitro independent of GPIIbIIIa, which we confirmed using platelets from Glanzmann thrombasthenia patients and transgenic mouse lines on C57BL/6 background (GPIbα-/-, IL4R-GPIbα-tg, and muMac1 mice). In vivo, inhibition or genetic deletion of CD11b and GPIbα induced a significant reduction of platelet-mediated DC adhesion to the injured arterial wall. Interestingly, only intravascular antiCD11b inhibited DC recruitment, suggesting a dynamic DC-platelet interaction. Indeed, we could show that activated platelets induced CD11b upregulation on Mg2+-preactivated DCs, which was related to protein kinase B (Akt) and dependent on P-selectin and P-selectin glycoprotein ligand 1. Importantly, specific pharmacological targeting of the GPIbα-Mac-1 interaction site blocked DC-platelet interaction in vitro and in vivo. These results demonstrate that cross-talk of platelets with DCs is mediated by GPIbα and Mac-1, which is upregulated on DCs by activated platelets in a P-selectin glycoprotein ligand 1-dependent manner.

Apolipoprotein E derived from CD11c+ cells ameliorates atherosclerosis.

Atherosclerosis is studied in models with dysfunctional lipid homeostasis-predominantly the ApoE-/- mouse. The role of antigen-presenting cells (APCs) for lipid homeostasis is not clear. Using a LacZ reporter mouse, we showed that CD11c+ cells were enriched in aortae of ApoE-/- mice. Systemic long-term depletion of CD11c+ cells in ApoE-/- mice resulted in significantly increased plaque formation associated with reduced serum ApoE levels. In CD11ccre+ApoEfl/fl and Albumincre+ApoEfl/fl mice, we could show that ≈70% of ApoE is liver-derived and ≈25% originates from CD11c+ cells associated with significantly increased atherosclerotic plaque burden in both strains. Exposure to acLDL promoted cholesterol efflux from CD11c+ cells and cell-specific deletion of ApoE resulted in increased inflammation reflected by increased IL-1ß serum levels. Our results determined for the first time the level of ApoE originating from CD11c+ cells and demonstrated that CD11c+ cells ameliorate atherosclerosis by the secretion of ApoE.

Inflammation in Metabolic and Cardiovascular Disorders-Role of Oxidative Stress.

Cardiovascular diseases (CVD) constitute the main cause of death worldwide. Both inflammation and oxidative stress have been reported to be involved in the progress of CVD. It is well known that generation of oxidative stress during the course of CVD is involved in tissue damage and inflammation, causing deleterious effects such as hypertension, dysfunctional metabolism, endothelial dysfunction, stroke, and myocardial infarction. Remarkably, natural antioxidant strategies have been increasingly discovered and are subject to current scientific investigations. Here, we addressed the activation of immune cells in the context of ROS production, as well as how their interaction with other cellular players and further (immune) mediators contribute to metabolic and cardiovascular disorders. We also highlight how a dysregulated complement system contributes to immune imbalance and tissue damage in the context of increases oxidative stress. Additionally, modulation of hypothalamic oxidative stress is discussed, which may offer novel treatment strategies for type-2 diabetes and obesity. Together, we provide new perspectives on therapy strategies for CVD caused by oxidative stress, with a focus on oxidative stress.

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.

Complement, inflammation and thrombosis.

A mutual relationship exists between immune activation and mechanisms of thrombus formation. In particular, elements of the innate immune response such as the complement system can modulate platelet activation and subsequently thrombus formation. Several components of the complement system including C3 or the membrane attack complex have been reported to be associated with platelets and become functionally active in the micromilieu of platelet activation. The exact mechanisms how this interplay is regulated and its consequences for tissue inflammation, damage or recovery remain to be defined. This review addresses the current state of knowledge on this topic and puts it into context with diseases featuring both thrombosis and complement activation. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.