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.

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.

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 (αMβ2, 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. Key Points Platelet GPIbα interacts with Mac-1 on DCs. For binding, P-selectin–PSGL1 interaction induces Mac-1 upregulation on DCs.

Diseases featuring tissue ischemia are wide-spread and among the main cause of death in the Western world.[1] To date, the exact mechanisms of revascularization of tissues after arterial obstruction, usually as a result of atherosclerosis,[2] remain incompletely understood. This is underscored by the fact that pharmacological approaches to enhance vessel growth in patients with chronic vascular occlusions are not successfully established for clinical use, yet.[3] Several reports implicate complement activation as a contributor to ischemic tissue injury via interaction with reactive oxygen species,[4] for example, the lectin[5] or the classical pathway.[6] [7] Thus, the complement system may be a possible therapeutic target in this disease setting (reviewed in Markiewski et al[8]). The complement system is a very well-preserved and phylogenetically old part of the immune system serving several functions from immune protection to tissue homeostasis and dysfunction.[9] Previously, the complement receptor C5aR1 was demonstrated to be pro-angiogenic,[10] but has also been attributed an inhibitory role for neovascularization.[11] Interestingly, platelet activation can colocalize with areas of increased complement activity and several functional links have been described connecting complement and platelets.[12] [13] [14] Platelets play a decisive role in cardiovascular diseases featuring thrombosis, where they are the decisive cellular part of any thrombus and contribute to several mechanisms of thrombus formation.[15] Beyond that, platelets were recently implicated in tissue remodeling processes such as apoptosis,[16] [17] immune patrolling,[18] or adaptive immunity.[19] Furthermore, platelets contribute to the immediate response after vascular injury by promoting vascular inflammation,[15] immunomodulation,[20] [21] [22] and atherosclerosis.[23] [24] Recently, we could show that complement receptors are expressed on platelets, and that the anaphylatoxin C3a receptor modulates primary hemostasis.[25] [26] Moreover, platelets express C5a receptor 1 (C5aR1).[27] Absence of this anaphylatoxin receptor, however, had no effect on in vivo thrombus formation.[25] Thus, it might be of alternative fictional relevance for platelet effects in tissue remodeling. The group of J. Italiano could visualize that platelets store pro- and antiangiogenic factors in distinct granules and can release them upon stimulation.[28] Recently, we demonstrated that C5aR1-induced CXCL4 release modulates revascularization.[14] Here, we present further data on the importance of C5aR1 on platelets for the modulation of tissue revascularization. First, we analyzed C5aR1 expression on murine platelets ([Fig. 1A]). Then, we assessed receptor expression levels on platelets in a cohort of peripheral artery disease patients, which were not symptomatic. Previously, this condition has been linked to improved formation of collateral vessels, and presumably thereby lack of pain symptoms.[29] Interestingly, we observed an increased expression of C5aR1 on platelets of patients with an asymptomatic disease ([Fig. 1B]). This led us to investigate the platelet C5aR1 in the hindlimb ischemia model. In this mouse model of ischemic disease, the femoral artery is ligated to induce tissue ischemia (see [Supplementary Materials] for further details). One week after induction of tissue ischemia, the distal gastrocnemius muscle was explanted and processed for immunofluorescence microscopy. Co-staining of CD42b (red, “a platelet marker”) and C5aR1 (green) revealed expression of C5aR1 on DAPI (blue)-negative platelets in the ischemic tissue ([Fig. 1C]). To decipher the role of platelets in a controlled proangiogenic environment, we used a further mouse model, the Matrigel plug assay, whereby a gel-like substance is injected into the skin of mice forming an extracellular-space-like matrix. If the matrix is supplemented with vascular growth factors such as bFGF, vessels grow into the plugs, which can then be quantified. We injected Matrigel supplemented with bFGF and freshly isolated platelets with (wild type [WT]) or without C5aR1 (C5aR1^−/−) into C57/Bl6 mice. Addition of platelets to the Matrigel resulted in reduced bFGF-mediated angiogenesis ([Fig. 1D]). Interestingly, presence of C5aR1 on WT platelets was associated with a reduced extent of vessel growth into the plugs and supplementation with C5aR1^−/− platelets resulted in significantly more revascularization ([Fig. 1E]). We then created a platelet-specific C5aR1-deficient mouse strain using the cre-lox system (for details please refer to the Material and Methods section in the [Supplementary Information] [14]). We assessed platelet reactivity by stimulating diluted whole blood with collagen-related protein and measured the platelet marker β₃ integrin (GPIIIa, CD61), which mediates binding to fibrinogen. There was no significant difference in between both strains ([Supplementary Fig. S1A]), suggesting that there are no effects on the fibrinogen receptor. However, addition of C5a induced some activation in platelets on the level of fibrinogen binding and activated GPIIbIIIa (measured by activation-specific PAC1 antibody), but not on P-selectin upregulation ([Supplementary Fig. S2]). These effects could be blocked by the C5aR1 antagonist PMX205 ([Supplementary Fig. S3]). In the absence of C5a ligation, WT and C5aR1^−/− platelets did not display differences in binding to fibrinogen ([Supplementary Fig. S4]). We monitored revascularization over 14 days post induction of hindlimb ischemia and observed that the C5aR1^flox/floxPF4cre⁻ mice show a lower degree of revascularization than the C5aR1^flox/floxPF4cre⁺ littermates, where C5aR1 is absent in platelets ([Fig. 1F]). Thus, the platelet C5aR1 is an important modulator of revascularization. As all previous data suggest a platelet-mediated effect of C5aR1 on platelets, we stimulated platelets from WT mice with C5a in vitro. Using an ELISA-based analysis, we could measure a release of antiangiogenic CXCL4 (platelet factor 4, PF4) from platelets upon C5a stimulation, which could not be observed when C5aR1 was blocked by the C5aR1 antagonist PMX205 ([Fig. 1G]). To verify that the observed effect is indeed mediated through CXCL4 secreted from platelets, we assessed endothelial tube formation in vitro and co-incubated cells with C5a-conditioned platelet supernatant ([Fig. 1H, I]). Confirming our hypothesis, we could show that C5a-conditioned platelet supernatant inhibited endothelial tube formation, which could be blocked by administering the C5aR1 antagonist PMX205 ([Fig. 1H]). Interestingly, a neutralizing antibody against CXCL4 did not have an additive effect on top of PMX205 in reversing the effect of C5a-conditioned platelet supernatant ([Fig. 1H]). As the collagen pathway of platelet activation has gained recent attention,[30] we excluded involvement of glycoprotein VI in C5a-mediated release of CXCL4 from platelets ([Supplementary Fig. S5]). Together, we demonstrate that C5a-mediated release of CXCL4 is one mechanism how platelets modulate vessel growth. Here, we identified a novel mechanism for inhibition of neovascularization via CXCL4 secretion induced by platelet C5aR1. Targeting the complement system may offer novel approaches to treat patients with diseases featuring tissue ischemia and inflammation. These diseases are largely caused by atherosclerosis.[2] Interestingly, the anaphylatoxin receptors C5aR1 and C3aR are expressed in atherosclerotic plaques.[31] Importantly, in patients with atherosclerosis, expression of C3aR and C5aR shows a significant correlation with platelet activation markers.[25] [27] [32] In a mouse model, anti-C5aR1 compounds have already proven effective in enhancing revascularization.[14] The potential for the treatment of human patients, however, will first have to be evaluated in future clinical studies. Recently, the C5a–C5aR1 axis was suggested to be an important marker of inflammation associated with COVID-19.[33] Interestingly, the platelet C5aR1 has been recognized as an important player in the complex pathophysiology of SARS-CoV-2 infection.[34] Thus, evaluating the C5aR1–CXCL4 axis in this context may improve our understanding of disease mechanisms and provide new treatment options for COVID-19 and other diseases featuring thromboinflammation and complement involvement. Complement-active drugs are available and already in use.[35] For instance, eculizumab, which inhibits the cleavage of C5 by the C5 convertase into C5a, has been clinically established for the treatment of aHUS (atypical hemolytic uremic syndrome) and for PNH (paroxysmal nocturnal hemoglobinuria).[36] Indeed, several novel pharmacological approaches have recently been introduced, i.e., Pegcetacoplan[37] or Avacopan,[38] a C5aR1 inhibitor, to the clinic. In light of our findings, implications of treatment regimen for vascular remodeling and regeneration should be assessed in future studies and should be taken into account when designing trials on complement therapeutics. In conclusion, understanding the crosstalk of platelets with the complement system is important to apprehend the exact role of this interplay for platelet and complement activation and resulting diseases featuring thromboinflammation. Accepted Manuscript online: 18 August 2023 Article published online: 19 October 2023 © 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

Platelet activation is a central driver of myocardial infarction. The complement anaphylatoxin C5a is abundantly generated during myocardial infarction, and its receptor C5aR1 is highly expressed on platelets. However, the functional role of platelet C5aR1 in myocardial infarction (MI) remains unknown. Here, we show that platelet&[ndash]expressed C5aR1 critically amplifies thromboinflammatory cardiac injury by promoting platelet&[ndash]mediated neutrophil activation after MI. Platelet&[ndash]specific C5aR1 deletion reduced infarct size, fibrosis, and adverse remodeling while enhancing neovascularization and preserving cardiac function. Mechanistically, cell&[ndash]specific C5aR1 deletion markedly reduced myocardial platelet&[ndash]neutrophil accumulation and neutrophil extracellular trap (NET) formation, while circulating platelet&[ndash]neutrophil complexes were increased. Ex vivo, C5a&[ndash]stimulated platelets robustly induced NET release from neutrophils in a platelet C5aR1&[ndash] and CXCL4&[ndash]dependent manner, whereas platelets lacking C5aR1 failed to trigger NET formation. Pharmacological C5aR1 inhibition with PMX205 phenocopied the genetic platelet&[ndash]specific deletion, resulting in comparable cardioprotection. Together, these findings identify platelet C5aR1 as a druggable target of platelet&[ndash]neutrophil&[ndash]NET signaling that exacerbates myocardial injury and limits reparative healing after MI, highlighting platelet C5aR1 as a potential therapeutic approach to restrain thromboinflammation.