Background: Most human disease definitions, except for rare and communicable diseases, are based on symptoms in specific organs, not on causal molecular mechanisms. This limits treatments to imprecise symptomatic approaches with high numbers needed to treat. Systems medicine, instead, has a holistic approach and defines diseases in an organ‐agnostic manner on the basis of associated risk genes, their encoded proteins, and protein–protein interactions. Dysregulation of such disease modules is best corrected by multitarget, synergistic network pharmacology. Here we test this principle in acute ischemic stroke, a highly unmet medical indication without any approved neuroprotective drug so far. Methods: We extend 3 validated risk genes, neuronal nitric oxide synthase ( NOS1 ), NADPH oxidase 5 ( NOX5 ), and soluble guanylate cyclase ( sGC ), to a single disease module. For preclinical validation, we used C57/Bl6 mice and humanized NOX5 –knock‐in mice because NOX5 is not present in the mouse genome despite playing a key role in early stroke. Because up to 70% of patients with stroke have diabetes or prediabetes as an aggravating comorbidity, we also induced diabetes in these mice to model the increased clinical risk for hemorrhagic transformation. Results: We found that a triple‐drug combination of a NOX inhibitor, a nitric oxide synthase inhibitor, and an sGC activator reduced infarct size and, in diabetic animals, also prevented hemorrhagic transformation. Reducing each individual compound dose to subthreshold levels still resulted in full protection when combined, typical for supra‐additive network pharmacology. To examine clinical safety, 3 drugs, either marketed for sGC or repurposed for nitric oxide synthase and NADPH oxidase, were administered to healthy volunteers in a phase I trial. Conclusions: Our data establish that a mechanism‐based network pharmacology approach is effective and clinically safe, warranting a currently ongoing first‐in‐class neuroprotective phase II interventional trial. Registration: URL: https://clinicaltrials.gov/study/NCT05762146?term=repo‐stroke&rank=1 ; Unique Identifier: NCT05762146.

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.

Background: Lysophosphatidic acid (LPA) is a pleiotropic lipid messenger that addresses at least six specific G-protein coupled receptors. Accumulating evidence indicates a significant involvement of LPA in immune cell regulation as well as Schwann cell physiology, with potential relevance for the pathophysiology of peripheral neuroinflammation. However, the role of LPA signaling in inflammatory neuropathies has remained completely undefined. Given the broad expression of LPA receptors on both Schwann cells and cells of the innate and adaptive immune system, we hypothesized that inhibition of LPA signaling may ameliorate the course of disease in experimental autoimmune neuritis (EAN). Methods: We induced active EAN by inoculation of myelin protein 2 peptide (P2_55–78) in female Lewis rats. Animals received the orally available LPA receptor antagonist AM095, specifically targeting the LPA₁ receptor subtype. AM095 was administered daily via oral gavage in a therapeutic regimen from 10 until 28 days post-immunization (dpi). Analyses were based on clinical testing, hemogram profiles, immunohistochemistry and morphometric assessment of myelination. Results: Lewis rats treated with AM095 displayed a significant improvement in clinical scores, most notably during the remission phase. Cellular infiltration of sciatic nerve was only discretely affected by AM095. Hemogram profiles indicated no impact on circulating leukocytes. However, sciatic nerve immunohistochemistry revealed a reduction in the number of Schwann cells expressing the dedifferentiation marker Sox2 paralleled by a corresponding increase in differentiating Sox10-positive Schwann cells. In line with this, morphometric analysis of sciatic nerve semi-thin sections identified a significant increase in large-caliber myelinated axons at 28 dpi. Myelin thickness was unaffected by AM095. Conclusion: Thus, LPA₁ signaling may present a novel therapeutic target for the treatment of inflammatory neuropathies, potentially affecting regenerative responses in the peripheral nerve by modulating Schwann cell differentiation.

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.

Nitric oxide (NO) is a key gasotransmitter that binds to soluble guanylate cyclase (sGC), thereby stimulating cGMP production. Under disease conditions, reactive oxygen species disrupt this signaling by either: (1) scavenging NO, or (2) promoting heme-free, NO-insensitive apo-sGC due to impaired heme incorporation or oxidative damage. To counteract these conditions, two pharmacological approaches have emerged: sGC stimulators, which allosterically enhance sGC sensitivity to low NO levels; and sGC activators, which directly activate apo-sGC (heme-free sGC) by binding to its empty heme pocket, inducing a conformation that mimics NO-bound sGC. Thus, sGC stimulators are thought to target only sGC. Instead, we here show both in vitro and in vivo that sGC stimulators also modulate apo-sGC and - together with sGC activators - exert additive effects on apo-sGC. This newly identified activity of sGC stimulators on apo-sGC appears to be redox-sensitive. Indeed, when inactive sGC (ferric heme sGC) is generated by the commonly used oxidant 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the stimulatory effect of sGC stimulators is abolished. The potential in vivo relevance of this mechanism was further demonstrated in a mouse model of ischemic stroke, wherein sGC is known to be predominantly NO-insensitive, yet sGC stimulators remain protective. Altogether, these findings challenge the current pharmacological paradigm of sGC modulation, revealing that both sGC and apo-sGC can be stimulated by sGC stimulators, whereas sGC activators remain specific to apo-sGC. This expanded understanding highlights the therapeutic potential of sGC stimulators, both as standalone treatments and in combination with sGC activators.

Nuclear erythroid 2-related factor 2 (Nrf2) and its downstream effector heme oxygenase 1 (HO-1) are commonly activated in response to cellular stresses. The elevated expression of HO-1 has been associated with markedly accelerated peripheral nerve regeneration. This study aimed to evaluate the impact of a naturally occurring dietary Nrf2/HO-1 activator—sulforaphane (SFN)—on regeneration in a murine sciatic nerve crush model. The beneficial safety profile of SFN has been thoroughly investigated and confirmed several times. Here, SFN was administered daily, starting immediately after C57BL/6 mice were subjected to sciatic nerve crush injury. Injured sciatic nerves were excised at various time points post injury for molecular, immunohistochemical and morphometric analyses. Moreover, functional assessment was performed by grip strength analysis and electrophysiology. Following SFN treatment, the early response to injury includes a modulation of autophagic pathways and marked upregulation of Nrf2/HO-1 expression. This enhancement of HO-1 expression was maintained throughout the regeneration phase and accompanied by a significant increase in repair Schwann cells. In these cells, elevated proliferation rates were observed. Significant improvements in grip strength test performance, nerve conduction velocity and remyelination were also noted following SFN treatment. Collectively, SFN modulates cytoprotective and autophagic pathways in the injured nerve, increasing the number of repair Schwann cells and contributing to effective nerve regeneration. Given the availability of SFN as a nutritional supplement, this compound might constitute a novel regenerative approach with broad patient accessibility and further studies on this topic are warranted.