The first major finding of this study is that the PPIs network of DEPs and DEPs/genes of death from IS versus stroke survival show a high connectivity and two interconnected subnetworks, namely, a first which is centered around immune genes and a second which is centered around hemostasis-coagulation genes. All query DEPs/genes participated in these networks except INADL. The backbone of this network consists of DEPs/genes which contribute to both subnetworks, namely IL6, TNF, TGFB1, VCAM, and SELE as authorities in the immune subnetwork, and ALB, SERPINE1, and VWF in the hemostasis subnetwork. Moreover, ALB ad VWF are important switches that connect both subnetworks because these DEPs show many significant interactions with proteins in both communities. Therefore, it appears that death due to IS predicted by an integrated response in a network which comprises strongly interconnected immune and hemostasis subnetworks. This indicates that both the immune response (as indicated by increased IL-6, IL-10, TNF-α, VCAM-1, SELE, and CRP but lowered TGF-β1 and ALB) and the activated hemostasis, thrombosis and coagulation pathways (as indicated by increased F8, VWF, and FBG but lowered PROC, PROS1, SERPINC1 and ALB levels) are intertwined phenomena leading to death from IS. The strong interconnections among the immune and hemostasis subnetworks have probably an evolutionary origin [47]. We will now discuss both the significant biological functions, paths, molecular complexes, and transcriptional factors enriched in the networks.

The second major finding of this study is that the most significant paths and functions enriched in the networks of death due to IS were the inflammatory and NF-κB signaling pathways, NFKB1/RELA transcriptional factors, TNF-α and IL-17 signaling, and the TLR, TGF-β1, nucleotide-binding and oligomerization domain (NOD) and JAK-STAT pathways. These results indicate that altered expression of these pathways, and transcriptional factors, may lead to death due to IS.

There is now evidence that in IS, NF-κB may be a key factor for the transcriptional induction of cellular adhesion molecules, including SELE and VCAM-1, proinflammatory cytokines, including IL-6 and TNF-α, CRP, and FTH1, growth factors including TGF-β1, and coagulation factors including F8, PAI1, uPA, tissue factor and fibronectin [47,48]. Transient focal cerebral ischemia is accompanied by NF-κB activation in the nuclei of striatal and cortical neurons in the ischemic hemisphere [49]. It is important to note that ischemia-induced astroglial NF-κB may have neurodegenerative effects, whereas constitutive neuronal nuclear factor kappa B subunit 1 (NFΚB1) may have neuroprotective effects [50].

In animal models of middle cerebral artery occlusion, activation of astroglial NF-κB is downstream of TLR2 activation and a deficiency in TLR2/4 reduces the neurodeficit and IS size [50]. Moreover, models of focal cerebral ischemia show that increased NF-κB is associated with IS severity and size, and that there may be a causative association between both factors, whereby induced reductions in NF-κB levels are accompanied with lowered IS size and neurodeficit [48,51]. Inhibition of NF-κB (through administration of caffeic acid phenethyl ester, mycophenolate, atorvastatin, and cephalexin may attenuate oxidative stress and neurodegeneration due to middle cerebral artery occlusion [52]. Moreover, the promoter variant (rs11940017, -1727 C>T) of the NFKB1 gene may affect IS susceptibility in the Korean population [53].

Furthermore, there is evidence that in IS the deleterious effects of increased NF-κB are associated with RELA activation [48], which is the second transcription factor which is highly significantly enriched in our PPI network. RELA plays a key role in the activation of NF-κB and the translocation of the latter to the nucleoplasm, while the RELA-NFKB1 complex mediates gene expression of many cytokines (UniPro UniProtKB—Q04206 (TF65_HUMAN) (uniprot.org (accessed on 19 September 2021). Moreover, the apoptotic responses caused by NF-κB are dictated by acetylation of RELA in Lys310, and RELA, but not p50, knockouts show a decreased infarct size [54,55].

The TLR2/4 complexes play a key role in IS with increased expression of both receptors being associated with the inflammatory response and progression of infarct volume and ischemic damage [56]. Both TLR levels (as assessed in peripheral blood monocytes using flow cytometry) are associated with increased plasma levels of IL-6, TNF-α, VCAM-1, and the clinical outcome and lesion volume [57]. It is interesting to note that in IS patients, serum levels of fibronectin, heat shock protein (HSP)60 and HSP70 are endogenous ligands leading to TLR2/4 activation [57]. Likewise, the inflammatory response during stroke is attenuated by blockade of the TLR2/4 complexes and cellular fibronectin. In TLR4 knockout mice, smaller stroke sizes and better neurocognitive functions are observed [58,59]. All TLR pathways activate NF-κB [56,60], indicating that activation of the TLR2/4 pathways and NF-κB signaling are interrelated phenomena. This suggests that the cumulative effects of both TLR2/4 and NF-κB signaling pathways may confer risk towards death due to IS. There are also reports that TLR4 genetic variants (e.g., TLR4-119A allele) are associated with an increased risk of IS [61]. Moreover, it is important to note that vitamin D3, which in our studies was associated with death due to IS, attenuates TLR2/4 expression and signaling, thereby preventing the translocation of p65 to the nucleus, and consequently, attenuating inflammatory responses [56].

Soon after IS onset, microglia cells and circulating monocytes are activated and consequently, increased release of TNF-α and IL-6 may be detected in the brain, cerebrospinal fluid, and bloodstream [8]. Increased TNF-α levels in CSF and blood are significantly associated with stroke outcome assessments including the Barthel Index and Scandinavian Stroke Scale (SSS) [62]. The plasma levels of TNF-α, which are observed in IS patients, may impact neuronal function and viability, even leading to neuronal death [63]. A recent meta-analysis shows that TNF-α is increased in Asian and Caucasian IS patients (overall SMD = 0.65, 95% CI = 0.29, 1.01), and that the TNF-α-308 G > A (rs1800629) genetic variant is associated with increased risk of IS [64]. IS is accompanied by an increased expression of IL-17 and IL-17RC in the serum in association with increased IL-6 and granulocyte-monocyte colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) [65]. It is thought that the IL-17-IL-17R pathway plays a key role in secondary poststroke damage via (a) effects on neutrophil infiltration in cerebral parenchyma, thereby promoting damage and thrombosis; (b) effects on tight junctions with blood-brain-barrier (BBB) breakdown and induction of neuronal apoptosis, and (c) synergistic effects with IL-6 [66]. Moreover, a polymorphism of the IL17RC gene is associated with a poorer prognosis of IS [65].

There are now some reports that in experimental models of IS, the JAK2-STAT3 pathway is activated and contributes to neuronal damage [67,68,69,70]. Middle cerebral artery occlusion is accompanied by increased concentrations of TNF-α, high mobility group box B1 (HMGB1), and phosphorylated JAK2/JAK2 and STAT3/STAT3 in the brain. Blocking JAK2, STAT3 and the JAK2/STAT3 signaling pathway significantly reduces IS-associated inflammatory responses [71]. Additionally, Wang and coworkers reported that in an IS model attenuation of the JAK-STAT pathway reduces apoptosis in neuronal cells and loss of neurological functions [72]. Cytokines including IL-6 and IL-10 activate Janus kinases causing translocation of STAT to the nucleus which, in turn, leads to changes in expression of a great number of immune and wound-healing genes [73].

NOD-like receptors are a family of cytoplasmic receptors which sense bacterial motifs and danger signals (including uric acid and ATP) and trigger an innate immune response by activating NF-κB and mitogen-activated protein kinase (MAPK) [74,75]. In the middle cerebral artery-occlusion model, the expression of NOD2 is significantly elevated, and ablation of the NOD2 gene reduces stroke size and inflammation as indicated by lowered expression of NF-κB, MAPK, IL-6, and TNF-α [75].

Our REACTOME path classifications revealed that our DEP network was highly significantly associated with IL-10, IL-4, and IL-13 pathways. We discussed before that increased activity of the IL-10 pathway in nonsurvivors may be part of a compensatory immune-regulatory mechanism which counterbalances an overzealous inflammatory response [8]. Elevated levels of IL-10 are often associated with a better functional outcome. For example, subjects with reduced plasma IL-10 in the first hours after IS have an increased risk of developing neurological symptoms two days later [76]. Some IL10 genetic variants are associated with increased risk of IS, including the IL-10 rs1800896 variant [77]. IL-4 also has anti-inflammatory effects and drives macrophages from a M1 proinflammatory phenotype to a M2 phenotype with homeostatic, repair and immune regulatory properties, explaining how IL-4 administration may improve recovery in a mouse stroke model [78]. IL4 KO mice show a worsened neurological outcome and increased brain damage following cerebral ischemia [79]. In IS, intracerebral delivery of IL-13, an anti-inflammatory cytokine, drives macrophages and microglia into the alternative activation state [80]. Nevertheless, high levels of anti-inflammatory cytokines such as IL-10 may sometimes be accompanied by a less favorable outcome, including increased risk of infections [81].

One of the major bottlenecks in the PPI network is TGF-β. This cytokine is elevated in the brain following IS and may exert anti-inflammatory, antiapoptotic and neuroprotective effects, thereby improving repair mechanisms and favoring nerve regeneration [82,83,84]. There are now some publications showing that genetic TGFB1 variants are associated with IS [85]. Moreover, the effects of TGF-β1 on multiple target genes (including ECM) are mediated via the SMAD signaling pathway [86,87], which was significantly enriched in the hemostasis subnetwork list, albeit SMAD/MAD did not make the top five. Importantly, the TGF-β/SMAD2/3 signaling pathway has neuroprotective properties, for example by elevating Bcl-2 and lowering caspase-3 expression and decreasing microglial activation via NF-κB inhibition [88,89].

In a rat model of cerebral ischemia/reperfusion, SMAD3 administration may downregulate inflammatory and proapoptotic genes, suggesting that the TGF-β/SMAD pathway is a possible drug target [88]. All in all, the lowered levels of TGF-β1 in IS nonsurvivors versus survivors may contribute to the pathways leading to death due to IS.

VCAM-1, another hotspot of the backbone of the PPI network, plays a critical role in the inflammatory response following IS, for example through adhesion of leukocytes to endothelial cells and transendothelial migration via interactions with integrin subunits [90,91]. Due to a fenestrated endothelium, the choroid plexus is the entry site for patrolling lymphocytes—mostly CD4⁺ central memory T cells—in the healthy brain [92]. To promote leukocyte trafficking, the choroid plexus epithelium constitutively expresses intercellular adhesion molecule (ICAM)-1 and VCAM-1, which, together with the mucosal vascular addressin cell-adhesion molecule, are upregulated in stroke [49]. In IS, intracranial VCAM-1 levels are associated with infarct and edema size [93]. Cell-adhesion molecules (CAM) KO models show a reduced infarct size, and administration of anti-CAM antibodies may decrease infarct size [94]. Increased levels of VCAM-1 and IL-6 predict a new vascular incident, and thus may increase risk of death due to IS [95].

The second most important subnetwork in death due to IS was centered around hemostasis, thrombosis and coagulation genes, which were enriched especially in fibrin clotting or the clotting cascade, TGF-β binding, the uPA and uPAR-mediated (PID-uPA-uPAR) signaling pathways, ECM proteoglycans, integrin cell-surface interactions and platelet activation, aggregation and degranulation. Although abnormalities in the hemostasis axis are not a common cause of IS [96,97,98], perturbations are seen following stroke onset, and these may aid as biomarkers in diagnosis and severity of IS and prediction of the treatment response. This is further substantiated by our findings that a response to wounding is over-represented in the hemostasis subnetwork. It could be argued that a combined deficiency in the PROC, PROS1 and SERPINC1 proteins in the hemostasis network may contribute to the response to wounding. Nevertheless, deficits in these proteins are not a common cause of IS [99], although our studies suggest that they may contribute to death due to IS.

One major hotspot of the backbone of the PPII network and a major connector of both subnetworks is VWF, which shows many interactions with genes from both the immune and hemostasis networks. Such data agree with the view that VWF mediates the crosstalk between immune cells and hemostasis mechanisms and contributes to inflammation, including vascular inflammation [100,101]. The VWB factor is released during the rupture of the endothelial layer of the vessels, whereby the consequent exposure of collagen to platelets leads to clot formation [102]. Consequently, endothelial cells release VWF, P-selectin, SELE, and inflammatory mediators [103]. VWF promotes platelet adhesion to the damaged site by forming a molecular bridge between the subendothelial collagen matrix and the platelet surface receptor complex GPIb-IX-V [101]. VWF levels are increased in IS patients and are associated with the cardioembolic and large-vessel disease subtypes [104]. Additionally, VWF levels are associated with severity of arterial thrombus formation and poor functional outcomes [105,106]. Furthermore, VWF may function as a biomarker of the response to thrombolytic or endovascular treatment in IS patients [106,107]. Variations in the VWF gene (Sma I) may be associated with an increased risk of IS [108]. Preclinical and clinical studies using VWF antagonists and combining the latter with tPA could prevent microvascular thrombus formation, thereby attenuating the progression of IS [106]. Additionally, other authors proposed that targeting VWF-mediated platelet activation and adhesion is a new drug target to treat IS [109].

Fibrinogen (FBG) is one of the hemostasis biomarkers with an essential role in the thrombosis process because it is related to platelet aggregation after injury and inflammation [110,111]. FBG is released from the liver into the bloodstream and is cleaved by thrombin at the damaged site, resulting in fibrin formation. Fibrin is one of the main constituents of blood clots and provides remarkable biochemical and mechanical stability [111]. FBG and CRP levels can independently predict the risk of early death in middle-aged IS patients, emphasizing the role of inflammation and coagulation in the evolution of IS. For each 10 mg/dL increase in FBG levels there was a 18% higher risk of dying, while for 1 mg/L increase in CRP the additive risk was 18%. FBG levels >490 mg/dL and CRP levels >18 mg/L were the optimal points that discriminated those who died from survivors [112]. Another study showed that hyperfibrinogenemia, defined as a plasma FBG concentration >350 mg/dL, predicts the long-term risk of death in IS patients [113]. Moreover, FBG has been used to evaluate the long-term outcome and the size of the clot burden in patients after IS [114,115]. Furthermore, an increase in FBG levels is observed after IS and is associated with infarct size and outcome [116,117,118]. Elevated hemostatic markers after acute IS stroke identify patients with increased risk for mortality independently of IS severity or stroke type [119]. Higher concentrations of fibrinopeptide A, b-thromboglobulin, prothrombin fragments 1 and 2, thrombin-antithrombin complexes, platelet factor four and VWB factor have all been associated with a worse clinical course in IS and increased mortality [119,120,121]. In addition, patients with high PAI-1 levels are less likely to achieve recanalization after receiving tPA, and experience poorer outcomes [122].

Another possible major pathway which was found using enrichment analysis is the uPA-uPAR pathway. This pathway is activated in the periphery of growth cones of the injured neurons in IS brains, where it induces repair mechanisms [123]. The binding of uPA to its receptor promotes β1-integrin recruitment to the plasma membrane with consequent activation of “small Rho GTPase Rac1 and Rac1-induced axonal regeneration” [123]. Since this process is regulated by the low-density lipoprotein receptor (LRP1), it was proposed that the uPA-uPAR-LRP1 axis is another possible drug target in IS and by inference may be a new drug target to prevent death due to IS. Moreover, recombinant uPA may protect the integrity of pre- and post-synaptic terminals and astrocytic elongations against the detrimental effects of IS [124]. Unfortunately, uPA also increases the production of reactive oxygen species and NADH oxidases (Nox1 and Nox4) and enhances superoxide production by neutrophils, findings which suggest that reducing uPA functions may be beneficial [125].

Our enrichment and annotation analyses revealed that the hemostasis subnetwork genes were significantly associated with ECM proteoglycans and integrin cell surface interactions, which are components of the BBB. These functions are impacted by IS, the consequent vasogenic edema, early angiogenesis, inflammation, and reperfusion injury [126,127,128,129]. These IS-induced processes strongly impact the BBB, resulting in breakdown of the tight junctions and paracellular barrier and increased BBB permeability, which may further worsen vasogenic edema and neuroinflammation through increased entry of inflammatory mediators and activated T cells into the brain [130,131]. As observed in our paper and previously, such processes may lead to increased mortality due to IS. Moreover, proteoglycans, integrins and fibronectin are key ECM adhesion receptors which integrate external and internal signals, and regulate global cellular processes, cellular signaling, and cell growth, proliferation, migration, and survival [126]. The ECM regulates the tight junctions, neurons, astrocytes, and the vasculature and plays a key role in wound healing, cell homeostasis, and tissue and neuronal regeneration [132]. Importantly, the proteoglycan-integrin-ECM complex and related BBB functions including tight junctions are now considered to be new drug targets in the treatment of IS, for example by targeting integrins and MMPs [128,129].

Our network analyses showed that not only the VWF (as discussed in the previous section) but also ALB is a major controller gene between both subnetworks of death due to IS and, consequently, that ALB may also be an important drug target. ALB has anti-inflammatory, antioxidant and neuroprotective capacities and thus plays a role in the immune subnetwork, although a second clustering analysis performed in our study allocated ALB into the hemostasis network. ALB also shows antiplatelet aggregability and is a carrier for two anticlotting compounds, namely heparin cofactor and SERPINC1 [133,134]. Subjects with low ALB display increased primary hemostasis, and enhanced platelet aggregation and clot formation, explaining how low ALB levels observed in nonsurvivor IS patients may increase the vulnerability to develop venous thromboembolism [133]. Importantly, reduced baseline ALB levels reflect a chronic inflammatory state, which is already present weeks before the IS (because the half-life of ALB is 21 days). All in all, lowered ALB levels may predispose patients towards interrelated aberrations in both the immune and hemostasis subnetworks, thereby, increasing risk of death due to IS.

In the enlarged giant network, VEGFA is another switch between the immune and hemostasis communalities. This gene is a member of the VEGF family and the protein acts as a mitogen, thereby activating endothelial cells and regulating neuronal cells [135]. VEGFA is implicated in the pathophysiology of atherosclerosis, and in stroke, VEGFA is elevated in the ischemic penumbra and mediates vessel and neuron repair and remote plasticity in ischemic brain regions [135]. Treatment with VEGFA coupled with stem cells may show therapeutic effects in animal stroke models. Moreover, there is an association between IS and different genetic variants of VEGF genes (e.g., -2578C>A and 936C>T variants) [136].

There are many more pathways and functions which link inflammation and coagulation, including TLRs (impact coagulation, platelet activity and aggregation, and thrombosis) [137], NOD2 signaling (enhances platelet activation and thrombosis) [138], TNF-α signaling (activates coagulation, fibrinolysis, neutrophil degranulation, and the release of secretory phospholipase A2, predominantly mediated by the p55 TNFR) [139], platelets (platelet degranulation causes increased cIMT and thus increased inflammatory atherosclerotic processes) [47,140], protease-activated receptors and thrombin, complement, neutrophil extracellular traps, and microparticles [141].

Moreover, our TTRUST enrichment analysis shows that not only NF-κB and RELA (discussed in the previous sections), but also SP1 is a major regulator of both the immune and hemostasis subnetworks. NF-κB is not only critically involved in modulating inflammatory processes but also in thrombotic responses [47]. Thus, NF-κB activation increases the thrombogenic potential, activates platelets, and promotes coagulation, microthrombi formation, immunothrombosis and thromboinflammatory disease [47]. In addition, F8, PAI1, uPA and F3 are target genes of NF-κB. SP1 or transcription factor SP1 (or specificity protein 1) regulates the expression of housekeeping genes which are involved in immune responses, apoptosis, chromatine remodelling, cell differentiation and cell growth [142]. This transcription factor regulates different subnetworks including the immune response (including TNF-α), MAPK and JAK-STAT pathways, platelet activation, ECM, and blood vessels [142].

Our REACTOME pathway and PANTHER process enrichment analyses performed on all markers including miRNA levels revealed the strong impact of translation, RNA, metabolism of mRNA, RNA splicing, and nonsense-mediated decay on the interactome of death due to IS, indicating that dysfunctions in postischemic translation regulation are involved. During ischemia and brain cell injury, translation may arrest due to lack of ATP, and changes in translational regulation at the mRNA level and the ribosomic network may develop, which may cause a multitude of aberrations in the downstream PPI and metabolic network modules [143,144]. The nonsense-mediated decay pathway degrades transcripts with a premature stop codon, thereby reducing errors in gene expression. Failures in this surveillance system may be accompanied by increased synthesis of abnormal, including toxic, proteins [145].

It is interesting to note that both miRNAs, which are altered in death due to IS, regulate immune functions, with hsa-mir-10a regulating IL-8, IL-6, TNF-α, GATA6 apoptotic pathways, and hsa-mir-34b regulating innate immunity (target mining in www.mirbase.org (accessed on 19 September 2021)). Our study also showed that the expression of the most important miRNA, which is over-represented in death due to IS (hsa-mir-16-5p), was found to be increased in IS and to regulate NF-κB transcription [146,147]. Moreover, also the second-most important miRNA (hsa-mir-26b-5p) is part of a long noncoding RNA (LncRNA)-miRNA-mRNA network of IS and inhibits NF-κB expression [148,149]. Some miRNAs are associated with IS risk factors including hypertension (miR-155), atherosclerosis (miR-21, miR-126, miR-143), atrial fibrillation (miR-26), diabetes mellitus (miR-124a, miR-126), and dyslipidemia (miR-33, miR-122), while some miRNA antagonists have the potential to act as neuroprotective molecules [150]. Elevated expression of miR-15a, miR-16, and miR-17-5p in the serum is strongly associated with IS [151].