Spleen tyrosine kinase (SYK) signals are implicated in cardio-cerebrovascular diseases

Abstract

Post-translational modifications regulate numerous biochemical reactions and functions through covalent attachment to proteins. Phosphorylation, acetylation and ubiquitination account for over 90% of all reported post-translational modifications. As one of the tyrosine protein kinases, spleen tyrosine kinase (SYK) plays crucial roles in many pathophysiological processes and affects the pathogenesis and progression of various diseases. SYK is expressed in tissues outside the hematopoietic system, especially the heart, and is involved in the progression of various cardio-cerebrovascular diseases, such as atherosclerosis, heart failure, diabetic cardiomyopathy, stroke and others. Knowledge on the role of SYK in the progress of cardio-cerebrovascular diseases is accumulating, and many related mechanisms have been discovered and validated. This review summarizes the role of SYK in the progression of various cardio-cerebrovascular diseases, and aims to provide a theoretical basis for future experimental and clinical research targeting SYK as a therapeutic option for these diseases.

Highlights

• •Besides traditional functions, SYK is also involved in the pathogenesis and progress of cardiovascular diseases.
• •BTK inhibitors may increase in the incidence of hypertension, atrial fibrillation and other adverse reactions.
• •Target SYK signals provide novel therapeutic option on prevention, slowdown and treatment of cardiovascular diseases.

1. Introduction

Protein post-translational modification (PTMs) [1] is an important area of protein chemistry research. PTMs, including phosphorylation [2], ubiquitination [[3], [4], [5], [6], [7]], acetylation [[8], [9], [10]] and etc., regulates numerous biochemical reactions and functions by covalently attaching to the protein. Phosphorylation [[11], [12], [13]] is one of the most common and important modifications, mainly occurs on three amino acids in the peptide chain [14], namely tyrosine [15] and serine and threonine residues. Generally, protein kinases could be divided into: serine/threonine (Ser/Thr) protein kinase, tyrosine (Tyr) protein kinase, histidine (His)/lysine (Lys)/arginine (Arg) protein kinase, cysteine (Cys) protein kinase and aspartate (Asp)/glutamate (Glu) protein kinase according to the difference of amino acid residues [16]. Among them, tyrosine protein kinases (TPK) [17,18] can be divided into three categories: ①receptor tyrosine kinases [19]; ②cytoplasmic tyrosine kinases, such as SYK family [20,21], Src family [[22], [23], [24], [25]], JAK family [26,27] and etc.; ③nuclear tyrosine kinases [28]. As a member of the non-receptor cytoplasmic tyrosine kinase family, the SYK family is mainly involved in the phosphorylation of protein amino acids. The SYK family includes two members [29]: spleen tyrosine kinase (SYK) and zeta-chain-associated protein of 70 kDa (ZAP70) [30,31].

SYK is a 72 kDa non-receptor tyrosine kinase containing two SRC homology 2 (SH2) structural domains and a C-terminal tyrosine kinase structural domain [32], which is highly expressed in hematopoietic cells and is a key molecule in signal transduction and differentiation, especially in B-cell signaling. SYK is known to play a crucial role in the hematological system, and is involved in the proto-oncogene of lymphocyte transformation and actively participates the pathogenesis of non-Hodgkin's lymphoma and chronic lymphocytic leukemia [33]. Meanwhile, SYK has been identified as a new therapeutic target of acute myeloid leukemia (AML). In addition, upregulated expression of SYK has been reported in sarcoid lymphoma, and both SYK and ZAP70 are upregulated in ALL [34,35].

SYK expression was also evidenced in other tissues, especially in myocardium, cardiac/cerebral vessels. SYK participates the pathology of atherosclerosis, stroke, heart failure, diabetic cardiomyopathy and other diseases. Several SYK inhibitors have been developed and shown initial effects on retarding the progression of pathologic diseases [36,37], Inhibition of SYK may slow down the progression of activated macrophage-mediated atherosclerosis [38]. Now, basic research on the role of SYK in cardiovascular and cerebrovascular-related diseases is still in its prototype, through understanding of the working mechanism of SYK might promote the development of clinical efficient SYK inhibitors or drugs in the treatment of cardiovascular and cerebrovascular diseases. This review comprehensively summarizes the role of the SYK signaling pathway in cardiovascular and cerebrovascular -related diseases, aiming to provide a theoretical basis for future experimental and clinical studies targeting SYK as a therapeutic option for patients with cardiovascular and cerebrovascular diseases.

2. Structure and function of SYK

In 1991, Taniguchi and colleagues detected the 40-kDa cytoplasmic protein tyrosine kinase [39,40] that had been discovered by Kobayashi and colleagues in 1990, but also a 72-kDa protein. The 40-kDa kinase was hydrolyzed by this newly discovered 72-kDa protein, which was found to be one of the members of the cytoplasmic tyrosine protein kinase [22] p72syk and named spleen tyrosine kinase (SYK). SYK belongs to the non-receptor type of cytoplasmic tyrosine kinases [41], is encoded by a gene located on human chromosome 9q22 [42] and comprises 629 amino acids [41]. Although SYK and ZAP70 belong to the same family [21], they differ in various respects [43]. ZAP70 is involved in the T-cell activation process [44], while SYK is considered the more important kinase, mainly regulating the signaling pathways of B cells, neutrophils and macrophages [[45], [46], [47]]. Generally, SYK binds to B-cell antigen receptors to activate itself, thereby exerting its molecular function [48].

At the molecular level, SYK has two adjacent SH2 domains and a tyrosine kinase domain at the C-terminus [41,49,50]. Generally, the helical coil-like region between two SH2s is called the A domain, and the segment between the kinase and SH2 is called the B domain. Reportedly, the kinase close to the C-terminal SH2 domain has a significant affinity for immunoreceptor tyrosine-based activation motifs (ITAMs), which are found in B-cell receptors (BCRs), T-cell receptors, natural killer-cell receptors and other cell-surface receptors; thus, the two SH2 domains and the A domain between them are considered to be ITAM-binding regions. The B domain, as a docking region [47], includes most of its phosphorylation sites, including Tyr290, Tyr317 (a binding site for c-Cbl), Tyr342 (a binding site for Vav1 and phospholipase C [PLC]-γ) and Tyr346 (a binding site for PLC-γ) Notably, the catalytic region contains Tyr620, which is a unique site in SYK (Fig. 1). Generally, phosphorylation can be classified into four categories, depending on the phosphorylated amino acid residues: O-linked, N-linked, phosphoanhydride and phosphorothioate phosphorylation. SYK phosphorylation belongs to the O-linked category. The phosphorylation process is essential for the activation and propagation of downstream signals [51].

Fig. 1.

The molecular structure of Spleen tyrosine kinase (SYK). This protein contains two adjacent Src homology 2 (SH2) domains at the N-terminus and a tyrosine kinase domain at the C-terminus. The helical coil-like region between two SH2s is called the A domain, and the segment between the kinase and SH2 is called the B domain. Two SH2 domain and the A domain are considered to be ITAM-binding regions. The B domain, as a docking region, includes most of its phosphorylation sites, including Tyr290, Tyr317 (a binding site for c-Cbl), Tyr342 (a binding site for Vav1 and PLC-γ), Tyr346 (a binding site for PLC-γ) and etc. Of note, the catalytic region contains Tyr620 which is a unique site in SYK.

SYK is expressed in multiple organs in mice, with the highest expression in spleen, heart, mammary gland, thymus and some lymphocyte lines [52]. SYK is also expressed in non-hematopoietic cells, including epithelial cells and vascular endothelial cells [53]. Information on the role of SYK in the field of cardiovascular diseases is accumulating. Studies show that SYK is involved in the pathogenesis and progress of hypertrophic cardiomyopathy (HCM), diabetic cardiomyopathy (DCM), coronary heart disease (CHD) and arterial thrombosis [54].

3. SYK in atherosclerosis

3.1. Platelet activation

AS is a chronic inflammatory process that mainly affects the walls of large- and medium-sized arteries, such as the aorta, carotid and coronary arteries [55]. When the endothelium is damaged, platelets can be activated by inflammatory triggers; the activated platelets then aggregate, adhere to each other and interact with the endothelium, contributing to endothelial activation, a critical initiation component of the atherosclerotic process and pathologic thrombosis [56,57]. SYK is involved in the platelet activation process via modulating the platelet glycoprotein VI (GPVI) signal [58]. GPVI has been regarded as a key signal [59] of platelet activation for decades. In pathological conditions (e.g. AS and vascular endothelial damage), a collagen-FCγ receptor (FCγR)-GPVI compound can be formed [[60], [61], [62]]. ITAMs are phosphorylated by Src family tyrosine kinase (SFKs), which are non-receptor tyrosine kinases that can transmit activation signals from platelet surfaces. The signal transmission of GPVI receptors mainly depends on SFKs [63,64]. The phosphorylated ITAM can then bind to the non-receptor tyrosine kinase SYK, form a collagen-FCγR-GPVI-SYK compound, activate several downstream key proteins (e.g. Bruton's tyrosine kinase [BTK] proteins and joint protein SLP76) and affect the activities of various enzymes (e.g. PLCγ2), thus promoting a series of couplet reactions, including calcium mobilization and the activation of protein kinase C [65]. These activated signals further promote platelet adherence to the endothelial injury site, eventually resulting in platelet aggregation and thrombosis [66,67]. SYK is also involved in the C-type lectin-like receptor 2 (CLEC2)-related signaling pathway on the platelet membrane. Similar to the above process (Fig. 2), after the ITAM motif of the CLEC2 receptor has been phosphorylated, SYK can bind to it, become phosphorylated and activate downstream key proteins, actively participating in the process of platelet activation and aggregation [68].

Fig. 2.

Diagram of the collagen-FCγR-GPVI-SYK pathway under high-shear stress. In the pathological conditions (atherosclerosis and vascular endothelial damage), collagen-FCγR-GPVI compound could be formed. ITAM will be phosphorylated by Src family tyrosine kinase（SFKs，a non-receptor tyrosine kinase which can transmit activation signals from platelet surfaces. The signal transmission of GPVI receptors mainly depends on SFKs）.The phosphorylated ITAM can then bind to the non-receptor tyrosine kinase SYK, forming the collagen-FCγR-GPVI-SYK compound and activate several downstream key proteins (BTK proteins, joint protein SLP76, and so on) and effecting enzymes (Phospholipase Cγ2 (PLCγ2), and so on), thus promotes a series of couplet reactions including calcium mobilization and the activation of PKC. These activated signals further promote platelets adherence to the endothelial injury site, eventually resulting in platelet aggregation and thrombosis. SYK is also involved in the C-type lectin-like receptor 2 (CLEC2)-related signaling pathway on the platelet membrane. Similar to the above process, after phosphorylation on the ITAM motif of the CLEC2 receptor, SYK could bind to it and be phosphorylated, then activate downstream key proteins, actively participate in the process of platelet activation and aggregation.

SYK hyperphosphorylation, which is caused by different factors, is believed to be associated with the pathogenesis of various cardio-cerebrovascular diseases. For example, hyperglycemia has been shown to increase the phosphorylation level of SYK four-fold. Phosphorylated SYK can mediate the activation of Plcγ2, which plays an important role in early collagen-induced platelet aggregation [69]. Chen et al. found that galectin-3, a β-galactoside-binding lectin, binds to and activates Dectin-1, then phosphorylates SYK, thus increasing Ca2+ influx, activating protein kinase C and promoting reactive oxygen species (ROS) production to enhance platelet hyperreactivity [70]. The aforementioned studies indicate that high phosphorylation levels of SYK are often associated with abnormal platelet activation and contribute to the hypercoagulable state of blood and thrombosis. Accordingly, reducing the phosphorylation level of SYK might have potential as a therapeutic tool for targeting the blood hypercoagulation process in various cardio-cerebrovascular diseases.

3.2. Lipid metabolism and calcification

Phosphorylation of SYK has been shown to be involved in the formation of foam cells, a characteristic pathological cell type of AS [71]. Guo et al. initially discovered that overexpression of SYK further increased the blood levels of total cholesterol, low-density and high-density lipoprotein cholesterol, and reduced superoxide dismutase levels, aggravating AS in ApoE^−/− mice fed a high-fat diet [72]. The evidence suggests that inhibiting SYK might also promote metabolic redistribution, upregulate cellular hydrogen sulfide production, and reduce oxidative phosphorylation and ROS levels [73]. Therefore, SYK may promote AS by increasing oxidative stress and cellular lipid accumulation. Additionally, studies show that very-low-density lipoprotein cholesterol is enriched in apolipoprotein C3, which can bind to Toll-like receptors 2/4 and upregulate Lyn (one of several Src-family tyrosine kinases in immune cells), thereby accelerating phosphorylation of SYK by Lyn, mediating calcium inward flow and activating nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammatory vesicles, and ultimately promoting AS [74].

3.3. Myocardial infarction

The collagen-FCγR-GPVI-SYK axis is the critical pathway involved in the formation of atherosclerotic plaque and arterial thrombosis [[75], [76], [77]]. Knockdown of the platelet collagen receptor FCγR/GPVI chain was shown to virtually entirely eradicate platelet thrombi formed after endothelial damage [78]. Furthermore, Takaya et al. showed that SYK activity was significantly downregulated and infarct size was significantly reduced after 30 min of occlusion and 24 h of reperfusion of the left anterior descending artery in FCγR^−/− mice. These findings suggest that downregulating the FCγR-GPVI-SYK pathway might be a promising option to attenuate myocardial insult post myocardial infarction [78].

Mechanistically, high-shear stress in the atherosclerotic vascular region can enhance the phosphorylation level of SYK. High phosphorylation levels of SYK may activate blood platelets and enhance thrombosis formation, which would in turn increase the risk of myocardial infarction. Speich and colleagues showed that pretreatment with SYK inhibitors not only reduced shear stress-induced platelet aggregation and SYK phosphorylation in isolated platelets, but also diminished, by up to 50%, the platelet-mediated thrombosis formation that occurred when whole blood was perfused over type-III collagen [58]. In a clinical study, Liang and colleagues found that SYK expression on peripheral blood monocytes was significantly higher in patients with non-ST segment elevation acute coronary syndrome, ST segment elevation, acute myocardial infarction or stable angina pectoris compared with that in control patients with non-congenital heart disease. Furthermore, the SYK expression level was positively related to the disease severity of CHD [54]. Such experimental and clinical studies highlight the role of upregulated SYK and its phosphorylation level in the pathogenesis of myocardial infarction.

4. SYK in stroke

Strokes can be divided into two main categories: ischemic [79] and hemorrhagic [80]. Platelet overactivation can enhance thrombus formation and block the cerebral blood vessels, causing ischemic stroke (IS) [81]. Considered a global public health problem, more than 25% of IS can be attributed to AS [72,82]. Ye and colleagues constructed an IS model in C57 mice [83]. They found that Dectin-1 receptors bound to necrotic substances after a stroke and promoted the activation of SYK, enhancing the expression of inflammatory genes and resulting in further brain tissue damage. The expression levels of SYK and phosphorylated (p)-SYK were significantly upregulated in this model. Piceatannol, an SYK inhibitor, reversed the upregulated expression of SYK and p-SYK and reduced the size of cerebral infarction [83,84]. Previous studies have confirmed that microRNAs (miRNAs) modulate SYK and are associated with stroke and AS [85,86]. Overexpressed miR-135b and miR-377 (which had the best combining capacity) bound to the 3′-untranslated region (UTR) of SYK mRNA, thereby downregulating the expression of SYK and attenuating its AS-promoting effect by decreasing CD68 numbers [72]. Additionally, Huang et al. found that the expression of miR-129-2-3p was negatively correlated with the occurrence of IS through negatively regulating the SYK 3′-UTR site [87].

Subarachnoid hemorrhage (SAH) is a severe subtype of hemorrhagic stroke, accounting for 5% of strokes [88,89]. SYK is involved in this injury via the macrophage-inducible C-type lectin (Mincle), which is expressed in both glial cells and neurons of SAH rats [90]. Mincle binds to SAP130 ligand (a subunit of histone deacetylase) released by necrotic cells, promotes SYK phosphorylation and activates inflammatory pathways, resulting in increased accumulation of inflammatory factors. He et al. found that the expression of Mincle, SYK and p-SYK was increased at 12 h and peaked at 24 h in the SAH rat model. Intervention with SYK inhibitor piceatannol reduced brain edema at 24 h after the SAH. Thus, targeting Mincle and SYK might also serve as a potential therapeutic option in the setting of hemorrhagic brain insult [91]. Thus, SYK is actively involved in the pathogenesis of IS.

5. SYK in cardiomyopathy

5.1. Diabetic cardiomyopathy

Persistent diabetes may induce cardiac insufficiency, and the pathological mechanisms of diabetic cardiomyopathy (DCM) include alterations in intracellular ion homeostasis (K⁺, Na⁺, Ca²⁺), disturbances in intracellular energy metabolism (mitochondrial damage), increased ROS production, upregulated accumulation of advanced glycation end products, and activation of signaling targets such as the nuclear factor (NF)-κB pathway and the renin–angiotensin system [92]. Diabetes mellitus damages cardiomyocytes through the above pathological processes, and can result in pathological myocardial remodeling and dysfunction, as well as heart failure (HF) [[93], [94], [95], [96]]. Through construction of a protein–protein interaction (PPI) network, SYK was found to be one of the pivotal genes in the pathogenesis of type 2 diabetes in patients with HF [97]. Konigsberger et al. reported that disruption of SYK expression altered BCR signaling quality [98]. A previous study found that hyperglycemia increased the phosphorylation of SYK and promoted platelet aggregation [69]. It is thus reasonable to assume that both platelet-related signaling pathways and BCR pathways are involved in the pathogenesis of DCM, and that SYK might play pivotal roles in these disease processes. The specific interactions between these signaling pathways through the hub of SYK remain to be further investigated.

Previous studies revealed that the NLRP3 inflammasome is involved in regulating the progression of DCM and changes in myocardial structure and function [[99], [100], [101]]. Li et al. recently found that SYK indirectly caused diabetic myocardial injury through activation of the NLRP3 inflammasome and that, compared with the findings in the control group, SYK phosphorylation was significantly increased in DCM rats. They summarized the role of SYK on DCM as follows: high glucose stimulation activated SYK, which further activated the NLRP3 inflammasome through the phosphorylation of c-Jun N-terminal kinase (JNK, a key transcriptional regulator of inflammation), thus indirectly lead to diabetic myocardial injury. As expected, SYK inhibitors attenuate diabetic myocardial injury by inhibiting NLRP3 activation. Thus, it is postulated that the SYK/JNK/NLRP3 signaling pathway plays a key role in the pathogenesis of DCM, making SYK inhibition a feasible option to attenuate cardiomyocyte injury caused by DCM [102]. In patients with DCM, inhibiting the expression of the SYK gene (e.g. via supplementation with melatonin) may block the SYK/COX-1/sarcoendoplasmic reticulum calcium ATPase (SERCA) signaling pathway to attenuate DCM. Specifically, once SYK is activated, it can indirectly lead to an increase of ROS in mitochondria; these superoxides then lead to intracellular calcium overloading, which in turn induces mitochondrial (caspase-9 related) and endoplasmic reticulum (caspase-12-related) apoptosis, thus jointly promoting high glucose-induced cardiomyocyte death. Blocking the SYK pathway might not only protect the myocardial function of patients with DCM, but also reduce myocardial fibrosis and maintain the activity of cardiomyocytes (Table 1) [103].

Table 1.

Summary of new findings of SYK in DCM-related events.

Glucose-state	Events	New findings of SYK	references
Hyperglycemia (DCM)	Heart failure and diabetes	being one of the pivot genes in the pathogenesis of HF and type 2 diabetes. disruption of SYK expression could alter BCR signaling quality.	[97] [98]
	Cardiomyocyte injury	activates the SYK/JNK/NLRP3 signaling pathway and ultimately indirectly leads to diabetic heart dysfunction.	[102]
	Myocardial fibrosis	inhibits SYK gene in DCM patients blocks the SYK/COX-1/SERCA signaling pathway thus reduces myocardial fibrosis and maintains cardiomyocytes activity.	[103]
	Thrombus	phosphorylated level increased.	[69]

5.2. Hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM), a common inherited heart disease, is the most common cause of nontraumatic cardiac death in adolescents [104]. Numerous studies have found that ubiquitination in protein PTM is closely related to HCM [105]. The ubiquitination process is generally carried out by a cascade reaction involving three enzymes: activating enzyme E1, conjugating enzyme E2 and ligase E3 [3]. Various E3 enzymes play important roles in HCM. The E3 MDM2 was found to be capable of inhibiting cardiac hypertrophy [106], while downregulation of Murf-1 promoted pressure overload-induced cardiac hypertrophy [107]. Tumor necrosis factor receptor-associated factor 2 promotes cardiac hypertrophy by modulating the NF-κB and JNK pathways [108]. It is worth mentioning that ubiquitination modification and other modification processes (e.g. phosphorylation) are actively involved in the pathogenesis of HCM. Using coexpression analysis of genes, Chen et al. found that the expression level of SYK was significantly increased in HCM rats and demonstrated that SYK played a central role in the disease process of HCM. Notably, the mechanism by which SYK affects cardiac hypertrophy is not fully understood [109], necessitating future studies.

6. SYK in heart failure

HF is one of the most common insidious diseases requiring hospital admission, burdening public health worldwide [110]. Cardiac fibrosis is a crucial mechanism of HF [111,112]. SYK might aggravate HF by promoting inflammatory responses and cardiac interstitial fibrosis.

Recently, the relationship between SYK and myocardial fibrosis has been explored. SYK can bind to and inhibit miR136, which can reduce epithelial–mesenchymal transition by targeting transforming growth factor (TGF)-β1/Smad3 signaling pathway, thus ultimately promoting fibrosis [113]. Li et al. investigated the association between disruptor of telomeric silencing 1-like (Dot1l), SYK and fibrosis in vivo and in vitro, showing that the knockout of Dot1l downregulated the modification level of histone 3 K79 dimethylation (H3K79me2) on the promoter of SYK, thereby reducing the interaction between SYK and miR136 and ultimately attenuating cardiac fibroblast proliferation and fibrosis. These effects were reversed by upregulating SYK via activation of the TGF-β1/Smad3 signaling pathway (Fig. 3) [114]. A very recent publication (2023) suggested that the SYK/NF-κB signaling pathway could be activated by angiotensin II (AngII)-induced Dectin-1 homodimerization, which in turn upregulated the expression of inflammation-related molecules including tumor necrosis factor-α, interleukin-1β and others. These altered pathways were found to be important players in AngII-induced cardiac remodeling and dysfunction [115].

Fig. 3.

SYK plays a role in mediating TGF-β1/Smad3 signaling pathway in myocardial fibrosis. Dot1l may activate the gene transcription of SYK. Dot1l-knockout could downregulate the modification level of H3K79me2 on the promoter of SYK, thereby reduce the expression of SYK. SYK was discovered to bind to and inhibit miR136, which can reduce epithelial–mesenchymal transition by targeting TGF-β1/Smad3 signaling pathway, thus ultimately promotes fibrosis. Abbreviations: Dot1l, Disruptor of Telomeric silencing 1-like; SYK, spleen tyrosine kinase; TGFβ, transforming growth factor-beta; SMAD2/3, Mothers against decapentaplegic homolog 2/3; SMAD4, Mothers against decapentaplegic homolog 4.

Additionally, SYK is a major immunological player in HF. In the pathogenesis of HF, the BCR-SYK pathway is known to play a major role. SYK is inactive and nonphosphorylated in resting B cells. In response to antigen stimulation [116], the BCR is activated, leading to phosphorylation of SYK at site Y348. The downstream substrate proteins, which include PLCγ2, B-cell linker protein and others, are subsequently activated [116], thereby promoting B-cell survival and proliferation [117]. It is believed that this immune response activation process could induce left ventricular dysfunction and related adverse cardiac remodeling in the setting of HF [118], indicating a strong connection between SYK-related innate and adaptive immunity and the development of HF [119]. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small-vessel disease that causes direct endothelial injuries and promotes diffuse AS, vascular stiffening and cardiac dysfunction [120,121]. Epidemiological studies have found that AAV patients have a high risk of developing chronic HF [122,123]. Recently, Prendecki et al. found that there p-SYK is increased in circulating neutrophils and monocytes from patients with active AAV. Mechanistically, ANCA immunoglobulin G could phosphorylate SYK at Y348. Accordingly, SYK inhibition could inhibit ANCA-mediated PLCγ/Vav/Ras/mitogen-activated protein kinases/NF-κB signaling [124,125]. Collectively, SYK is involved in the HF process by inducing myocardial fibrosis via activation of the TGF-β1/Smad3 pathway in cardiac tissues and modulating immune responses.

7. Research on inhibitors of the SYK signaling pathway

The above experimental and clinical studies illustrated a fundamental role of activation of platelets in promoting the occurrence of AS [126]. Accordingly, antiplatelet drugs, including SYK inhibitors, have been tested and widely applied for the prevention and treatment of AS and related diseases [[127], [128], [129], [130]]. Many pharmaceutical companies, including Rigel, Pfizer, Bayer and ZaBeCor, are actively involved in the development of small molecule inhibitors. Four structurally related analogs of competitive SYK inhibitors, namely SYK R406, R788, R112 and R343, are currently being developed and evaluated for the treatment of cardiovascular-related inflammatory diseases [47,131].

Previous studies have demonstrated that the highly selective SYK inhibitors fostamatinib (R406) [[132], [133], [134]] and entospletinib (GS-9973) inhibit platelet aggregation without increasing bleeding events [116,135]. Lindau and colleagues showed that fostamatinib significantly reduced the occurrence of early neo-atherosclerotic plaques in Apoe^−/− mice fed a high-fat diet (inhibition rate >70%). However, for mice with established AS, fostamatinib was only able to reduce the infiltration and differentiation of monocytes, and did not affect cell proliferation or cell death of macrophages in the plaque [136]. Because the selective SYK inhibitor fostamatinib completely prevented FcγR chain and CLCE-2 receptor activation-induced human platelet aggregation and reduced platelet spreading on fibrinogen, its mitigating effects on cardiovascular diseases such as thrombotic wandering-induced myocardial infarction are being investigated intensively [137].

Andre and colleagues found that PRT060318, a highly specific SYK inhibitor, inhibited the formation of arterial thrombus in vivo in mouse, rabbit and pig models [138]. Irfan et al. explored the role of gintonin, a glycolipoprotein extracted from ginseng. This team established a mouse model of pulmonary thromboembolism and found that gintonin significantly inhibited collagen-induced platelet aggregation and inhibited related signaling molecules in the GPVI signaling pathway, including SYK and PLCγ2, in this model [139]. Gintonin also downregulated the early signals of platelet activation, thereby reducing the formation of arterial thrombi [139,140]. Wang and coworkers analyzed the antithrombotic effect of Xinmailong (XML), and revealed that treatment with XML (10 mg/mL) inhibited collagen-induced phosphorylation of signaling molecules such as SYK and PLCγ2 [141]. Additionally, infarct size was reduced by the SYK inhibitor BI1002494 in mice [76,142]. The SYK inhibitor OXSI-2 dose dependently inhibited thrombosis formation on type-III collagen under high shear stress [58]. All of these results imply that inhibiting SYK could be a potentially effective antithrombotic option in various clinical settings, including cardiovascular disorders. Natural SYK inhibitors, such as curcumin and piceatannol, have also been reported to inhibit the SYK signaling pathway. Several oral SYK inhibitors, including fostamatinib (R788), entospletinib (GS-9973), cerdulatinib (PRT062070) and TAK-659, are being evaluated in clinical trials [143]. Among them, fostamatinib is also used in the treatment of ischemia-reperfusion injury, particularly for improving coronary flow after myocardial infarction [144,145].

Inhibition of SYK might be able to attenuate brain tissue damage following an IS [[146], [147], [148]]. Piceatannol, an SYK inhibitor derived from passion fruit seeds, rescued cerebral ischemic injury by reducing infarct volume and brain edema [90]. Piceatannol also reduced the ischemia-induced release of inflammatory cytokines, thereby partially reversing the inflammatory response after IS, in mice [83]. In fact, BI1002494, another SYK inhibitor, was also found to slow down the progression of stroke and improve prognosis in a mouse IS model [76,82]. Many studies have found that the overexpression of miR-148a-3p downregulates T-cell ubiquitin ligand-2 (a tyrosine kinase detectable in platelets), resulting in increased SYK phosphorylation, while targeted inhibition of miR-148a-3p reduces thrombus formation after platelet activation [149]. Clinically, targeting miRNA-SYK-related pathways may be a promising option in the field of stroke prevention and treatment.

Moreover, activated SYK is capable of promoting phosphorylation of BTK, which is an essential downstream signal of SYK, thereby enhancing platelet-related pathological processes. Studies have shown that low-dose irreversible BTK inhibitor ibrutinib (PCI-32765), which is generally used to treat patients with chronic lymphocytic leukemia, can inhibit CLEC2 activation of the tyrosine pathway, resulting in reduced platelet aggregation and activation, thus protecting against atherosclerotic plaque formation. Similar results were seen with another irreversible BTK inhibitor, acalabrutinib [150]. Recent studies have also demonstrated that reversible inhibitors of BTK (e.g. fenebrutinib) are more effective at inhibiting platelet activation than ibrutinib [151]. Payrastre and colleagues found that the use of low-dose (140 mg/day) ibrutinib effectively prevented thombus formation in mice [150]. These data imply that targeting BTK might be a promising and potentially meaningful therapeutic strategy for treating or preventing the formation of platelet-related thrombi (Table 2).

Table 2.

Researches on inhibitors of SYK signaling pathway.

Inhibitors	Medicine name	Biological mechanism	references
SYK-related inhibitors	Fostamatinib（R406）	●inhibits platelet aggregation and has no adverse bleeding events. ●reduces the incidence of early atherosclerosis, but could not effectively affect established atherosclerosis. ●improving coronary flow after myocardial infarction	[[132], [133], [134],136,144,145]
	Entospletinib (GS-9973)	●inhibits platelet aggregation and has no adverse bleeding events.	[116,135]
	PRT060318	●inhibits arterial thrombus formation.	[138]
	Gintonin	●significantly inhibits collagen-induced platelet aggregation and inhibits related signaling molecules in GPVI signaling pathway including SYK, PLCγ2. ●reduces the formation of arterial thrombi.	[139,140]
	Xinmailong (XML)	●inhibits collagen-induced phosphorylation of signaling molecules such as SYK and PLCγ2.	[141]
	BI1002494	●alleviates the infarct size. ●slows down the progression of stroke and improve prognosis	[76,142]
	OXSI-2	●inhibits thrombosis formation (dosage dependence) of type-III collagen under high shear stress.	[58]
	Piceatanol	●rescues cerebral ischemic injury by reducing infarct volume and brain edema. ●reduces ischemia-induced releasing of inflammatory cytokines, thereby partially reversed the inflammatory response after ischemic stroke. ●inhibits the SYK signaling pathway.	[83,90]
BTK-related inhibitors	Ibrutinib (PCI-32765); Acalabrutinib	●reduces platelet aggregation and activation, thus protect against atherosclerotic plaque formation.	[150]
	Fenebrutinib	●inhibits platelet activation more effectively than Ibrutinib.	[151]

Notably, in addition to its potential beneficial effects, ibrutinib also has unexpected off-target side effects [152] that can lead to increases in the incidence of hypertension and atrial fibrillation (AF), and other adverse reactions including diarrhea and fatigue [153]. Additionally, the prevalence of hypertension increases in proportion to the duration of prolonged application of ibrutinib [154,155]. A recent study found that the incidence of new-onset hypertension was as high as 70% in 562 patients receiving ibrutinib treatment [156]. In another retrospective study of 144 patients with B-cell malignancies, the authors reported significantly higher systolic and diastolic blood pressure in patients treated with ibrutinib chemotherapy than in the control group [155]. New-onset AF is another common side effect of ibrutinib treatment, with clinical studies confirming an increased incidence of new-onset AF in patients with chronic myeloid leukemia post ibrutinib treatment [154,[157], [158], [159], [160], [161]]. Therefore, to achieve the best clinical benefit, the decision-making process in the use of ibrutinib should include an assessment of the clinical benefit–risk balance; namely, the consideration of dose-dependent effects.

8. Conclusion

As one of the non-receptor tyrosine kinases, in addition to the key role in hematologic oncologic diseases, SYK also plays important roles in the pathogenesis of various cardiovascular-related diseases. Activation of SYK-related pathways largely contribute to the pathogenesis of various cardio-cerebrovascular diseases, evidence is accumulating on the application of SYK inhibitors, including BTK inhibitors, for the prevention and treatment of related cardio-cerebrovascular diseases. Ongoing researches on SYK in the cardiovascular and cerebrovascular field will deepen our understanding at the molecular basis on the pathological mechanism of various cardio-cerebrovascular diseases, which might pave the way of developing more effective target medication for the prevention and treatment of cardio-cerebrovascular diseases. At present, the role of various SYK inhibitors on the clinical treatment of cardiovascular diseases are in the stage of in-depth researching and special attention is needed to keep an eye on the related progresses.

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

The present study was funded by the National Natural Science Foundation of China (82171572, 82171571 and 81970211). Naijin Zhang was supported by National Natural Science Foundation of China {82171572}.

Ying Zhang was supported by National Natural Science Foundation of China {82171571}.

Yingxian Sun was supported by National Natural Science Foundation of China { 81970211}.

Data availability statement

Data used to support the findings of this study are available from the corresponding author upon request.

Declaration of interest's statement

The authors declare no conflict of interest.