Proteomic investigations of acute ischemic stroke in animal models: a narrative review : Journal of Bio-X Research

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Proteomic investigations of acute ischemic stroke in animal models: a narrative review

Sun, Fangxu; Gu, Rong-Fang; Wei, Ru*

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Journal of Bio-XResearch 6(1):p 15-22, March 2023. | DOI: 10.1097/JBR.0000000000000134
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Approximately 87% of all strokes are ischemic strokes. Acute ischemic stroke (AIS) is the second leading cause of death worldwide. Over half of affected patients suffer from persistent long-term disability. Although significant improvements have been made in stroke care and prevention, there is very limited progress in therapeutic development and biomarker identification. Recombinant tissue plasminogen activator (rtPA) is still the only FDA-approved drug and mechanical thrombectomy is the major technique for treating AIS. Both are only accessible to a small fraction of patients due to the strictly eligible criteria and the narrow therapeutic window of a few hours, albeit the accessibility and treatment effect have improved via advanced medical devices.[1] The urgent need for new treatments, particularly for limiting cell death and recovering neuronal function post AIS, has kept great research interests in the field. Indeed, a PubMed search of “(ischemic OR ischemia) AND stroke” in [Title/Abstract] (August 2022) included ~75,000 articles (excluding reviews), indicating a big gap from enormous preclinical research efforts to a very low success rate of clinical therapy. Among many factors contributing to this disparity, the intrinsic complexity of AIS and the lack of novel therapeutic targets and biomarkers are the leading causes. AIS is a circulatory system-linked disorder that can occur in the whole brain (global AIS, usually triggered by cardiac arrest) or in part of the brain (focal AIS). Focal AIS is the typical clinical presentation caused by locally formed blood clots or remotely developed and traveled plaques. The onset of AIS is triggered unpredictably and multifariously, which can result in death or a series of significant injuries in the brain within hours or days. Although many pathophysiological and cellular processes, including edema, brain–blood barrier disruption, neuroinflammation, immune responses, cell death, neuronal remodeling, and spontaneous recovery, have been deciphered, the underlying molecular mechanisms are largely unknown, which impedes the identification of novel targets and biomarkers and thereafter therapeutic development. In addition, the fundamental understanding of the etiology of stroke has primarily focused on a single target, which is not sufficient to clarify various stroke-induced injuries. Adjunctive therapies paired with reperfusion approaches (eg, mechanical thrombectomy) and cocktail therapies (eg, cell therapy) targeting multiple key pathways to simultaneously reduce neuronal cell death and restore neuroprotection may be necessitated, further demanding to uncover AIS mechanism at the molecular level.

To delineate molecular mechanisms and identify novel AIS targets and biomarkers, the systems biology (ie, Omics) approach is the best and the most efficient tool. With the rapid development of instrumentation and bioinformatics, proteomics is capable of globally identifying and quantifying thousands of proteins in a given state of complex biological systems and has become a valuable tool to divulge molecular pathways and intracellular signaling cascades leading to new therapeutic targets and biomarkers. Compared with other omics approaches, proteomics has several advantages: proteins perform main and most cellular functions, only via proteomics, one can at omics-scale study protein posttranslational modifications (PTMs), interactions and complexes, the crucial players of molecular mechanisms, and the majority of drug targets are proteins. Owing to their unbiased nature, in-depth proteome coverage, and suitability for nearly all biological matrices, mass spectrometry (MS)-based discovery (or unbiased) proteomics platforms have been widely used in the systematic investigation of disease mechanisms, target identification, drug mode of action (MoA) and biomarker discovery studies. Proteomics study of AIS is, however, scarce, fewer than 150 studies were published in the past 20 years and only a small portion of these have relatively in-depth proteome coverage (>5000 proteins).

Research on human AIS is limited owing to high death rate, unexpected onset, and severity, mainly focusing on diagnostic/prognostic biomarker discovery.[2–12] Therapeutic targets are commonly generated and characterized preclinically using various in vitro and in vivo models. Simple systems like cell models are suitable for high-throughput screenings and function interrogation. Only animal models are capable of closely mimicking pathophysiological conditions in humans and comprehensively assessing responses of therapeutic intervention, empowering modern drug discovery.[13] Testing animals typically have the same genetic background, “perfectly” matched age/sex and good survival rates, enabling the use of a small number of biological replicates without confounding. Practically, reproducible stroke lesion size can be only achievable in animals. Accessing various tissues and biofluids is particularly important for simultaneously investigating AIS effects extending beyond stroke site(s). Indeed, a variety of AIS in vivo models, mostly mouse or rat and also cynomolgus or rhesus monkey, have been developed using diverse AIS induction methods, such as intraluminal suture middle cerebral artery occlusion (MCAO),[14–17] to recapitulate transient or permanent artery occlusion with complete, partial, or no reperfusion, enabling acute, chronic or longitudinal injury, or recovery from AIS. Detailed discussions about different stroke animal models and applications are out of scope here and can be found elsewhere.[18–24]

Proteomic investigations using animal models become vital to map molecular events and build knowledge bases of AIS-induced pathophysiological processes as well as to decipher the MoA of therapeutic interventions. Recent publications have reviewed proteomics approaches and their general applications for studying AIS.[25–29] Herein, this review summarizes the main findings of proteomics studies of AIS conducted in animal models and provides a snapshot of insights into molecular mechanisms and repeatedly observed target/biomarker candidates, serving as a knowledge resource for future AIS-related research, demonstrating the potential of proteomic approaches, and thus calling for its broad application.

Database retrieval strategy

Literature review was performed using the PubMed database. The search keywords were “acute ischemic stroke,” “AIS,” “ischemic stroke, proteomics,” “therapeutic target and intervention,” “stroke biomarker,” “MCAO model,” “animal model,” and “ischemic molecular mechanism.” The literature search was performed up to 2022. The articles included in this review were selected based on their relevance to the topic. The results were further screened according to the title and abstract. Data extraction focused on information about the proteomic studies of acute ischemic stroke in animal models.

Proteomics studies of acute ischemic stroke in animal models

Global proteomic investigation of AIS progression

Advances in instrumentation and bioinformatic tools have made MS-based proteomics a valuable tool to globally quantify AIS-induced changes in protein abundance to allow for the characterization of the molecular mechanisms underlying AIS progression and facilitation of biomarker and therapeutic target discovery. This section focuses on studies of AIS without therapeutic intervention.

Many pathological changes occur following AIS including neuronal death, inflammation, and oxidative stress.[30] Proteomics has been used to explore the molecular mechanisms associated with AIS-related damages. A comparison of brain tissues from MCAO and sham rats resulted in identification of 282 differentially expressed proteins enriched in energy metabolism and neurodegenerative disease-related pathways. Sod1 and Syn1, which are associated with neurodegeneration and synaptic plasticity, were downregulated in ischemic rats, as determined using proteomics and western blotting. This finding indicated that these proteins might be potential therapeutic targets for stroke-induced brain injury.[31] Another study showed that Eno1, a key protein in the glycolytic pathway, was identified using gel-based MS as a potential target to alleviate the ischemic injury.[32] Using a rat model, Chen et al[33] found that ischemia induced oxidative and endoplasmic reticulum stress in brain neuron cells by downregulating Comt and Ctsd and upregulating Calb2. Comt and Ctsd were found to be associated with oxidative stress, inflammatory response, and apoptosis. Calb2 was involved in endoplasmic reticulum stress-induced neuronal apoptosis. These findings were further validated in vivo and in vitro using immunohistochemistry. Label-free proteomics was used to compare brain injuries caused by ischemic and hemorrhagic stroke. The results showed that 38 and 86 proteins were differentially expressed in ischemic and hemorrhagic stroke, respectively. Distinct pathological processes were identified with oxidative stress and caspase pathways in AIS and autophagy, necrosis, and calpain activation associated with hemorrhagic stroke, while inflammatory and apoptotic effects were associated with both hemorrhagic and ischemic strokes.[34] A study used isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics to show that 61 proteins were dysregulated in the acute and/or subacute phase(s) poststroke. These proteins were primarily associated with energy metabolism, glutamate excitotoxicity, synaptic plasticity, and inflammation.[35] Data independent acquisition (DIA)-based proteomics along with transcriptomics showed that the immune response and inflammation played a key role in stroke progression in the acute phase (6–24 hours).[36] Proteomics analysis using tandem mass tag (TMT) showed that proteins located on organelle outer membranes were downregulated, and those involved in cytosolic the ribosome and spliceosomal complex were upregulated, in mice following stroke and 1 hour of reperfusion. These findings suggested that the underlying molecular changes in stroke-related damage were associated with the recovery process after stroke.[37] PTM proteomics has been increasingly applied in AIS research to understand the molecular mechanisms. Recently, phosphoproteomics performed on the hippocampus of MCAO mice showed that dysregulated phosphoproteins were closely correlated with the synapse and neurotransmission. Phosphorylation of Syt1 at Thr112, and its interaction with Anxa6, played a key role in ischemia-induced cerebral injury in MCAO mice. The results were validated in an oxygen-glucose deprivation cell model.[38] Through lysine-lactylation proteomics analysis on rat cerebral after ischemia followed by reperfusion, Yao et al[39] identified 1003 lactylation sites on 469 proteins, including 54 upregulated and 54 downregulated lysine-lactylation sites (vs controls) from 49 and 99 proteins, respectively. The authors further validated altered lactylation on the important Ca2+ signaling proteins (Scl25a4, Slc25a5, Vdac1, and Vdac2) and proposed lactylation involvement in the underlying mechanism of cerebral ischemia–reperfusion injury via mediating mitochondrial apoptosis and neuronal death.[39] Using targeted proteomics, molecular mechanisms that drive the brain-lung interaction poststroke were studied in a MCAO mouse model. A 92 multiplex protein panel developed by Olink Proteomics® was used to analyze the content in bronchoalveolar lavage fluid and lung homogenates, Hgf, Tgf-α, and Ccl2 were identified and further validated by the enzyme-linked immunosorbent assay as dysregulated proteins in the lungs after cerebral ischemia, suggesting a potential important role in stroke-induced lung damage.[40]

Many patients with AIS develop chronic neurological and functional disabilities. Therefore, understanding the dynamic molecular mechanisms associated with AIS is necessary for the development of novel therapies to prevent further neuronal damage and to promote recovery. Proteomics has been used to investigate proteome changes in the weeks following AIS. Label-free proteomic analysis of the cortices of MCAO rats from the subacute to the chronic phase (days 1, 7, and 14 poststroke) found that the expression of 1305 proteins changed during this period, and cytoskeleton and synaptic structures, energy metabolism, and inflammatory response were significantly disrupted in the subacute phase. However, in the long-term phase, recovery of the cytoskeleton was detected, and inflammation pathways different from those activated during the subacute phase were activated.[41] Using a distal hypoxic (DH)-MCAO mouse model and TMT-based proteomics, our laboratory quantified over 7600 proteins and found nearly half of them changed in abundance in MCAO mice during a 28-day poststroke period. Of these, 309 were temporally associated with stroke, and underwent relatively large and sustained increases. These proteins were largely associated with the immune response. In addition, proteins involved in cytoskeleton remodeling and synaptic signaling underwent small stroke-induced changes. On day 28 poststroke, most of the proteins returned to normal levels, indicating spontaneous poststroke recovery.[42] A chronic-phase investigation of AIS in cynomolgus monkeys found 55 dysregulated proteins in cortices with elevated or low infarct volumes on day 28 poststroke. These proteins were associated with tissue injury and recovery-related cellular processes including inflammation, neurogenesis, and synaptogenesis.[43] The dysregulated proteins identified poststroke at multiple time intervals largely overlapped across several studies despite use of different AIS animal models. For example, 604 dysregulated proteins overlapped between the aforementioned rat MCAO[41] and our mouse DH-MCAO[42] studies, and over 72% of these proteins changed in the same direction. The cytoskeleton and synaptic remodeling, the adaptive immune response, and possible later-phase recovery were observed in both studies. A study by Ren et al[34] showed consistent upregulation of 3 proteins (Cdc42, Eef1a1, and Alb) and downregulation of 13 proteins (Camk2a, Cntn1, Glud1, Arpc5l, Map6, Ndrg2, Ptpn11, Prkcb, Pgk1, Prkcg, Srgap3, Uba1, and Nsf) in rats subjected to stroke. In the previously described monkey study,[43] of 55 stroke-changed proteins, 16 (CAMK2A, CRMP1, GDA, GPM6A, SYP, A2M, ALB, ANXA5, TUBB2A, TUBB4A, COTL1, FLNA, MYH9, TAGLN2, LDHB, YWHAZ) from the elevated infarct volume group and 14 (FABP7, GSN, PFN1, RAB3A, CNN3, S100A11, ATP1A1, TUBA4A, RNH1, PLEC, CALM1, CKB, MDH2, PCP4) from the low infarct volume group changed in the same direction as those in our DH-MCAO mouse study. Furthermore, a human proteomic study of patients who survived AIS[44] reported changes in the expression of 43 proteins in plasma collected at 3 and 12 months poststroke. Nineteen (A2M, APOA2, APOA4, APOD, C4BPA, C8A, CFD, CFI, CLEC3B, CSPG4, FBLN1, FETUB, FGA, GPLD1, HPX, RBP4, TF, and VTN) changed at the same direction as those reported in our mouse stroke study.[42]

Breakdown of the brain–blood barrier during ischemia leads to leakage of brain-specific proteins into the general circulation. These brain-derived proteins could be potential biomarkers of disease. Some promising protein biomarker candidates such as MMP9, S100B, and CSTA have been proposed for diagnosis and prognosis of AIS in humans. Efforts have been made to discover additional molecular mechanism-informed AIS biomarkers using animal models. The expression levels of Rhoa and Cdc42 increased gradually in the tissues and serum of rats during prolonged ischemia (up to 4 hours), as determined using iTRAQ-based proteomics and validated using western blot, which indicated that these proteins may be biomarkers of AIS in the acute phases.[45] Proteomics results from the serum of ischemic rats showed differential expression of A2m, Itih3, C3, Alb, Hp, and Ttr, which are known to be associated with ischemia. This finding may facilitate future studies aimed at identification of clinical biomarkers.[46] Datta et al[35] found upregulated brain-specific proteins including Gfap, Uchl1, and S100b in the poststroke acute and subacute phase. Li et al[36] reported that C3, Apoa4, and S100a9 might be potential biomarkers of ischemia based on increased mRNA and protein expression levels during stroke progression. Proteomic, immunoblotting, and immunohistological studies showed that Hsp72 was a specific biomarker in the peri-infarct region of rats with permanent focal cerebral ischemia. As a result, an anti-Hsp72 vectorized stealth immunoliposome was developed for theranostics of AIS.[47] The 309 sustainably changed proteins identified in our DH-MCAO study,[42] including 182 annotated as secreted proteins, could serve as candidate pharmacodynamic and diagnostic biomarkers. Increased abundance of some of the 182 proteins was observed in humans. For example, in a longitudinal biomarker and drug target study, increases in CFB, AHSG, FN1, and APOA1 were observed in human serum with the progression of AIS.[5] Integration of transcriptomics and proteomics identified 76 proteins that were differentially expressed in the ischemic brain. Of these, Gadd45g and Ctnnd2 were identified as promising blood biomarkers for AIS prognosis and diagnosis after validation using Parallel Reaction Monitoring (PRM)-targeted proteomics and the Nanostring nCounter assay in a new cohort of MCAO mice.[48] Overall, proteomics has identified a pool of biomarker candidates for various poststroke phases. Many biomarker candidates were reported in multiple studies, including A2m, Ahsg, Alb, Apoa1, Apoa4, cdc42, Ctsd, Gfap, Hpx, Mug1, Rhoa, S100b, Tf, and Ttr.[28,33,34,36,41–45,49–56] Considering the complex physiological changes induced by ischemia, a panel of proteins might be the most effective AIS biomarker.

MS-based proteomic analysis has identified many known and novel dysregulated proteins from the acute to the long-term phase in AIS, which has allowed for the characterization of the molecular mechanisms underlying AIS-induced pathological changes such as excitotoxicity, ionic imbalance, oxidative stress, apoptosis, and inflammation. Proteomics results have also provided valuable insights into biomarker and drug target identification.

Proteomic analysis of AIS with therapeutic interventions

In addition to research efforts on drug discovery, studies have also focused on other avenues such as dietary supplements, metabolites, and cell therapy to determine potential therapies for stroke. Several approaches have been shown to mitigate ischemia-induced brain injury. Modulating energy metabolism and ameliorating oxidative stress have been shown to confer neuroprotection through proteomics analysis. It has been found that ferulic acid, a natural antioxidant, exerts a neuroprotective effect via attenuation of ischemia-induced changes in expression of multiple proteins such as Ahcy, Idh3a, Gapdh, Ptpa, Hpcal1, Prdx2, and Prdx1.[57–60] This research group also showed that resveratrol, another antioxidative and anti-inflammatory agent, protected against AIS by modulating oxidative stress and energy metabolism through increased expression of Prdx5, Idh3a, Apoa1, and Uchl1, and decreased expression Dpysl2.[49] Similarly, retinoic acid was found to perform neuroprotective function by regulating various proteins that mediate cell metabolism and function such as Ahcy, Idh3a, and Gpd1.[61] A label-free proteomic study found that the tyrosine metabolism and dopaminergic synapse signaling pathways were disrupted in MCAO rats, and glutathione treatment restored protein expression to normal levels. Western blot validation and metabolite quantification showed that the mechanism underlying the therapeutic effects of glutathione on AIS was to increase intrastriatal dopamine through reversal of AIS-induced downregulation of tyrosine hydroxylase. Increased dopamine would in turn result in increased glutathione through upregulation of glutathione synthetase and homocysteine levels.[62] Proteomic analyses using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that Crmp2, Hsp60, Eno2, Trx, and Pp2a, which are involved in energy metabolism, homeostasis, axonal growth, and oxidative stress, were dysregulated in MCAO rats. The levels of these proteins were restored to normal following treatment with melatonin, a sleep hormone and antioxidant. Further analysis via WB, immunofluorescence, and molecular docking confirmed that melatonin attenuated AIS-induced brain damage via regulation multiple protein targets listed above.[63] Recent proteomics results suggested that Bexarotene, a FDA-approved drug for cutaneous lymphoma, also displays neuroprotective benefits and inhibits the JIP3/ASK1/JNK/Caspase 3 signaling pathway via downregulating JNK-activated scaffolding protein JIP3 to decrease neuronal apoptosis.[64]

Chinese traditional medicines have been frequently used to alleviate brain injury. MS-based proteomics is an effective approach to investigate the MoA of traditional Chinese medicines because they have complex compositions. Proteomics analysis using iTRAQ showed that Buyang Huanwu Decoction and rtPA treatment altered 15 and 23 proteins in mice, respectively. Functional analysis showed that Buyang Huanwu Decoction treatment led to prevention of brain–blood barrier breakdown as indicated by restoration of Alb, Fga, and Trf levels, minimization of excitotoxicity through modulation of Gnai1, Gnai2, Gdi1, and Gdi2, which are associated with GABAB receptor activation, and enhancement of energy metabolism through inactivation of Gsk-3 and reduced tau activity. In contrast, rtPA induced brain–blood barrier breakdown, as evidenced by upregulation of Alb, Fga, and Trf, which is a major side effect of rtPA treatment.[50] To further explore the neuroprotective mechanisms of Buyang Huanwu Decoction, this research group combined proteomics and metabolomics to comprehensively study the cerebrospinal fluid from Buyang Huanwu Decoction-treated MCAO mice. The results showed that disruption of brain–blood barrier integrity, inflammation, and dysregulated energy homeostasis contributed to ischemia-induced brain injury, and Buyang Huanwu Decoction treatment reversed levels of most inflammation and neurodegeneration-associated proteins including A2m, Serpinb5, Ces1c, Hspa1a, Jup, and Nptxr in the cerebrospinal fluid.[51] Li et al[52] identified 3216 proteins associated with MCAO in mice using iTRAQ-based proteomics, of which 21 were differentially expressed following NaoMaiTong treatment. Enrichment analysis indicated that NaoMaiTong treatment might exert its neuroprotective effects through modulation of three pathways: ribosome function (Rpl26, Rpl17, Rpl39, and Rps13), tight junctions, and regulation of actin cytoskeleton (Tuba, Wasl, and Rac1). The expression changes in Rpl17, Tuba, and Rac1 were validated using western blot assay.[52] Proteomics results showed that the therapeutic effects of rhubarb, another Chinese traditional medicine, might result from modulation of various pathways such as cGMP-PKG signaling and the synaptic vesicle cycle via restoration of the expression levels of multiple proteins including Mapk1, Syn1, and Calm1 in MCAO rats.[53] Furthermore, Eftud2, mTOR, Rab11, Ppp2r5e, Hk1, and Eno2, which are associated with innate immune regulation, mTOR signaling, membrane trafficking, cell growth, and HIF-1 signaling pathways, were identified as key hub proteins underlying the effects of Hydroxysafflor Yellow A against ischemia-induced injury in rats using proteomic analysis.[54] A proteomics study found the tetrandrine isolated from a Chinese analgesic medicine alleviated neurological deficits, brain water content, and infarct volume by normalizing the expression of Grp78, DJ-1, and Hyou1 in MCAO mice.[65]Huanglian Jiedu Decoction exerts neuroprotective effects via mediating the expressions of Grin1, Rap1a, Actb and Akt in the RAP1 signaling pathway, while the beneficial effects of QishenYiqi for stroke recovery are closely associated with regulating lysosome pathway and galectin-3-mediated inflammation.[66,67] Muscone, an active component of musk, ameliorates the neurological damage mainly through remedying neuronal synaptic connections.[68] DIA proteomics results indicated that Alb, mTOR, Stxbp1, Cltc, Dync1h1, and Sptan1 may be the potential targets of sodium tanshinone IIA sulfonate, a key ingredient from Salvia miltiorrhiza Bunge for treating stroke injuries.[69] Scutellarin alleviates stroke-induced oxidative stress injury via downregulating aldose reductase and its downstream targets Nox1, Nox2, and Nox4 that lead to reactive oxygen species-induced oxidative damage.[70]

Preventive neuroprotection against cerebral ischemia has been studied using MS-based proteomics. The results indicated that intermittent hypobaric hypoxia preconditioning (6 hours/day) may have conferred protection in permanent MCAO rats via activation of the clathrin-dependent endocytosis pathway (upregulation of Chmp1a, Rabep1, Arpc5, and Hspa2) to promote transport of neuroprotective factors into cells.[55] Using TMT-based proteomics, 16 proteins associated with neuroprotective effects were found to be upregulated in MCAO Rhesus monkeys preconditioned with the immune activator D192935 (a TLR9 agonist). Specifically, F13A1, ORM1/AGP1, and STAB1 are associated with M2 macrophages, and MMRN2, STAB1, HS6ST1, GALNT3, LOXL1 are involved in angiogenesis and tissue repair.[71] Pretreatment with an agonist of PPARα, which plays a key role in modulating energy metabolism and vascular homeostasis, has also been shown to induce a neuroprotective effect. A gel-based proteomics study identified 26 dysregulated proteins in MCAO rats, and PPARα agonist pretreatment preserved the expression of several proteins such as Ywhaz, Dpysl2, and Snca, which are associated with homeostasis, signal transduction, and synaptic plasticity, and modulated the expression of Pdia3.[72] Pretreatment with red wine polyphenol compounds (RWPC) might lead to the neuroprotective effect through multiple biological pathways in rats such as energy metabolism, proteolytic pathways, mitochondrial function, and apoptosis.[56]

Several other types of interventions also confer neuroprotection. Exogenous cell therapy has been evaluated in ischemic animal models using proteomics. Analysis using MALDI-TOF identified 14 proteins that were differentially expressed in stroke rats with cerebral endothelial cell transplantation compared to the sham and ischemia groups. Further analysis indicated that the neuroprotective effects of this procedure may have resulted from controlling neuroinflammation via decreased Spg7 and Prdx6, inhibition of a transcriptional repressor via downregulation of Zfp90, and modulation of damaged vasculature via upregulation of Clic4.[73] Mesenchymal stem cells (MSCs)-derived microvesicles (MVs) have shown the ability to ameliorate functional deficits in permanent MCAO rats. Proteomics results showed that treatment with MSC-MVs might have exerted therapeutic effects on AIS rats through regulation of tissue repair pathways including angiogenesis, neurogenesis, anti-inflammation, and apoptosis.[74] Comprehensive proteomic profiling of conditioned media of hiPSC-derived glial and neuronal progenitor cells (GPCs and NPCs) showed unique protein expression patterns in each medium, which might explain their differential therapeutic effects on AIS rats.[75] Mild hypothermia is another promising therapeutic strategy for mitigating ischemic injury. A proteomics study found that 26 proteins were differentially expressed in ischemic rats with and without hypothermia treatment. These proteins were involved in cellular assembly and organization (Dpysl2, Tuba1a, and Actb), signal transduction (Map2k1, Phb, and Gnao1), and metabolism (Ndufv2, Bpnt1, and Mdh1), which indicated that hypothermia exerted neuroprotective effects via regulation of multiple molecular targets and cellular pathways. The authors proposed Baiap2l1 and A1at as novel therapeutic targets based on their pivotal roles in actin cytoskeleton remodeling and protease inhibition.[76] In another study, label-free proteomics identified 28 upregulated and 22 downregulated proteins in the plasma of AIS rhesus monkeys before and after remote ischemic conditioning. Pathway analysis showed that remote ischemic conditioning attenuated brain injury via modulation of multiple pathways related to regulation of lipid metabolism (APOA2 and APOC2), anticoagulation (FGA and SERPINA1), complement activation (C3 and C1), and endovascular homeostasis (HSPG2).[77]

MS-based proteomics has enabled comprehensive investigations of the molecular mechanisms underlying different types of therapeutic interventions for AIS, resulting in increased understanding of AIS. Revealing neuroprotective MoA of unconventional therapeutics, such as Chinese traditional medicine, MSC-MV, iPSC-derived GPC, and NPC, and cell conditioning medium, supports their clinical benefits for reducing ischemic and reperfusion damage and recovering from AIS, strengthens the therapeutic strategy of targeting multiple targets/pathways, and warrants more continuous efforts in the development of cocktail therapies for treating AIS.

Prestroke prognosis with MS-based proteomics

As the number of individuals affected by stroke is expected to increase, improving the prognosis associated with ischemia is critical in stroke prevention. Multiple risk factors including diabetes, atrial fibrillation, hypertension, and dysfunctional glucose metabolism have been associated with increased risk of AIS. In addition to preclinical studies, proteomics investigations can be conducted using available human samples from other studies or biobanks to identify prognostic biomarker candidates. Two human studies are reviewed in this manuscript to motivate biomedical researchers to engage in this area.

Bergerat et al[78] reported global proteomic analysis of cerebral cortical microvessels from stroke-prone and nonstroke-prone rats, and identified about 2000 proteins. Metaproteomic analysis resulted in identification of differentially regulated proteins associated with ischemia, brain–blood barrier integrity, and angiogenesis and these molecular changes might be related to stroke susceptibility in the prestroke stage. Age and sex were associated with pathway differences in glycolysis, cell–microenvironment interactions, and transendothelial migration.[77] In a spontaneously hypertensive stroke-prone rats (SHRSP) model, proteins including Tf, Hpx, Alb, Ashg, Abp, Serpina1, Gc, Ttr, and thiostatin were found in the urine and serum weeks before stroke onset.[79] Mitaki et al[80] isolated extracellular vesicles in serum from patients that later developed symptomatic ischemic stroke in health checkups and compared the proteomes from these vessels against those from sex-matched healthy controls using iTRAQ-based proteomics. The results showed increased expression of several proteins including A2MG, C1QB, C1R, and HRG, which are involved in the inflammatory and immune response. These proteins may be potential biomarkers for prediction of future ischemic events.[80] Proteomic analysis of serum from patients with AIS and healthy controls using DIA resulted in identification of 11 potential protein biomarkers, including F2, VTN, and HRG, that were correlated with one or more stroke risk factors such as hypertension, cardiovascular disease, high cholesterol, and diabetes.[81]

Multiple proteins have been identified as promising indicators of stroke risk, but proteomics research on prestroke prognosis is underrepresented. This is likely due to the difficulty in prediction of stroke onset and a lack of appropriate animal models. Establishment of biobanks and increased access to human samples suitable for investigation of prestroke prognosis will allow for increased numbers of proteomics studies.

Challenges and strategies

Pharmaceutical industry and AIS research field face mounting pressure of developing therapeutics to save lives and prevent long-term disability and identifying prognostic and diagnostic biomarkers to enable stroke prevention screening. From a drug discovery perspective, the lack of comprehensive and in-depth understanding of molecular mechanisms and novel targets and biomarkers of AIS is the fundamental and most challenging scientific task. Shifting therapeutic strategies to target multiple pathophysiological processes to limit cell death and damage and to promote neuronal recovery are in progress. While it is promising, developing cell therapy has been challenging. In addition to improving sustained efficacy, safety and distribution of transplanted cells, systematic understanding of treatment effects and MoA at the molecular level would be crucial for validation and further development of such therapy. The research in this area would largely benefit from extensive proteomics investigation using animal models. Additionally, clinical validation of AIS therapeutics proves to be very difficult, as it requires a very large and diverse population and so representative of extremely heterogenous clinical presentations and intervention responses of stroke patients. Recruitment of study subjects is however a big challenge due to high death and disability rates and short initial treatment time window. To this end, rigorous validation of therapeutics and MoA preclinically and thorough characterization of target and biomarker candidates via advanced technologies like proteomics in various preclinical models, particularly in animal models, are essential.

On the other hand, proteomics investigation presents its own challenges. Proteomics is a sophisticated technology that generates large data sets and multidimensional biological information. To achieve excellence in proteomics studies requires a multidisciplinary team of scientists with expertise in biology, proteomics, and bioinformatics to correctly design a statistically powered study, meticulously execute the sample analysis, comprehensively analyze data, and properly interpret the results. Lack of proper follow-up for omics studies is a common challenge in drug discovery, mostly due to limited resources and access to advanced technologies. Many proteomics studies have generated lists of differentially expressed proteins as potential target and biomarker candidates, but few of these studies have been able to be used to determine relevant biological information. Broad orthogonal validation of the findings of proteomics studies is crucial. Several technologies can be used to facilitate multitarget validation. Targeted proteomics, like PRM, inherently with the highest sensitivity and quantitative accuracy among different data acquisition strategies used in MS-based proteomics, are readily accessible in most proteomics laboratories and can be used to accurately quantify tens to hundreds of proteins at relatively low cost. SomaScan (SomaLogic),[82] proximity extension assay (Olink),[83,84] and conventional antibody-based multiplex protein assays/arrays[85,86] are excellent tools for quantification of tens to thousands of proteins. Additional challenges include intrinsic lower sample analysis throughput in unbiased proteomics analysis compared to transcriptomics technologies (eg, RNAseq), low resolution in identifying and distinguishing proteoforms, and relatively low sensitivity in quantifying PTM proteins. Technology advancements in these areas are steadily progressing but are yet to be revolutionized. Proteomics scientists thus need to vet analytical options and have clearly defined study aims for a proteomic study. Furthermore, the number of reported AIS proteomics studies is still small and they are likely conducted on different preclinical models (eg, different species and stroke induction methods) with different study designs (eg, sampling scheme) and various proteomics platforms. Comparison of identified target and/or biomarker candidates across studies is difficult. More proteomics investigation of AIS is needed, and it is also of great importance to follow the best laboratory practice in this field ranging from sample preparation, instrumentation to bioinformatics. To take advantage of available proteomics data, performing a meta-analysis at the pathway level based on those differentially expressed proteins might generate interesting biological insights.

Conclusion and prospective

AIS preclinical research embraces proteomics technologies. Molecular mechanisms underlying the pathophysiological processes of AIS and MoA of therapeutic intervention are being delineated and novel therapeutic target and biomarker candidates are emerging. Promisingly, enriched molecular pathways and significantly changed proteins in response to ischemic induction have been repeatedly revealed in independent investigations with different study designs and/or animal models. Proteomics is a right approach and an effective path forward to address many challenges in current preclinical research and to build knowledge bases regarding AIS, thus improving and accelerating AIS drug discovery on multiple targets and pathways. To facilitate its broad application, a throughput leap in proteomics sample analysis, standardization and automation of sample process workflow, as well as an efficient and scalable data mining pipeline are urgently needed. To achieve the full potential of proteomics and thereby efficiently promote AIS therapeutic development, advanced proteomics technologies, including PTM proteomics (eg, phosphorylation and ubiquitination), interaction proteomics (eg, IP-LC-MS/MS), and chemoproteomics, can be applied to elucidate molecular signaling cascades, validate targets, and evaluate intervention effects. With the accretion of proteomics data, artificial intelligence will become an invaluable tool to mining data and unveiling vital biological insights.[87] Together with other omics, proteomics will accelerate the development of targets, biomarkers, and therapeutics, and significantly increase the translational significance of preclinical findings in stroke research.



Author contributions

FS, RFG, and RW conceived the content and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

Financial support

This work was supported by Biogen. The funder did not participate in data collection and analysis, article writing or submission.

Data availability statement

Not applicable.

Conflicts of interest

There are no conflicts of interest. No conflicts of interest exist between Biogen and publication of this paper.


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acute ischemic stroke; ischemic molecular mechanism; MCAO model; proteomics; stroke biomarker

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