Introduction:Stroke prognosis. (3) Many efforts have addressed identifying

Introduction:Stroke is a leading cause of death and long-term adult disability worldwide. (1) Stroke include two main types, ischemic stroke (IS) and hemorrhagic stroke (HS).

IS is the major type of the stroke, responsible for more than 80% of cases.(2) (3) Stroke mortality is mainly through causing neurological damage and cardiovascular complications. (4)Stroke is a complex and diverse disease and a single biomarker may not be able to reflect this complexity. A number of markers are available but none could be used efficiently in clinical practice. (5) The diagnosis of stroke is mainly done clinical and later confirmed by neuroimaging techniques (6)(7)(3) like Computed Tomography (CT) or MR imaging examination.

Despite that, it is difficult to discriminate stroke from other neurological and non-neurological diseases when the symptoms are similar. Till the present, no reliable biomarkers for stroke risk prediction, diagnosis, and prognosis. (3) Many efforts have addressed identifying stroke biomarkers to reach better diagnosis that is (7)(8) fast, cost effective, specific, and sensitive.(3)The vascular endothelium constitutes the endothelial barrier between blood and the surrounding tissues. They are first line of defense against inflammatory, hypoxic and chronic stress. They react to stress signals such as hypoxia and inflammation by changing cellular physiology and secretion of growth factors and cytokines that recruit endothelial progenitor cells or cells that play a role in the innate immune response.(9) Exosomes are 30–100 nm membrane-derived vesicles released by most cell types and exist in most body fluids.

They carry different proteins, nucleic acids and lipids from their host cell. Exosomes function cell-to-cell communication through their miRNAs shipments. (10) They are key elements in acquisition pathogenic information and recognizing biomarkers, especially for non-accessible tissues like the central nervous system. (1) This review aim to outline the potential use of endothelial exosomes as biomarker for stroke, addressed by several studies.Stroke Biomarkers:Biomarkers are important in drug development by providing more accurate and complete information related to drug performance, disease progression, or response to a specific drug therapy. Stroke biomarkers include protein, genetic, extracellular vesicle (EV), and metabolomics associated biomarkers.(11)Markers for stroke can include that of brain tissue damage, inflammation, and coagulation /thrombosis, such as C-reactive protein, interleukin-6, and D-dimer. (3) Among which, IL-6 is an early marker for outcome in acute ischemic stroke.

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(30) Given that heterogeneity of IS, the successful translation to a protein biomarker useful in clinical practice has been provenGhada Yousif Mohammed Student Number: 2100080632 | P a g edifficult.(3) A biomarker panel may be able to better reflect the diverse pathophysiology involved in stroke and thereby distinguish ischemic stroke from hemorrhage, predict which TIAs proceed to stroke, and predict causes of stroke.(5)Blood biomarkers might provide useful information to improve the prediction of outcome after acute ischemic stroke.

Although some markers had some predictive ability, none of the current biomarker added predictive power to a validated clinical model. The clinical usefulness of current blood biomarkers for predicting prognosis in the setting of ischemic stroke has yet to be established.(12)(13)(14)Exosome role in stroke pathophysiology:The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis.(9)Notable progress has been made toward understanding the physiopathology and biochemistry of stroke.

Number of ischemic injury-related proteins have been identified, such as calcium binding protein B (S100B), neuron-specific enolase (NSE),myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP)(1)Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for “acquired” proteins or micro RNAs (miRNAs) repressing “resident” mRNA translation suggests that they can influence the physiological behavior of recipient cells.(15)In acute ischemic stroke the mechanisms of brain ischemia are based on endothelial, vascular and inflammatory factors.

Endothelial membrane microparticles (EMP) in plasma are elevated in several vascular diseases. Certain circulating EMP were found to be associated with severity, lesion volume and outcome of acute ischemic stroke. EMP analysis contribution to understanding stroke pathophysiology.(16)Exosome-Based biomarker for stroke diagnosis and prognosis:Several factors have increased the interest in exosomes as a source for stroke biomarkers. First, exosomes are accessible, they are present in plasma and can circulate in the body using blood.(17) Second, endothelium-derived exosomes are involved in the endothelial response to cellular stress like hypoxia or inflammation, (9) that are known to be associated with stroke.

(18) Third, the RNA and protein content of exosomes can be used to evaluate the physiological condition of their producing cells. (9)Ghada Yousif Mohammed Student Number: 2100080633 | P a g eMicroRNAs are promising disease markers due to their cell type–specific expression patterns, stability in peripheral blood (19)(20) and less prone to minor differences in sample processing. (20) MicroRNAs (miRNAs) are small, non-coding RNAs that function in regulating different biological processes by influencing messenger RNAs (20)(21)Extracellular miRNAs in serum, plasma, saliva, and urine were found to be associated with pathological conditions including cancer.(21)Their expression patterns reflect the pathophysiological status of a tissue and particular disease states.(20) The presence of miRNAs in the circulatory system is mediated via exosome.(22) miRNA signatures from plasma, serum and whole blood are not significantly different and they are abundant inside exosomes.

Overall, Exosome isolation improves the sensitivity of miRNA detection and reduces false negative. (20)Exosomal-miRNAs associated with stroke:MiRNAs related to endothelial/vascular function, erythropoiesis, angiogenesis and neural function had charachteristic expression profile in cerebral ischaemic stroke. This, included miRNAs that are involved in hypoxic conditions. Suggesting that, they could be used as biomarkers in diagnosis and prognosis of stroke. (23)From these, miR-214 regulates endothelial cell function and angiogenesis, plays a key role in exosome-mediated signaling between endothelial cells. Endothelial cells release miR-214-containing exosomes to stimulate angiogenesis was reported in human and mouse endothelial cells.(15) Furthermore, the serum concentration and levels of exosomal miR-124 and miR-9 were reported to be significantly higher in acute ischemic stroke (AIS) patients than control non-stroke.

Their levels were positively correlated with NIHSS scores, infarct volumes and serum concentrations of IL-6. (1) Indicating that, exosomal miR-9 and miR-124 are promising biomarkers for diagnosing AIS and evaluating the degree of damage caused by ischemic injury (1)Down-regulation of serum brain specific microRNA is associated with inflammation and infarct volume in acute ischemic stroke. Serum miR-124 was significantly decreased within 24 hours after stroke onset and serum miR-9 was decreased in patients with larger stroke. There were no significant changes in serum miR-219. Both serum miR-124 and miR-9 levels within 24 hours were negatively correlated with infarct volume and plasma high-sensitivity C-reactive protein levels. So, serum miR-124, miR-9 and miR-219 are suppressed in acute ischemic stroke thus facilitating neuroinflammation and brain injury. (29)Also, microRNA-126 (miR-126), which is endothelial cell specific and regulate EC function, controlling angiogenesis, and maintaining vascular integrity, significantly decreased in ischemic stroke.

MiR- 126 facilitates vascular remodeling and decreases fibrosis. Moreover, decreased miR- 126, derived from endothelial cells, expression were found to associate withGhada Yousif Mohammed Student Number: 2100080634 | P a g ecardiac dysfunction after stroke in mice.(4) In addition, the significant reductions in serum miR-126 were detected at 3 h after permanent ischemia but not transient ischemia in mice model. Indicating, the potential of using the changes in miR-126 level in differentiating severe permanent ischemia from milder injury after transient ischemia.(24) Research conducted in stroke patients showed, miR-30a and miR-126 levels were markedly decreased until 24 weeks.

Among all patients, circulating miRNAs levels returned to normal 48 weeks after symptom onset.(6)Also, miRNA-221-3p and miRNA-382-5p levels were significantly lower in patients with ischemic stroke compared to healthy control subjects, whereas there was no significant difference in the serum levels of miRNA- 4271 between the stroke patients and healthy controls. Serum circulating miRNA-221-3p and miRNA-382-5p might be used as potential noninvasive biomarkers for the diagnosis of ischemic stroke. significant difference of serum miRNA-221-3p and miRNA-382-5p between AIS and healthy control subjects, and serum miRNA-4271 in- creased in majority of IS patients but it need further confirmation due to the small sample size(2)On the other hand, the let-7 family ( let-7a, b, c, and e) found to be elevated within 48 h in brain from a rat stroke model. (22) This increase in miR-17 level was further detected inin acute ischemic stroke and found to be associated with future stroke recurrence.(25) Specifically, let-7c and miR-221-3p in levels serum were up-regulated that are related to stroke and let-7e expression (in blood and CSF) was markedly increased at the acute stage of IS but not in the IS patients at recover stage. No significant association between serum let-7e levels and NIHSS scores in IS patients at acute stage but positively correlated with the serum CRP levels.

Also, miR- 338 was found to be stably expressed in peripheral circulation during the different IS courses.(22)Also, circulatory microRNA-145 expression was reported to be significantly higher in ischemic stroke patients than in control subjects.(27) (26)Exosomal microRNA-223 is associated with acute ischemic stroke and plays a role in stroke through up-regulating growth factor such as insulin-like growth factor-1 gene.

(28) MiR-223 in acute ischemic stroke patients was significantly increased compared to control group. Exosomal miR-223 level was positively correlated with NIHSS scores. It expression in stroke patients with poor outcomes was higher than those with good outcomes. Overall, increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. (7)Comparison of miR-124-3p and miR-16 for early diagnosis of hemorrhagic and ischemic stroke. The primary outcome was the differentiation of hemorrhagic and ischemic stroke. Median plasma124-3p concentrations taken within 24 h of symptom onset were higher in HS patients than that in IS patients, while median miR-16 concentration in IS patients were higher than that in HS patients. Both miR-124-3p and miR-16 are diagnostic markers to discriminate HS and IS.

(31)Ghada Yousif Mohammed Student Number: 2100080635 | P a g eupregulated in patients with IS compared with both healthy control subjects (miR-125a-5p , miR-125b-5p and miR-143-3p and patients with transient ischemic attack. miR-143-3p has been linked to the phenotype and function of vascular smooth muscle cells and endothelial cells. Whereas miR-125a-5p has been linked to supporting endothelial barrier properties in the context of the blood–brain barrier and to the differentiation of inflammatory cell smiR-125b-5p regulates synaptic morphology and function. In conclusion, circulating microRNAs (miR-125a-5p, miR-125b-5p, and miR-143-3p) associates with acute IS and might have clinical utility as an early diagnostic marker. (19)Moreover, the impact of age and sex on miRNA expression following ischaemic stroke in an animal model was investigated. Adult and middle-aged female and male rats. At 2 days post-stroke, 21 circulating miRNAs were differentially regulated and the variance was due to age.

At 5 days post-stroke, 78 circulating miRNAs exhibited significantly different regulation, and most of the variance was associated with sex. miRNAs, miR-15a, miR-19b, miR-32 miR-136 and miR-199a-3p, were found to be highly expressed exclusively in adult females compared with middle-aged females, adult males and middle-aged males. The pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity.

(32)Also. the characterization of serum miRNAs profile from middle-old-aged patients with acute IS revealed 115 miRNAs were differentially expressed in IS, among which miR-32-3p, miR-106-5p, and miR-532-5p were first found to be associated with IS.(3) In conclusion, large number of exosomal miRNA found to be associated with stroke, either decreased or increased, and have the potential to be an efficient biomarker to stroke.Conclusion and Future Directions:Biomarkers in stroke represent a possible challenge in the diagnostic and prognostic evaluation of stroke onset and pathogenesis and in poststroke recovery.(33) Biomarker panel may add valuable and time-sensitive diagnostic information in the early evaluation of stroke.

(14)Exosome have the potential to be a biomarker with clinical utility for stroke.Cells release at least two major subpopulations of exosomes with distinct molecular compositions and biological properties, this is consistent across several cell types as well as for plasma, indicating the heterogeneity within exosomes. (34)In addition, better methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) have been developed. Which provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs. (35)(36)Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics. (34)Further studies are needed to explore the potential roles of the exosomes released from brain tissues in post stroke complications.(1) Large sample are needed to assess the clinicalGhada Yousif Mohammed Student Number: 2100080636 | P a g eapplication of exosomal miRNA as a novel biomarker for ischemic stroke diagnosis.

(7) its target in ischemic pathogenesis are also essential.(7)Confirm their role and the sensitivity and specificity should be analyzed in larger, long term studies. And the reasons behind varying expressions of some miRNA among various types of ischemic stroke deserves further investigation.

(6)New question and hypothesis:Panel of Exosomal miRNAs could be used as effective biomarker for early diagnosis of stroke, distinguish ischemic stroke from hemorrhage, and predict causes of stroke.Could Exosomal miRNAs panels be used as stroke biomarker for early diagnosis and prognosis?Potential ethical issue:Since the objective of the future research is finding a diagnostic biomarker for stroke, it will involve human subjects and the regulation that governs enrolling human in biomedical research should be followed.(37)(38)(39)Potential intellectual property issue:There is a patent on methods and systems that use exosomes for determining phenotypes with the number US 20100203529 A1.(40)References:1.

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