Circulating Biomarkers For Collateral And Disease Progression In Symptomatic Intracranial Atherosclerosis

BackgroundIntracranial atherosclerotic stenosis(ICAS),as a leading cause of stroke and death worldwide,contributes to the incidence of acute ischemic stroke(AIS)predominantly in China.Several recent studies show that collaterals dramatically alter stroke risk and functional outcome in intracranial arterial steno-occlusive disease(ICASD).However,the extent of collaterals in ICAS is limited as seventy percent of patients with significant stenosis absent of collaterals,which potentially accounts for the worse outcome compared to other stroke etiologies.The annual rate of recurrent stroke and death in symptomatic ICAS(sICAS)is above 10%,although the first-line intensive medical management(IMM)characterized by antiplatelet treatment in conjunction with aggressive control of vascular risk factors has been optimized in the last decade.The high prevalence,poor prognosis,and lack of treatment options for sICAS necessitate the identification of meaningful markers of disease evolution.Serological studies regain attention recently,which attributes to the discovery of novel biomarkers and the improvement of detection methods.The investigation of serological biomarker in sICAS may uncover the relevant mechanism underlying disease progression and new targets for development of improved therapeutic strategies.Objectives1)assessing the association between collateral development in ICASD and the serological composition of two alternative splicing products of vascular endothelial growth factor-A165(VEGF-A165),namely VEGF-A165 a and VEGF-A165b.2)exploring the angiogenesis-related circulating cytokines in relation to IMM failure and recurrent ischemic events in sICAS.3)exploring the circulating exosomal microRNAs(e-miRNAs)in relation to IMM failure and recurrent ischemic events in sICAS,and evaluating regulatory effects of the relevant e-miRNAs on angiogenesis-related biological functions and the significant circulating cytokines found in Objective 2MethodsAngiogram-diagnosed severe sICAS(more than 70%stenosis)were enrolled in this cohort study.Immediately upon enrollment,blood samples were collected,processed and stored in-80℃ freezer for subsequent measurement.For objective 1,patients with moyamoya disease(MMD)and epilepsy were recruited simultaneously as positive and negative control of collaterals,respectively.Blood samples were also collected at baseline.The plasma levels of VEGF-A165a and VEGF-A165b were measured using enzyme-linked immunosorbent assay(ELISA)and compared among the three groups.For objective 2,the enrolled sICAS patients were categorized into two groups based on the incidence of recurrent ischemic events within 6 months after IMM initiation.Those who failed IMM and had recurrent ischemic events were termed as Non-responders while the others as Responders.Thirteen pro-angiogenic factors and eight anti-angiogenic factors were measured using ELISA.Principle component analysis(PCA)was conducted to determine the association between a broad spectrum of angiogenic factors and recurrent ischemic events in patients with sICAS.For objective 3,Responders and Non-responders in Objectives 2 were further matched in 1:1 ratio through propensity score matching,considering age,gender,and compliance to IMM.Next Generation Sequencing(NGS)technology was applied to quantify the expression levels of e-miRNAs in the matched cohort.PCA was carried out for selecting significant e-miRNAs in association with recurrent ischemic events.The regulatory effects of the significant e-miRNAs on angiogenesis-related functions and the meaningful angiogenic factors identified in Objective 2 were further evaluated in Ingenuity Pathway Analysis(IPA)environment.ResultsA total of 74 sICAS patients,6 MMD patients,and 5 epilepsy patients were enrolled.Among patients with sICAS,66 had The American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology Score(ASITN/SIR)less than 2,indicating poor collaterals.MMD patients had a similar mean plasma level of VEGF-A165 but significantly higher VEGF-A165a/b ratio comparing to sICAS(p=0.015)and epilepsy(p=0.043).Twenty-nine patients with sICAS experienced recurrent ischemic events during 6-month follow up.PCA transformed 21 angiogenic factors into 7 angiogenic profiles(AP)retaining 75%of original data variation.Univariable and multivariable binary logistic regression analysis revealed that only AP4 was significantly associated with recurrent ischemic events in patients with sICAS(p=0.02),even after adjustment for clinical variables including age,gender,previous stroke,hypertension,hypercholesterolemia,diabetes mellitus,and obesity(p=0.003).The dominant elements of AP4 containing soluble VEGF receptor 1(sVEGFR1),endostatin,angiostatin,hepatocyte growth factor(HGF),and VEGF-A165a exerts a net effect of anti-angiogenesis.Eleven responders and 11 non-responders were further matched by PSM.NGS generated a total of 122 e-miRNAs that were mapped to reference genome and isolated after noise elimination.After PCA,13 principle components(PC)were produced and only PC3 was significantly associated with recurrent ischemic events in patients with sICAS(p=0.0052).Thirteen significant e-miRNAs with the absolute coefficients above 75%quantile were identified as the major contributors of the PC3,including miR-27b-3p,miR-122-5p,miR-16-5p,miR-30c-5p,miR-486-5p,miR-10a-5p,miR-10b-5p,miR-101-3p,miR-24-3p,miR-192-5p,miR-30c-5p,miR-425-5p,and miR-191-5p.The modified PC3 with only these 13 e-miRNAs as components was highly correlated with the original PC3(R2=0.940),and was able to predict recurrent ischemic event with 90%sensitivity and 100%specificity.IPA demonstrated that seven of the 13 meaningful e-miRNAs had in the functional analysis statistically significant associations with angiogenesis-related functions(p<0.025),and 5 of them were upstream modulators of VEGFA,VEGFR1,HGF,and MMP7 as a main proteinase for production of angiostatin and endostatin,which have been shown association with recurrent ischemic events.Furthermore,the regulatory effects of the distinctive e-miRNA expression profile in non-responders were predicted via MAP,which revealed a net effect of inhibition of all the angiogenesis-related functions.In addition,the predicted expression of VEGFA,VEGFR1,HGF,angiostatin,and endostatin in accordance to the specific e-miRNA expression profile in non-responders was consistent with the actually observed expression of these angiogenic factors in Objective 2.ConclusionAntiangiogenesis may play a pivotal role on inhibition of ischemia-induced collateral development and disease progression despite IMM in sICAS.The anti-angiogenic phenotype presented by circulating angiogenic factors and e-miRNAs suggests unfavorable outcomes in sICAS.