Current Neurovascular Research - Volume 14, Issue 3, 2017

Background: Sclerostin is an inhibitor of the wingless-type mouse mammary tumor virus integration site family/β-catenin signalling pathway (WβcSP), which plays an important role in bone metabolism and in vascular biology. It could act protective regarding atherosclerosis development through its effect on WβcSP in vascular cells. Nevertheless, results of studies analyzing association between circulating sclerostin level (CSL) and atherosclerotic diseases (AD) are showing conflicting results. The aim of this study is to test the value of CSL as a biomarker of subclinical carotid atherosclerosis (SCA) in obese persons. Methods: The cross-sectional study included 50 obese persons without previous history of diabetes and AD. Participants underwent adequate anthropometrical, ultrasound and laboratory examinations, including 2h 75 g oral glucose tolerance test (OGTT). Results: Only the presence of SCA significantly indirectly correlated with CSL (p<0.05). Based on the median value of CSL, we formed two groups: low CSL (CSL7.9 pmol/l) and high CSL (CSL>7.9 pmol/l). There were no statistically significant differences in general (gender, age and current smoking) and anthropometrical characteristics (body mass index, waist circumference, systolic and diastolic blood pressure), inflammatory (total white blood cell count, erythrocyte sedimentation rate, fibrinogen, C-reactive protein and uric acid), glucose metabolism (fasting and 2h OGTT blood glucose, glycated hemoglobin and presence of dysglycemia) and lipid metabolism (low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B and lipoprotein (a)) parameters between low and high CSL groups. Low CSL group had significantly higher incidence of SCA (p<0.05). Conclusion: CSL could serve as a useful biomarker of early atherosclerosis in obese persons without previous history of cardiometabolic disorders but the final conclusion requires further testing.

Background: Autophagy is an intracellular bulk self-degrading process in which cytoplasmic contents of abnormal proteins and excess or damaged organelles are sequestered into autophagosomes, and degraded upon fusion with lysosomes. Although autophagy is generally considered to be pro-survival, it also functions in cell death processes. We recently reported on the hippocampal, higher vulnerability to cerebral ischemia in mice lacking the circadian clock protein PERIOD1 (PER1), a phenomenon we found to be linked to a PER1-dependent modulation of the expression patterns of apoptotic/autophagic markers. Methods: To exclude the contribution of vascular or glial factors to the innate vulnerability of Per1 knockout-mice (Per1^{136;’/136;’-}mice) to cerebral ischemia in vivo, we compared the autophagic machinery between primary hippocampal cultures from wild-type (WT)- and Per1^{136;’/136;’-}mice, using the lipophilic macrolide antibiotic, Rapamycin to induce autophagy. Results: Development of autophagy in WT cells involved an increased LC3-II-to-LC3-I ratio (microtubule-associated protein 1 light chain 3) and an overall increase in the level of LC3-II. In addition, immunostaining of LC3 in WT cells revealed the typical transformation of LC3 localization from a diffused staining to a dot- and ring-like pattern. In contrast, Per1^{136;’/136;’-}hippocampal cells were resistant to Rapamycin induced alterations of autophagy hallmarks. Conclusion: Our in vitro data suggests that basal activity of autophagy seems to be modulated by PER1, and confirms the in vivo data by showing that the autophagic machinery is depressed in Per1^{136;’/136;’-}hippocampal neurons.The implication of both autophagy and circadian dysfunction in the pathogenesis of cerebral ischemia suggests that a functional connection between the two processes may exist. may exist.

Background: Early neurological deterioration (END) was common in single small subcortical infarction (SSSI). Distal type of SSSI (dSSSI) was reported to have a lower risk of END than proximal type of SSSI (pSSSI) in lenticulostriate artery(LSA) territory. However, dSSSIs with different lesion thickness might have different risks of END. Objective: In this prospective cohort study, we aimed to investigate whether dSSSIs visible on ≥3 serial axial diffusion weighted imaging (DWI) slices were also imaging markers for END. Methods: Patients of SSSIs in the LSA territory admitted within 72 hours from the onset were selected in a prospective stroke database. Clinical characteristics including the occurrence of END after admission were recorded. The lowest slice (LS), total number of slices (TNS) involved and the maximum axial diameter were evaluated for lesion location and size on axial plane of DWI images. Lesion patterns were categorized according to LS and TNS. Multivariate logistic analysis was performed to determine the imaging pattern that associated with END. Results: A total of 201 out of 1,158 patients were analyzed. END occurred in 32(15.9%) patients after admission. SSSI was categorized to pSSSI (LS≤2), distal and large SSSI (dl-SSSI, LS>2, TNS≥3), distal and small SSSI (ds-SSSI, LS>2, TNS<3) respectively. Multivariate logistic analysis showed that ds-SSSI patients had a significantly lower rate of END(OR 0.20, 95% CI 0.06-0.71, P=0.013) comparing to dl-SSSI patients; pSSSI patients, however, had a similar rate of END (OR 1.27, 95% CI 0.50-3.21, P=0.611) to dl-SSSI patients. Conclusion: Except for pSSSI, dl-SSSI was also an imaging marker for END in the territory of LSA.

Background: Somatic mutations of Janus kinase 2 (JAK2V617F), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) are the major clonal molecules that drive the pathogenesis of myeloproliferative neoplasms (MPN). It is well recognized that MPN patients carry an excessive risk of thrombohemorrhagic complications. However, little is known about the prevalence of these clonal markers in patients with cerebral vascular disease. Methods: To address this issue, 153 consecutive stroke patients in Taiwan were enrolled in the study. Allele-specific PCR (AS-PCR), real-time AS-PCR, and Illumina paired-end sequencing were employed to detect the presence of MPL, JAK2V617F, and CALR exon 9 mutations, respectively. Results: JAK2V617F mutation was detectable in 13 samples (8.5%), but the allele burdens (AB) were greater than 1% in only six (3.9%) of them. Compared to JAK2-unmutated patients, those with JAK2V617F AB > 1% had significantly higher white blood count (p = 0.01), although four of the six did not exhibit MPN phenotypes. Two patients had a heterozygous CALR exon9 mutation locating outside the coding region and did not alter the amino acid sequence of this protein. On the other hand, there were no patients carrying the MPL mutations. Using patient age, baseline hemogram, and stroke-relevant risk factors, we developed a predictive model that could successfully identify stroke patients at risk of carrying clonal JAK2V617F mutation. Conclusion: The prevalence of JAK2V617F mutation in stroke patients was higher than that seen in general population. Based on our newly developed probability stratification model, genotyping of JAK2V617F mutation in selected patients with stroke might be warranted.

Background: Diabetic macular edema (DME) is resulted from the retinal microvascular leakage that accompanies the breakdown of blood-retinal barrier. Triamcinolone acetonide (TA) is a therapeutic agent for DME, but since the detailed mechanism of action of TA is not known, part of its action was examined. Methods: In vitro model to enhance the permeability of human retinal microvascular endothelial cells (HRMECs) was constructed by using DME-related cytokines such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The efficacy of TA and anti-VEGF antibody against retinal permeability was evaluated. Furthermore, the localization of ZO-1 in HRMECs was examined using immunofluorescence staining and the expression level of tight junction proteins (ZO-1, Occludin, and Claudin-5) was examined using immunoblot. Results: TA and anti-VEGF antibody showed inhibitory effects against VEGF-induced permeability enhancement, and TA also inhibited the increase in permeability induced by TNF-α and IL-1β. In addition to the inhibitory effects against cytokine-induced hyperpermeability, TA enhanced the barrier function of HRMECs and reduced the vascular leakage. TA altered the localization of tight junction proteins, but did not increase the upregulation of tight junction proteins. Moreover, the enhancement of barrier function by TA was inhibited by 17-AAG (glucocorticoid receptor inhibitor). Conclusion: It was suggested that TA reinforced the barrier function through the glucocorticoid receptor. In this study, we found that TA suppressed the inflammation caused by VEGF, TNF-α and IL-1β, and decreased the retinal vascular hyperpermeability.

Background: The impact of hyperfibrinogenemia on short-term outcomes after acute ischemic stroke (AIS) is still not well understood. Objective: We investigated the association between hyperfibrinogenemia upon hospital admission and the short-term prognosis of AIS patients. Methods: A total of 3,212 AIS patients enrolled from December 2013 to May 2014 across 22 hospitals in Suzhou city were included in the present study. Hyperfibrinogenemia was defined as having a serum fibrinogen>4.0g/L. Cox proportional hazard and logistic regression models were used to estimate the effect of hyperfibrinogenemia on all-cause in-hospital mortality and poor discharge outcome (modified Rankin Scale score≥3) in AIS patients. Results: During hospitalization, 106 patients (3.3%) died from all-cause and 1226 (38.2%) patients experienced poor functional outcome at discharge. Multivariable model adjusted for age, sex, baseline National Institutes of Health Stroke Scale score, white blood cell count and other covariates, showed that hyperfibrinogenemia was associated with a 1.76-fold increase in the risk of in-hospital mortality (hazard ratio [HR] 1.76; 95% confidence interval [CI], 1.10-2.81; P-value=0.019). However, there was no significant association between hyperfibrinogenemia and poor outcome at discharge (adjusted odds ratios[OR]1.15; 95% CI 0.86–1.53; P-value=0.338). Sensitivity and subgroup analyses also confirmed a significant association between hyperfibrinogenemia and in-hospital mortality. Conclusion: In patients with AIS, hyperfibrinogenemia at the time of admission was independently associated with increased in-hospital mortality.

Background: The most valid model for vascular cognitive impairment (VCI) is the mouse bilateral common carotid artery stenosis (BCAS), whose behavioral outcomes are supposedly affected by the remote ischemic conditioning (RIC) through the induction of autophgy. We hope to determine whether RIC contributes to the neuroprotection through the induction of autophagy. Methods: Wastar male mice were randomized into three groups including the Sham, the BCAS and the RIC+BCAS groups. All the animals were submitted to 4 cycles of 5 min occlusion and 5 min reperfusion of both the femoral vessels performing RIC. Then the animal behaviors were recorded as well as the expression of proteins and the mRNA levels. Notably, the expression of proteins relates to autophagy. By this means, it is possible to estimate the cell death, the severity of pathology and the expression of proteins under different groups. Results: Compared with the sham group, the expression of proteins increased for ATG7, Beclin-1, LC3, ATG5-ATG12 while decreased for P62 in the BCAS group. These changes were further promoted in the RIC+BCAS group, which indicates that the RIC can improve the cognitive function in the BCAS group. Moreover, RT-PCR demonstrated that the mRNA level of BECN1, Atg5, Atg7 in white matter (WM) and Hippocampus in BCAS group was higher than the sham group, while it was much greater in the RIC+BCAS group. This confirmed that the autophagy was activated in the BCAS and the RIC+BCAS groups, especially the RIC+BCAS group. Conclusion: Improved cognition during vascular injury and RIC was associated with increased activity of autophagy.

Background: Carotid artery stenosis (CAS) may induce cerebral hypoperfusion. Early-phase ¹⁸F-Florbetapir (AV-45/Amyvid, ¹⁸F-AV-45) positron emission tomography (PET) imaging can provide perfusion-like property (pAV-45) for the estimation of cerebral blood flow (CBF). Supra-tentorial lesions may cause decreased blood flow and metabolism in the contralateral cerebellum known as crossed cerebellar diaschisis (CCD). Objective: The aim was to study the occurrence of CCD after CAS using pAV-45 PET. Methods: Eleven healthy controls and 21 patients with unilateral CAS were studied. All subjects underwent ¹⁸F-AV-45 PET imaging and arterial spin labeling (ASL) CBF magnetic resonance perfusion imaging. The pAV-45 and ASL CBF values were first correlated. Then, cerebral and cerebellar hypoperfusion volume was analyzed. The cerebral and cerebellar perfusion asymmetry indices (AIs) were calculated from the pAV-45 standard uptake value ratios (SUVRs) of bilateral cerebral and cerebellar cortices, respectively. Results: We found that pAV-45 SUVR was significantly correlated to ASL CBF (p<0.0001, r=0.5731). The AI of cerebellar perfusion was negatively correlated to that of cerebral perfusion (p<0.0001, r=-0.8751). Multiple linear regression showed the cerebral AI (p<0.0001) and hypoperfusion volume (p=0.02) but not the infarction severity and CAS degree significantly correlated to cerebellar AI. If the lower limit of 95% confidence interval of cerebellar AI in healthy controls was set as cut-off for positive CCD, the occurrence of CCD correlated to infarction severity in CAS patients (p=0.03). Conclusion: Our results suggest pAV-45 is reliable to study CBF change. Under unilateral CAS, cerebral AI and hypoperfusion severity may determine the occurrence of CCD.

Background: An impaired cerebrospinal venous drainage was postulated to be a cofactor in the multifactorial pathogenesis of multiple sclerosis (MS). Chronic cerebrospinal venous insufficiency (CCSVI) is characterized by abnormalities of the main extracranial cerebrospinal venous outflow routes, which can be detected by color Doppler Ultrasound (CDUS) using 5 venous hemodynamic (VH) criteria. Discrepant results between different investigators were reported in the past, therefore the usefulness and applicability of the CCSVI CDUS-based diagnosis in clinical research and practice has been questioned. The reproducibility of proposed criteria for CCSVI detection depends on the blinding, training level, skills of the operator and interpretation of VH criteria. Objectives: To assess agreement between centralized and local reading of CDUS examination for diagnosis of CCSVI in trained Doppler sonologists. Methods: This study was performed in 78 MS patients and 28 age- and sex-matched healthy controls (HCs). Extracranial and transcranial CDUS venous hemodynamic assessment was conducted, according to International Society of Neurovascular Disease (ISNVD) recommended criteria, by a single CCSVI-trained expert sonologist blinded to the subject disease status. After the local Doppler sonologist performed the investigation, all images and video clips of the CDUS examination were sent to the centralized reading center, where a second blinded reading was performed by two CCSVI-trained expert sonologists. Statistical analyses were performed comparing accuracy of CCSVI diagnosis (≥2 VH criteria) and each of the 5 individual VH criteria using Cohen kappa statistic, along with positive/negative agreement and Odds ratio (OR) with 95% confidence intervals (95% CI). Results: Diagnosis of CCSVI was obtained in 59.7% of local and 64.3% centralized readers (Kappa, 0.67, p<0.001). Similar Kappa values were obtained for CCSVI diagnosis and individual CCSVI criteria in both MS patients and HCs. The highest Kappa between local and centralized readers was observed for VH criteria 5 (0.93) followed by VH criteria 4 (0.70), VH criteria 1 (0.66), VH criteria 2 (0.64) and VH criteria 3 (0.58). The positive predictive value (PPV) and negative predictive value (NPV) for CCSVI diagnosis were 82.7% and 86,7%, respectively with an OR of 31.1 (95% CI 11.1-87.5, p<0.001). The highest agreement between local and centralized readers was observed for VH criteria 4 (OR 98.7, 95% CI 17.1-569.9, p<0.001) with 72.7% PPV and 97.3% NPV followed by VH criteria 5 (53, 95% CI 13.4-209.2, p<0.001) with 98.1% PPV and 100% NPV value. Conclusion: Centralized reading of the CDUS examination for the diagnosis of CCSVI is feasible with high accuracy in CCSVI-trained Doppler sonologists. The most reproducible VH criteria between local and centralized readers were VH criteria 4 and 5.

Background: Promoting angiogenesis provides a possible therapeutic approach in treating spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a pro-angiogenic substance that is involved in endothelial cell (EC) proliferation, migration, and survival. Exogenous administration of VEGF to the lesion epicenter of the spinal cord has been recently revealed as a potential method for promoting the blood vessel sprouting. Methods: Spinal cord hemisection in a rat model was established and angiogenesis was studied through implant of an acellular spinal cord scaffold (ASCS) with sustained delivery of VEGF165. The poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating VEGF165 were fabricated on basis of an emulsion and solvent evaporation method and conjugated to ASCS by a Genipin (GP) crosslinking technology. The resultant scaffolds were marked as V-ASCS. VEGF165 entrapment efficiency (EE) and released kinetics were determined by an ultraviolet absorption measurement. Angiogenesis and vascular remodeling were observed via a high-resolution micro-CT and analyzed quantitatively by vascular morphometric parameters. Spinal cord histology and Basso, Beattie, and Bresnahan (BBB) locomotor rating scale were further studied. Results: VEGF165 was entrapped with high efficiency (90.8±3.1) %. In vitro VEGF165 release kinetics study showed an initial burst of 1.966 µg mg NPs-1 and 1.045µg mg V-ASCS-1 respectively in the first 24 hours. In the phase of sustained release, approximately 0.040µg mg NPs-1 and 0.022µg mg V-ASCS-1 per day was on-going until 720h. In the rat spinal cord hemisection model, implant of V-ASCS at the injured site showed a promotion of angiogenesis and vascular remodeling following SCI. A better outcome can be confirmed histologically. However, functional improvement is limited in the animal model. Conclusion: The results indicate that progress of vascular reconstruction is accelerated in the V-ASCS implanted SCI rats.

Background: Alzheimer’s disease is a severe neurodegenerative brain disorder, showing severe beta-amyloid depositions in the brain (plaques) and in vessels (cerebral amyloid angiopathy, CAA), tau pathology, neurodegeneration (and loss of acetylcholine), inflammation with reactive astrocytes and microglia and cerebrovascular damage, all resulting in memory loss. Methods And Results: In this review, I present a hypothesis that chronic vascular lesions and bleedings cause platelet overactivation and repair. Platelets express large amounts of amyloid precursor protein (APP) and release beta-amyloid, possibly playing a role as a clotting substance. As the number of bleedings increases over lifetime, the function of platelets diminishes until they are dysfunctional. Dysfunctional processing of APP in platelets and subsequent inflammatory processes may play a role in the formation of CAA. Local lesions and acidosis may transfer a pathological cascade including silent strokes into the brain, causing irreversible APP dysfunction, tau dysregulation and beta-amyloid deposition. Conclusion: Platelets may play a central role in the processing of plaque deposition in the Alzheimer brain and may be of interest for diagnostic as well as therapeutic strategies.

Background: The mammalian circadian clock and its associated clock genes are increasingly been recognized as critical components for a number of physiological and disease processes that extend beyond hormone release, thermal regulation, and sleep-wake cycles. New evidence suggests that clinical behavior disruptions that involve prolonged shift work and even space travel may negatively impact circadian rhythm and lead to multi-system disease. Methods: In light of the significant role circadian rhythm can hold over the body's normal physiology as well as disease processes, we examined and discussed the impact circadian rhythm and clock genes hold over lifespan, neurodegenerative disorders, and tumorigenesis. Results: In experimental models, lifespan is significantly reduced with the introduction of arrhythmic mutants and leads to an increase in oxidative stress exposure. Interestingly, patients with Alzheimer's disease and Parkinson's disease may suffer disease onset or progression as a result of alterations in the DNA methylation of clock genes as well as prolonged pharmacological treatment for these disorders that may lead to impairment of circadian rhythm function. Tumorigenesis also can occur with the loss of a maintained circadian rhythm and lead to an increased risk for nasopharyngeal carcinoma, breast cancer, and metastatic colorectal cancer. Interestingly, the circadian clock system relies upon the regulation of the critical pathways of autophagy, the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), and silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) as well as proliferative mechanisms that involve the wingless pathway of Wnt/β-catenin pathway to foster cell survival during injury and block tumor cell growth. Conclusion: Future targeting of the pathways of autophagy, mTOR, SIRT1, and Wnt that control mammalian circadian rhythm may hold the key for the development of novel and effective therapies against aging- related disorders, neurodegenerative disease, and tumorigenesis.