Acute ischemic stroke in Tsutsugamushi: understanding the underlying mechanisms and risk factors.

BACKGROUND: Tsutsugamushi (scrub typhus) is an acute infectious febrile disease common in the Asia-Pacific region. Common symptoms of tsutsugamushi include lymphadenopathy, fever, and myalgia, and it rarely causes acute ischemic stroke (AIS). However, we hypothesized that tsutsugamushi infection could trigger AIS. METHOD: We retrospectively examined patients diagnosed with AIS within 2 weeks of tsutsugamushi diagnosis at three hospitals over a 15-year period. We categorized patients who developed AIS while being treated for tsutsugamushi as the case group and those (of similar age and sex) who did not develop AIS as the control group. The case and control groups consisted of 22 and 66 participants, respectively. When a scattered pattern was observed or lesions were found in two or more vascular territories on diffusion-weighted imaging, the pattern was defined as embolic. Other patterns were defined as nonembolic. RESULTS: Among the 19 patients, excluding three with transient ischemic stroke, 15 (78.9%) showed an embolic pattern. Although fever was common in the control group, it was less common in the case group. A higher D-dimer level at the time of hospitalization was associated with the development of AIS in patients with tsutsugamushi. CONCLUSIONS: AIS in patients with tsutsugamushi showed an embolic rather than a non-embolic pattern on brain magnetic resonance imaging. It was more likely to occur in patients with risk factors for stroke. Tsutsugamushi patients with AIS were likely to have no fever or high D-dimer levels. We hypothesized that D-dimers play an important role in the pathophysiology, where tsutsugamushi infection increases the likelihood of AIS.

Circulating Extracellular Vesicles in Stroke Patients Treated With Mesenchymal Stem Cells: A Biomarker Analysis of a Randomized Trial.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete trophic factors and extracellular vesicles (EVs). However, the level and role of EVs after MSC therapy in patients with stroke are unknown. We investigated whether circulating EVs and trophic factors are increased after MSCs and are related to the therapeutic benefits in the STARTING-2 trial (Stem Cell Application Researches and Trials in Neurology-2) participants. METHODS: In this prospective randomized controlled trial, patients with chronic major stroke were assigned, in a 2:1 ratio, to receive autologous MSC intravenous injection (MSC group, n=39) or standard treatment (control group, n=15) and followed for 3 months. Detailed clinical assessment and neuroplasticity on diffusion tensor image and resting-state functional magnetic resonance imaging were evaluated. Serial samples were collected, before/after MSCs therapy. The primary outcome measure was circulating factors that are associated with the clinical improvement in the Fugl-Meyer Assessment (secondary end point of the trial) and neuroplasticity on diffusion tensor image and resting-state functional magnetic resonance imaging. Additional outcome measures were microRNAs and trophic factors enriched in the plasma EVs, obtained using quantitative polymerase chain reaction and ELISA, respectively. RESULTS: Circulating EV levels were increased ≈5-fold (mean±SD, from 2.7×109±2.2×109 to 1.3×1010±1.7×1010 EVs/mL) within 24 hours after injection of MSCs (P=0.001). After adjustment of age, sex, baseline stroke severity, and the time interval from stroke onset to treatment, only the EV number was independently associated with improvement in motor function (odds ratio, 5.718 for EV numberLog [95% CI, 1.144-28.589]; P=0.034). Diffusion tensor image and resting-state functional magnetic resonance imaging showed that integrity of the ipsilesional corticospinal tract and intrahemispheric motor network were significantly correlated with circulating EV levels, respectively (P<0.05). MicroRNAs related to neurogenesis/neuroplasticity (eg, microRNA-18a-5p) were significantly increased in circulating EVs after MSC therapy (P=0.0479). In contrast, trophic factor levels were not changed after MSC therapy. CONCLUSIONS: This trial is the first to show that treatment of ischemic stroke patients with MSCs significantly increases circulating EVs, which were significantly correlated with improvement in motor function and magnetic resonance imaging indices of plasticity. REGISTRATION: URL: https://www. CLINICAL TRIALS: gov; Unique identifier: NCT01716481.

Circulating DNAs, a Marker of Neutrophil Extracellular Traposis and Cancer-Related Stroke: The OASIS-Cancer Study.

Background and Purpose- The role of circulating neutrophil extracellular traps (NETs) in cancer-related stroke is unknown. Methods- We conducted a prospective cohort study to test whether NETs are increased in cancer-related stroke and whether elevated NETs levels are associated with coagulopathy, assessed using D-dimer levels (≥2 µg/mL). Plasma DNA and nucleosome were assessed as NET-specific biomarkers. Results- In total, 138 patients were recruited; 38 patients had cancer-related stroke (active cancer and acute cryptogenic embolic stroke), 33 patients were healthy-controls, 27 patients were cancer-controls (active cancer but no stroke), and 40 patients were stroke-controls (acute ischemic stroke but no cancer). Plasma DNA and nucleosome levels were significantly elevated in cancer-related stroke patients than in healthy-controls (P<0.05). These levels were correlated with the D-dimer levels (P<0.01). In multiple regression analyses, increased plasma DNA levels were associated with cancer-related stroke (odds ratio=11.65 for highest quartile; 95% CI, 3.199-42.46) and D-dimer levels of ≥2 µg/mL (odds ratio=19.09 for highest quartile; 95% CI, 4.143-87.95) after adjusting for possible confounders. Conclusions- Increased circulating DNA levels were associated with cancer-related stroke, suggesting that NETosis is one of the molecular mechanisms of cancer-related stroke. Further long-term follow-up studies in large cohorts are needed to confirm the role of NET-specific biomarkers.

Association of Cancer Cell Type and Extracellular Vesicles With Coagulopathy in Patients With Lung Cancer and Stroke.

BACKGROUND AND PURPOSE: Coagulopathy is an important cause of stroke in cancer patients. However, underlying mechanisms and clinical factors related to coagulopathy remain unclear. We hypothesized that certain characteristics of cancer affect coagulopathy in patients with lung cancer and ischemic stroke. METHODS: Consecutive patients with active lung cancer and acute ischemic stroke were prospectively studied. Volume and pattern of acute brain infarcts and plasma levels of circulating tumor extracellular vesicles (EVs) were measured using flow cytometry. In vitro experiments investigated the pathophysiological mechanisms underlying cancer-associated coagulopathy. RESULTS: Of 114 patients, 95 (83.3%) had an adenocarcinoma cell type and 95 (83.3%) had distant metastasis. Acute brain infarct volumes were larger and circulating EV levels were higher in patients with an adenocarcinoma cell type than in those with other cell types. The presence of metastasis was not associated with infarct volume or circulating EV levels. Coagulation assays demonstrated dose-dependent shorter clotting times after treatment with EVs from adenocarcinoma cell lines than with the use of EVs from squamous cell carcinoma. These findings were confirmed by coagulation assays using circulating EVs from patients with adenocarcinoma and stroke and from those with conventional stroke mechanisms. CONCLUSIONS: Our findings indicate that cancer cell type is associated with circulating EV levels and coagulopathy in patients with lung cancer and stroke.

Cav-1 (Caveolin-1) and Arterial Remodeling in Adult Moyamoya Disease.

Background and Purpose- Moyamoya disease (MMD) is a unique cerebrovascular occlusive disease characterized by progressive stenosis and negative remodeling of the distal internal carotid artery (ICA). We hypothesized that cav-1 (caveolin-1)-a protein that controls the regulation of endothelial vesicular trafficking and signal transduction-is associated with negative remodeling in MMD. Methods- We prospectively recruited 77 consecutive patients with MMD diagnosed via conventional angiography. Seventeen patients with intracranial atherosclerotic stroke and no RNF213 mutation served as controls. The outer distal ICA diameters were examined using high-resolution magnetic resonance imaging. We evaluated whether the degree of negative remodeling in the patients with MMD was associated with RNF213 polymorphism, cav-1 levels, or various clinical and vascular risk factors. We also investigated whether the derived factor was associated with negative remodeling at the cellular level using the tube formation and apoptosis assays. Results- The serum cav-1 level was lower in the patients with MMD than in the controls (0.47±0.29 versus 0.86±0.68 ng/mL; P=0.034). The mean ICA diameter was 2.48±0.98 mm for the 126 affected distal ICAs in patients with MMD and 3.84±0.42 mm for the asymptomatic ICAs in the controls ( P<0.001). After adjusting for confounders, cav-1 levels (coefficient, 1.018; P<0.001) were independently associated with the distal ICA diameter in patients with MMD. In vitro analysis showed that cav-1 downregulation suppressed angiogenesis in the endothelial cells and induced apoptosis in the smooth muscle cells. Conclusions- Our findings suggest that cav-1 may play a major role in negative arterial remodeling in MMD.

Distinct Roles of Endothelial Dysfunction and Inflammation in Intracranial Atherosclerotic Stroke.

BACKGROUND/AIMS: The aim of the study was to evaluate the differential roles of endothelial dysfunction and inflammation in intracranial atherosclerotic stroke (ICAS). METHODS: We prospectively recruited 262 patients with acute cerebral infarcts caused by ICAS and 75 individuals with no history of stroke as controls. Markers of endothelial dysfunction (asymmetric dimethylarginine, ADMA) and inflammation (lipoprotein-associated phospholipase A2, Lp-PLA2) were measured. Acute ischemic lesions were measured in terms of their size, composition, and patterns. Subclinical microangiopathy (degree of leukoaraiosis) and macroangiopathy (presence/number of asymptomatic stenoses) were graded in each patient. RESULTS: Compared to normal controls, serum levels of ADMA (0.69 ± 0.14 vs. 0.47 ± 0.10, p < 0.001) and Lp-PLA2 (138.1 ± 116.8 vs. 19.0 ± 58.0, p < 0.001) were elevated in patients with ICAS. A high ADMA serum level was associated with greater prevalence of preclinical microangiopathy and macroangiopathy. Contrastingly, an elevated serum Lp-PLA2 level was associated with larger ischemic lesions, a greater number of lesions, and a larger cortical pattern. CONCLUSIONS: Endothelial dysfunction and inflammation have distinct effects in ICAS patents; endothelial dysfunction is associated with the underlying micro- and macro-atherosclerotic burden, whereas inflammation is associated with acute infarct volume and pattern.

Trunk Impairment Scale for Predicting Lumbar Spine Bone Mineral Density in Young Male Patients With Subacute Stroke.

OBJECTIVE: To investigate the relationship between Trunk Impairment Scale (TIS) and lumbar spine bone mineral density (BMD) in subacute stroke patients. METHODS: Twenty-three subacute male stroke patients under the age of 65 were prospectively enrolled to exclude both postmenopausal and senile effects on BMD. The TIS, Berg Balance Scale, the Korean version of the Modified Barthel Index, and manual muscle test were measured at admission and 3 months after stroke onset. BMD of the bilateral lower extremities and lumbar vertebrae was measured by dual-energy X-ray absorptiometry 3 months after stroke onset. RESULTS: TIS at baseline (TIS_B) and TIS at 3 months after stroke (TIS_3m) showed significant correlations with lumbar BMD (TIS_B, r=0.522; TIS_3m, r=0.517). Through multiple regression analysis, the TIS_B was associated with lumbar BMD (adjusted R2=0.474). However, BMD of the bilateral lower extremities was not correlated with any clinical measurements except body mass index. CONCLUSION: We found a relationship between TIS_B and lumbar BMD in subacute young male stroke patients. Stroke patients with poor trunk control in the early subacute stage would have low BMD of vertebral bones at 3 months. The TIS can be useful for estimating bone fragility in the lumbar vertebrae of subacute stroke patients.

Treatment with Light-Emitting Diodes of Wavelength 863 nm Delays DMBA/TPA-Induced Skin Tumor Formation and Decreases Proinflammatory Cytokine Levels in ICR Mice.

The popularity of light/energy devices for cosmetic purposes (e.g., skin care) is increasing. However, the effects and underlying mechanisms remain poorly understood. Commencing in the 1960s, various studies have evaluated the beneficial effects of a light source on cells and tissues. The techniques evaluated include low-level light (laser) therapy and photobiomodulation (PBM). Most studies on PBM used red light sources, but, recently, many studies have employed near-infrared light sources including those of wavelength 800 nm. Here, we used a light-emitting diode (LED) array with a wavelength of 863 nm to treat DMBA/TPA-induced mouse skin tumors; treatment with the array delayed tumor development and reduced the levels of systemic inflammatory cytokines. These results suggest that light therapy could be beneficial. However, the effects were small. Further studies on different skin tumors using an optimized LED setup are required. Combination therapies (conventional methods and an LED array) may be useful.

The Role of Factor Xa-Independent Pathway and Anticoagulant Therapies in Cancer-Related Stroke.

BACKGROUND: The optimal strategy for stroke prevention in cancer patients is unknown. We compared the underlying mechanisms of coagulopathy and the effects of anticoagulants in patients with active cancer and atrial fibrillation (AF). METHODS: We retrospectively enrolled 46 consecutive patients with embolic stroke of unknown source and active cancer (cancer stroke). We consecutively screened patients with cancer patients without stroke (n = 29), AF stroke (n = 52), and healthy subjects (n = 28), which served as controls. Patients with cancer stroke were treated with either enoxaparin (a low-molecular-weight heparin) or a factor Xa inhibitor, and those with AF stroke were treated with factor Xa inhibitors. D-dimer, factor Xa, and circulating cell-free DNA (cfDNA), a marker of neutrophil extracellular traposis, were measured at both before and after anticoagulation. RESULTS: In AF stroke, factor Xa activity and cfDNA and D-dimer levels were decreased by treatment with factor Xa inhibitors. In contrast, in cancer stroke, factor Xa activity was decreased, D-dimer levels were unchanged, and cfDNA levels were increased by treatment with factor Xa inhibitors. In cancer stroke patients treated with enoxaparin, D-dimer levels were decreased (p = 0.011) and cfDNA levels were unchanged. CONCLUSION: The anticoagulation effects of factor Xa inhibitors differed between cancer stroke and AF stroke.

Brain morphological and connectivity changes on MRI after stem cell therapy in a rat stroke model.

In animal models of stroke, behavioral assessments could be complemented by a variety of neuroimaging studies to correlate them with recovery and better understand mechanisms of improvement after stem cell therapy. We evaluated morphological and connectivity changes after treatment with human mesenchymal stem cells (hMSCs) in a rat stroke model, through quantitative measurement of T2-weighted images and diffusion tensor imaging (DTI). Transient middle cerebral artery occlusion rats randomly received PBS (PBS-only), FBS cultured hMSCs (FBS-hMSCs), or stroke patients' serum cultured hMSCs (SS-hMSCs). Functional improvement was assessed using a modified neurological severity score (mNSS). Quantitative analyses of T2-weighted ischemic lesion and ventricular volume changes were performed. Brain microstructure/connectivity changes were evaluated in the ischemic recovery area by DTI-derived microstructural indices such as relative fractional anisotropy (rFA), relative axial diffusivity (rAD), and relative radial diffusivity (rRD), and relative fiber density (rFD) analyses. According to mNSS results, the SS-hMSCs group showed the most prominent functional improvement. Infarct lesion volume of the SS-hMSCs group was significantly decreased at 2 weeks when compared to the PBS-only groups, but there were no differences between the FBS-hMSCs and SS-hMSCs groups. Brain atrophy was significantly decreased in the SS-hMSCs group compared to the other groups. In DTI, rFA and rFD values were significantly higher and rRD value was significant lower in the SS-hMSCs group and these microstructure/connectivity changes were correlated with T2-weighted morphological changes. T2-weighted volume alterations (ischemic lesion and brain atrophy), and DTI microstructural indices and rFD changes, were well matched with the results of behavioral assessment. These quantitative MRI measurements could be potential outcome predictors of functional recovery after treatment with stem cells for stroke.

Application of Mesenchymal Stem Cell-Derived Extracellular Vesicles for Stroke: Biodistribution and MicroRNA Study.

Mesenchymal stem cells (MSCs) exert their therapeutic capability through a variety of bioactive substances, including trophic factors, microRNAs, and extracellular vesicles (EVs) in infarcted tissues. We therefore hypothesized that MSC-derived EVs (MSC-EVs) possess therapeutic molecules similar to MSCs. Moreover, given their nature as nanosized and lipid-shielded particles, the intravenous infusion of MSC-EVs would be advantageous over MSCs as a safer therapeutic approach. In this study, we investigated the biodistribution, therapeutic efficacy, and mode of action of MSC-EVs in a rat stroke model. MSC-EVs successfully stimulated neurogenesis and angiogenesis in vivo. When compared to the MSC-treated group, rats treated with MSC-EVs exhibited greater behavioral improvements than the control group (p < 0.05). Our biodistribution study using fluorescence-labeled MSC-EVs and MSCs demonstrated that the amounts of MSC-EVs in the infarcted hemisphere increased in a dose-dependent manner, and were rarely found in the lung and liver. In addition, MSC-EVs were highly inclusive of various proteins and microRNAs (miRNAs) associated with neurogenesis and/or angiogenesis compared to fibro-EVs. We further analyzed those miRNAs and found that miRNA-184 and miRNA-210 were essential for promoting neurogenesis and angiogenesis of MSC-EVs, respectively. MSC-EVs represent an ideal alternative to MSCs for stroke treatment, with similar medicinal capacity but an improved safety profile that overcomes cell-associated limitations in stem cell therapy.

Efficient scalable production of therapeutic microvesicles derived from human mesenchymal stem cells.

Microvesicles (MVs) released by cells are involved in a multitude of physiological events as important mediators of intercellular communication. MVs derived from mesenchymal stem cells (MSCs) contain various paracrine factors from the cells that primarily contribute to their therapeutic efficacy observed in numerous clinical trials. As nano-sized and bi-lipid layered vesicles retaining therapeutic potency equivalent to that of MSCs, MSC-derived MVs have been in focus as ideal medicinal candidates for regenerative medicine, and are preferred over MSC infusion therapy with their improved safety profiles. However, technical challenges in obtaining sufficient amounts of MVs have limited further progress in studies and clinical application. Of the multiple efforts to reinforce the therapeutic capacity of MSCs, few studies have reportedly examined the scale-up of MSC-derived MV production. In this study, we successfully amplified MV secretion from MSCs compared to the conventional culture method using a simple and efficient 3D-bioprocessing method. The MSC-derived MVs produced in our dynamic 3D-culture contained numerous therapeutic factors such as cytokines and micro-RNAs, and showed their therapeutic potency in in vitro efficacy evaluation. Our results may facilitate diverse applications of MSC-derived MVs from the bench to the bedside, which requires the large-scale production of MVs.

Serum-mediated Activation of Bone Marrow-derived Mesenchymal Stem Cells in Ischemic Stroke Patients: A Novel Preconditioning Method.

Stroke induces complex and dynamic, local and systemic changes including inflammatory reactions, immune responses, and repair and recovery processes. Mesenchymal stem cells (MSCs) have been shown to enhance neurological recovery after stroke. We hypothesized that serum factors play a critical role in the activation of bone marrow (BM) MSCs after stroke such as by increasing proliferation, paracrine effects, and rejuvenation. Human MSCs (hMSCs) were grown in fetal bovine serum (FBS), normal healthy control serum (NS), or stroke patient serum (SS). MSCs cultured in growth medium with 10% SS or NS exhibited higher proliferation indices than those cultured with FBS ( P < 0.01). FBS-, NS-, and SS-hMSCs showed differences in the expression of trophic factors; vascular endothelial growth factor, glial cell-derived neurotrophic factor, and fibroblast growth factor were densely expressed in samples cultured with SS ( P < 0.01). In addition, SS-MSCs revealed different cell cycle- or aging-associated messenger RNA expression in a later passage, and ß-galactosidase staining showed the senescence of MSCs observed during culture expansion was lower in MSCs cultured with SS than those cultured with NS or FBS ( P < 0.01). Several proteins related to the activity of receptors, growth factors, and cytokines were more prevalent in the serum of stroke patients than in that of normal subjects. Neurogenesis and angiogenesis were markedly increased in rats that had received SS-MSCs ( P < 0.05), and these rats showed significant behavioral improvements ( P < 0.01). Our results indicate that stroke induces a process of recovery via the activation of MSCs. Culture methods for MSCs using SS obtained during the acute phase of a stroke could constitute a novel MSC activation method that is feasible and efficient for the neurorestoration of stroke.

Stroke Induces Mesenchymal Stem Cell Migration to Infarcted Brain Areas Via CXCR4 and C-Met Signaling.

Mesenchymal stem cells circulate between organs to repair and maintain tissues. Mesenchymal stem cells cultured with fetal bovine serum have therapeutic effects when intravenously administered after stroke. However, only a small number of mesenchymal stem cells reach the brain. We hypothesized that the serum from stroke patients increases mesenchymal stem cells trophism toward the infarcted brain area. Mesenchymal stem cells were grown in fetal bovine serum, normal serum from normal rats, or stroke serum from ischemic stroke rats. Compared to the fetal bovine serum group, the stroke serum group but not the normal serum group showed significantly greater migration toward the infarcted brain area in the in vitro and in vivo models (p < 0.05). Both C-X-C chemokine receptor type 4 and c-Met expression levels significantly increased in the stroke serum group than the others. The enhanced mesenchymal stem cells migration of the stroke serum group was abolished by inhibition of signaling. Serum levels of chemokines, cytokines, matrix metalloproteinase, and growth factors were higher in stroke serum than in normal serum. Behavioral tests showed a significant improvement in the recovery after stroke in the stroke serum group than the others. Stroke induces mesenchymal stem cells migration to the infarcted brain area via C-X-C chemokine receptor type 4 and c-Met signaling. Culture expansion using the serum from stroke patients could constitute a novel preconditioning method to enhance the therapeutic efficiency of mesenchymal stem cells.

Cancer Cell-Derived Extracellular Vesicles Are Associated with Coagulopathy Causing Ischemic Stroke via Tissue Factor-Independent Way: The OASIS-CANCER Study.

BACKGROUND: Cancer and stroke, which are known to be associated with one another, are the most common causes of death in the elderly. However, the pathomechanisms that lead to stroke in cancer patients are not well known. Circulating extracellular vesicles (EVs) play a role in cancer-associated thrombosis and tumor progression. Therefore, we hypothesized that cancer cell-derived EVs cause cancer-related coagulopathy resulting in ischemic stroke. METHODS: Serum levels of D-dimer and EVs expressing markers for cancer cells (epithelial cell adhesion molecule [CD326]), tissue factor (TF [CD142]), endothelial cells (CD31+CD42b-), and platelets (CD62P) were measured using flow cytometry in (a) 155 patients with ischemic stroke and active cancer (116 - cancer-related, 39 - conventional stroke mechanisms), (b) 25 patients with ischemic stroke without cancer, (c) 32 cancer patients without stroke, and (d) 101 healthy subjects. RESULTS: The levels of cancer cell-derived EVs correlated with the levels of D-dimer and TF+ EVs. The levels of cancer cell-derived EVs (CD326+ and CD326+CD142+) were higher in cancer-related stroke than in other groups (P<0.05 in all the cases). Path analysis showed that cancer cell-derived EVs are related to stroke via coagulopathy as measured by D-dimer levels. Poor correlation was observed between TF+ EV and D-dimer, and path analysis demonstrated that cancer cell-derived EVs may cause cancer-related coagulopathy independent of the levels of TF+ EVs. CONCLUSIONS: Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms.

Caveolin-1, Ring finger protein 213, and endothelial function in Moyamoya disease.

BACKGROUND: Moyamoya disease is a unique cerebrovascular occlusive disease of unknown etiology. Ring finger protein 213 (RNF213) was identified as a susceptibility gene for Moyamoya disease in East Asian countries. However, the pathogenesis of Moyamoya disease remains unclear. METHODS: We prospectively analyzed clinical data for 139 patients with Moyamoya disease (108 bilateral Moyamoya disease, 31 unilateral Moyamoya disease), 61 patients with intracranial atherosclerotic stroke, and 68 healthy subjects. We compared the genetic (RNF213 variant) and protein biomarkers for caveolae (caveolin-1), angiogenesis (vascular endothelial growth factor (VEGF) and receptor (VEGFR2), and antagonizing cytokine (endostatin)) and endothelial dysfunction (asymmetric dimethylarginine (ADMA), and nitric oxide and its metabolites (nitrite and nitrate)) between patients with Moyamoya disease and intracranial atherosclerotic stroke. We then performed path analysis to evaluate whether a certain protein biomarker mediates the association between genes and Moyamoya disease. RESULTS: Caveolin-1 level was decreased in patients with Moyamoya disease and markedly decreased in RNF213 variant carriers. Circulating factors such as VEGF and VEGFR2 did not differ among the groups. Markers for endothelial dysfunction were significantly higher in patients with intracranial atherosclerotic stroke but normal in those with Moyamoya disease. Path analysis showed that the presence of the RNF213 variant was associated with caveolin-1 levels that could lead to Moyamoya disease. The level of combined marker of Moyamoya disease (caveolin-1) and intracranial atherosclerotic stroke (ADMA, an endothelial dysfunction marker) predicted Moyamoya disease with good sensitivity and specificity. CONCLUSION: Our results suggest that Moyamoya disease is a caveolae disorder but is not related to endothelial dysfunction or dysregulation of circulating cytokines.

Somatic VHL alteration and its impact on prognosis in patients with clear cell renal cell carcinoma.

Somatic inactivation of the von Hippel-Lindau (VHL) gene is the most frequent genetic event observed in clear cell renal cell carcinoma (CC-RCC). However, the prognostic relevance of somatic VHL alteration and its target, hypoxia inducible factor (HIF)-1alpha has not been defined. We investigated the genetic changes in the VHL gene and HIF-1alpha and studied their clinical implications in patients with sporadic CC-RCC. Patients who underwent nephrectomy were eligible if they had pathologically confirmed CC-RCC not associated with VHL disease or familial RCC. Tumor tissues were selected from paraffin blocks on the basis of hematoxylin and eosin (H&E)-stained sections. Polymerase chain reaction-single strand conformation polymorphism analysis was performed to detect VHL mutations and genetic changes in HIF-1alpha, which were followed by automated direct sequencing. VHL hypermethylation was examined by methylation-specific PCR. A total of 56 patients were enrolled and somatic VHL alterations were detected in 16 patients (29%); intragenic mutation in eight, hypermethylation in five, both alterations in three. The mutation types were missense in five patients, silent in three, nonsense in two, and frameshift in one. Somatic VHL alterations were not significantly associated with progression-free survival (PFS) or overall survival (OS). However, patients with 'loss-of-function' VHL mutation showed significantly decreased PFS (P=0.016) and OS (P=0.046). Although the association between VHL alteration and response to immunotherapy was not significant (P=0.486), patients with missense mutation seem to have better response to immunotherapy. The Pro582Ser change in HIF-1alpha was detected in six patients (11%) and was positively correlated with the development of metastases (P=0.023). This study did not show an association between somatic VHL alteration and prognosis in patients with sporadic CC-RCC. However, it suggests that the therapeutic and prognostic implication of somatic VHL alteration may be different according to the mutational subtype and that the Pro582Ser change in HIF-1alpha may contribute to the development of metastases.

Antioxidant effects of statins in patients with atherosclerotic cerebrovascular disease.

BACKGROUND AND PURPOSE: Oxidative stress is involved in the pathophysiological mechanisms of stroke (e.g., atherosclerosis) and brain injury after ischemic stroke. Statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, have both pleiotropic and low-density lipoprotein (LDL)-lowering properties. Recent trials have shown that high-dose statins reduce the risk of cerebrovascular events. However, there is a paucity of data regarding the changes in the oxidative stress markers in patients with atherosclerotic stroke after statin use. This study evaluated changes in oxidative stress markers after short-term use of a high-dose statin in patients with atherosclerotic stroke. METHODS: Rosuvastatin was administered at a dose of 20 mg/day to 99 patients who had suffered an atherosclerotic stroke and no prior statin use. Blood samples were collected before and 1 month after dosing, and the serum levels of four oxidative stress markers-malondialdehyde (MDA), oxidized LDL (oxLDL), protein carbonyl content (PCO), and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-were evaluated to determine the oxidation of MDA and lipids, proteins, and DNA, respectively, at both of those time points. RESULTS: The baseline levels and the degrees of reduction after statin use differed among the oxidative stress markers measured. MDA and PCO levels were associated with infarct volumes on diffusion-weighted imaging (r=0.551, p<0.05, and r=0.444, p=0.05, respectively). Statin use decreased MDA and oxLDL levels (both p<0.05) but not the PCO or 8-OHdG level. While the reduction in MDA levels after statin use was not associated with changes in cholesterol, that in oxLDL levels was proportional to the reductions in cholesterol (r=0.479, p<0.01), LDL (r=0.459, p<0.01), and apolipoprotein B (r=0.444, p<0.05). CONCLUSIONS: The impact of individual oxidative stress markers differs with time after ischemic stroke, suggesting that different oxidative markers reflect different aspects of oxidative stress. In addition, short-term use of a statin exerts antioxidant effects against lipid peroxidation via lipid-lowering-dependent and -independent mechanisms, but not against protein or DNA oxidation in atherosclerotic stroke patients.

Circulating mesenchymal stem cells microparticles in patients with cerebrovascular disease.

Preclinical and clinical studies have shown that the application of CD105(+) mesenchymal stem cells (MSCs) is feasible and may lead to recovery after stroke. In addition, circulating microparticles are reportedly functional in various disease conditions. We tested the levels of circulating CD105(+) microparticles in patients with acute ischemic stroke. The expression of CD105 (a surface marker of MSCs) and CXCR4 (a CXC chemokine receptor for MSC homing) on circulating microparticles was evaluated by flow cytometry of samples from 111 patients and 50 healthy subjects. The percentage of apoptotic CD105 microparticles was determined based on annexin V (AV) expression. The relationship between serum levels of CD105(+)/AV(-) microparticles, stromal cells derived factor-1α (SDF-1α), and the extensiveness of cerebral infarcts was also evaluated. CD105(+)/AV(-) microparticles were higher in stroke patients than control subjects. Correlation analysis showed that the levels of CD105(+)/AV(-) microparticles increased as the baseline stroke severity increased. Multivariate testing showed that the initial severity of stroke was independently associated with circulating CD105(+)/AV(-) microparticles (OR, 1.103 for 1 point increase in the NIHSS score on admission; 95% CI, 1.032-1.178) after adjusting for other variables. The levels of CD105(+)/CXCR4(+)/AV(-) microparticles were also increased in patients with severe disability (r = 0.192, p = 0.046 for NIHSS score on admission), but were decreased with time after stroke onset (r = -0.204, p = 0.036). Risk factor profiles were not associated with the levels of circulating microparticles or SDF-1α. In conclusion, our data showed that stroke triggers the mobilization of MSC-derived microparticles, especially in patients with extensive ischemic stroke.