High homocysteine is associated with idiopathic normal pressure hydrocephalus in deep perforating arteriopathy: a cross-sectional study.

BACKGROUND AND OBJECTIVE: The pathogenesis and pathophysiology of idiopathic normal pressure hydrocephalus (iNPH) remain unclear. Homocysteine may reduce the compliance of intracranial arteries and damage the endothelial function of the blood-brain barrier (BBB), which may be the underlying mechanism of iNPH. The overlap cases between deep perforating arteriopathy (DPA) and iNPH were not rare for the shared risk factors. We aimed to investigate the relationship between serum homocysteine and iNPH in DPA. METHODS: A total of 41 DPA patients with iNPH and 49 DPA patients without iNPH were included. Demographic characteristics, vascular risk factors, laboratory results, and neuroimaging data were collected. Multivariable logistic regression analysis was performed to investigate the relationship between serum homocysteine and iNPH in DPA patients. RESULTS: Patients with iNPH had significantly higher homocysteine levels than those without iNPH (median, 16.34 mmol/L versus 14.28 mmol/L; P = 0.002). There was no significant difference in CSVD burden scores between patients with iNPH and patients without iNPH. Univariate logistic regression analysis demonstrated that patients with homocysteine levels in the Tertile3 were more likely to have iNPH than those in the Tertile1 (OR, 4.929; 95% CI, 1.612-15.071; P = 0.005). The association remained significant after multivariable adjustment for potential confounders, including age, male, hypertension, diabetes mellitus, atherosclerotic cardiovascular disease (ASCVD) or hypercholesterolemia, and eGFR level. CONCLUSION: Our study indicated that high serum homocysteine levels were independently associated with iNPH in DPA. However, further research is needed to determine the predictive value of homocysteine and to confirm the underlying mechanism between homocysteine and iNPH.

Circulating endothelial microvesicles and their carried miR-125a-5p: potential biomarkers for ischaemic stroke.

BACKGROUND: Endothelial microvesicles (EMVs) are closely associated with the status of endothelial cells (ECs). Our earlier study has shown that EMVs could exert protective roles in ECs by transferring their carried miR-125a-5p. However, whether circulating EMVs and their carried miR-125a-5p can be used as biomarkers in ischaemic stroke (IS) are remain unknown. METHODS: We recruited 72 subjects with IS, 60 subjects with high stroke risk and 56 age-matched controls. The circulating EMVs and their carried miR-125a-5p (EMV-miR-125a-5p) levels were detected. We used microRNA (miR) array to study expression changes of miRs in plasma EMVs samples of three IS patients and three matched healthy controls. Transient middle cerebral artery occlusion (tMCAO) was used to establish IS mouse model. RESULTS: EMVs level was obviously elevated in IS patients, with the highest level in acute stage, and was positively related to carotid plaque, carotid intima-media thickness (IMT), National Institutes of Health Stroke Scale (NIHSS), infarct volume. On the contrary, we observed that EMV-miR-125a-5p level was obviously reduced in IS, with the lowest level in acute stage, and was negatively correlated with carotid plaque, IMT, NIHSS scores, infarct volume. EMVs and EMV-miR-125a-5p levels were closely related with large artery atherosclerosis subgroup. Importantly, EMVs and EMV-miR-125a-5p levels could serve as independent risk factors, and receiver operating characteristic curve achieved an area under curve (AUC) of 0.720 and 0.832 for IS, respectively, and elevated to 0.881 after their combination. In IS mouse model, control EMVs or n-EMVs administration could decrease the infarct volume and neurological deficit score, while increase the cerebral blood flow of IS mice compared with vehicle group, while IS EMVs or oxygen and glucose deprivation (OGD)-EMVs administration aggravated the tMCAO induced ischaemic injury. In addition, we observed that OGD EMVmiR-125a-5p could partially ameliorate the OGD EMVs induced brain injury after IS. CONCLUSIONS: These findings demonstrate that circulating EMVs and EMV-miR-125a-5p are closely related with the occurrence, progress, subtypes and severity of IS, and they can serve as innovative biomarkers and therapeutic targets for IS, especially when they are combined.

Microvesicles-mediated communication between endothelial cells modulates, endothelial survival, and angiogenic function via transferring of miR-125a-5p.

Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.

MicroRNA-126 Priming Enhances Functions of Endothelial Progenitor Cells under Physiological and Hypoxic Conditions and Their Therapeutic Efficacy in Cerebral Ischemic Damage.

Endothelial progenitor cells (EPCs) have shown the potential for treating ischemic stroke (IS), while microRNA-126 (miR-126) is reported to have beneficial effects on endothelial function and angiogenesis. In this study, we investigated the effects of miR-126 overexpression on EPCs and explore the efficacy of miR-126-primed EPCs (EPCmiR-126) in treating IS. The effects of miR-126 overexpression on EPC proliferation, migratory, tube formation capacity, reactive oxygen species (ROS) production, and nitric oxide (NO) generation were determined. In in vivo study, the effects of EPCmiR-126 on the cerebral blood flow (CBF), neurological deficit score (NDS), infarct volume, cerebral microvascular density (cMVD), and angiogenesis were determined. Moreover, the levels of circulating EPCs (cEPCs) and their contained miR-126 were measured. We found (1) miR-126 overexpression promoted the proliferation, migration, and tube formation abilities of EPCs; decreased ROS; and increased NO production of EPCs via activation of PI3K/Akt/eNOS pathway; (2) EPCmiR-126 was more effective than EPCs in attenuating infarct volume and NDS and enhancing cMVD, CBF, and angiogenesis; and (3) infusion of EPCmiR-126 increased the number and the level of miR-126 in cEPCs. Our data indicate that miR-126 overexpression enhanced the function of EPCs in vitro and in vivo.

MicroRNA-125a-5p alleviates the deleterious effects of ox-LDL on multiple functions of human brain microvessel endothelial cells.

MicroRNA-125a-5p (miR-125a-5p) could participate in the pathogenesis of vascular diseases. In this study, we investigated the role of miR-125a-5p in oxidized low-density lipoprotein (ox-LDL)-induced functional changes in human brain microvessel endothelial cells (HBMEC). The reactive oxygen species (ROS) production, nitric oxide (NO) generation, senescence, apoptosis, and functions of HBMEC were analyzed. For mechanism study, the epidermal growth factor receptor (EGFR)/extracellular signal-regulated protein kinase (ERK)/p38 mitogen-activated protein kinase (p38 MAPK) pathway and phosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase (Akt)/endothelial nitric oxide synthase (eNOS) pathway were analyzed. Results showed the following: 1) Expression of miR-125a-5p was reduced in ox-LDL-treated HBMEC. 2) Overexpression of miR-125a-5p protected HBMEC from ox-LDL-induced apoptosis, senescence, ROS production, and NO reduction. 3) Overexpression of miR-125a-5p increased HBMEC proliferation, migration, and tube formation, while decreasing HBMEC adhesion to leukocytes, as well as counteracting the effects of ox-LDL on those functions. 4) The levels of EGFR/ERK/p38 MAPK pathway, PI3K/Akt/eNOS pathway, cleaved caspase-3, and adherent molecular ICAM-1 and VCAM-1 were associated with the effects of ox-LDL on these HBMEC functions. In conclusion, miR-125a-5p could counteract the effects of ox-LDL on various HBMEC functions via regulating the EGFR/ERK/p38 MAPK and PI3K/Akt/eNOS pathways and cleaved caspase-3, ICAM-1, and VCAM-1 expression.

Microvascular endothelial cells-derived microvesicles imply in ischemic stroke by modulating astrocyte and blood brain barrier function and cerebral blood flow.

BACKGROUND: Endothelial cell (EC) released microvesicles (EMVs) can affect various target cells by transferring carried genetic information. Astrocytes are the main components of the blood brain barrier (BBB) structure in the brain and participate in regulating BBB integrity and blood flow. The interactions between ECs and astrocytes are essential for BBB integrity in homeostasis and pathological conditions. Here, we studied the effects of human brain microvascular ECs released EMVs on astrocyte functions. Additionally, we investigated the effects of EMVs treated astrocytes on regulating BBB function and cerebral ischemic damage. RESULTS: EMVs prepared from ECs cultured in normal condition (n-EMVs) or oxygen and glucose deprivation (OGD-EMVs) condition had diverse effects on astrocytes. The n-EMVs promoted, while the OGD-EMVs inhibited the proliferation of astrocytes via regulating PI3K/Akt pathway. Glial fibrillary acidic protein (GFAP) expression (marker of astrocyte activation) was up-regulated by n-EMVs, while down-regulated by OGD-EMVs. Meanwhile, n-EMVs inhibited but OGD-EMVs promoted the apoptosis of astrocytes accompanied by up/down-regulating the expression of Caspase-9 and Bcl-2. In the BBB model of ECs-astrocytes co-culture, the n-EMVs, conversely to OGD-EMVs, decreased the permeability of BBB accompanied with up-regulation of zonula occudens-1(ZO-1) and Claudin-5. In a transient cerebral ischemia mouse model, n-EMVs ameliorated, while OGD-EMVs aggravated, BBB disruption, local cerebral blood flow (CBF) reduction, infarct volume and neurological deficit score. CONCLUSIONS: Our data suggest that EMVs diversely modulate astrocyte functions, BBB integrity and CBF, and could serve as a novel therapeutic target for ischemic stroke.

Multilayer Membranes of Glycosaminoglycans and Collagen I Biomaterials Modulate the Function and Microvesicle Release of Endothelial Progenitor Cells.

Multilayer composite membrane of biomaterials can increase the function of adipose stem cells or osteoprogenitor cells. Recent evidence indicates endothelial progenitor cells (EPCs) and EPCs released microvesicles (MVs) play important roles in angiogenesis and vascular repair. Here, we investigated the effects of biomaterial multilayer membranes of hyaluronic acid (HA) or chondroitin sulfate (CS) and Collagen I (Col I) on the functions and MVs release of EPCs. Layer-by-layer (LBL) technology was applied to construct the multilayer composite membranes. Four types of the membranes constructed by adsorbing either HA or CS and Col I alternatively with different top layers were studied. The results showed that all four types of multilayer composite membranes could promote EPCs proliferation and migration and inhibit cell senility, apoptosis, and the expression of activated caspase-3. Interestingly, these biomaterials increased the release and the miR-126 level of EPCs-MVs. Moreover, the CS-Col I membrane with CS on the top layer showed the most effects on promoting EPCs proliferation, EPCs-MV release, and miR-126 level in EPCs-MVs. In conclusion, HA/CS and Collagen I composed multilayer composite membranes can promote EPCs functions and release of miR-126 riched EPCs-MVs, which provides a novel strategy for tissue repair treatment.

Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2.

Hepatic stellate cells (HSCs), previously described for liver-specific mesenchymal stem cells (MSCs), appear to contribute to liver regeneration. Microvesicles (MVs) are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP) or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in culture medium were also assessed. Results showed that (1) HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2) HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3) HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions.

Purpose: Microvesicles (MV) can modulate the function of recipient cells by transferring their contents. Our previous study highlighted that MV released from tumor necrosis factor-α (TNF-α) plus serum deprivation (SD)-stimulated endothelial progenitor cells, induce detrimental effects on endothelial cells. In this study, we investigated the potential effects of endothelial MV (EMV) on proliferation, migration, and apoptosis of human brain vascular smooth cells (HBVSMC). Methods: EMV were prepared from human brain microvascular endothelial cells (HBMEC) cultured in a TNF-α plus SD medium. RNase-EMV were made by treating EMV with RNase A for RNA depletion. The proliferation, apoptosis and migration abilities of HBVSMC were determined after co-culture with EMV or RNase-EMV. The Mek1/2 inhibitor, PD0325901, was used for pathway analysis. Western blot was used for analyzing the proteins of Mek1/2, Erk1/2, phosphorylation Erk1/2, activated caspase-3 and Bcl-2. The level of miR-146a-5p was measured by qRT-PCR. Results: (1) EMV significantly promoted the proliferation and migration of HBVSMC. The effects were accompanied by an increase in Mek1/2 and p-Erk1/2, which could be abolished by PD0325901; (2) EMV decreased the apoptotic rate of HBVSMC by approximately 35%, which was accompanied by cleaved caspase-3 down-regulation and Bcl-2 up-regulation; (3) EMV increased miR-146a-5p level in HBVSMC by about 2-folds; (4) RNase-treated EMV were less effective than EMV on HBVSMC activities and miR-146a-5p expression. Conclusion: EMV generated under inflammation challenge can modulate HBVSMC function and fate via their carried RNA. This is associated with activation of theMek1/2/Erk1/2 pathway and caspase-3/Bcl-2 regulation, during which miR-146a-5p may play an important role. The data suggest that EMV derived from inflammation-challenged endothelial cells are detrimental to HBVSMC homeostatic functions, highlighting potential novel therapeutic targets for vascular diseases.