Protects Brain Microvascular Endothelial Cells Research Articles

Intervention of exogenous VEGF protect brain microvascular endothelial cells from hypoxia-induced injury by regulating PLCγ/RAS/ERK and PI3K/AKT pathways

Ischemic stroke rapidly increases the expression level of vascular endothelial growth factor (VEGF), which promotes neovascularization during hypoxia. However, the effect and mechanism of VEGF intervention on cerebrovascular formation remain unclear.Therefore, our research discussed the protective effect of exogenous VEGF on cells in hypoxia environment in cerebral microvascular endothelial cells, simulating ischemic stroke in hypoxic environment. Firstly, we detected the proliferation and apoptosis of cerebral microvascular endothelial cells under hypoxia environment, as well the expression levels of VEGF-E, vascular endothelial growth factor re-ceptor-2 (VEGFR-2), BCL2, PRKCE and PINK1. Moreover, immunofluorescence and western blotting were used to verify the regulation of exogenous VEGF-E on VEGFR-2 expression in hypoxic or normal oxygen environment. Lastly, we manipulated the concentration of VEGF-E in the culture medium to investigate its impact on phospholipase Cγ1 (PLCγ1)/extracellular signaling regulatory protein kinase (ERK) -1/2 and protein kinase B (AKT) pathways. Additionally, we employed a PLCγ1 inhibitor (U73122) to investigate its impact on proliferation and PLCγ1/ERK pathways.The results show that hypoxia inhibited the proliferation of cerebral microvascular endothelial cells, promoted cell apoptosis, significantly up-regulated the expression of VEGF-E, VEGFR-2, PRKCE and PINK1, but down-regulated the expression of BCL2. Interference from exogenous VEGF-E activated PLCγ1/ERK-1/2 and AKT pathways, promoting cell proliferation and inhibiting apoptosis of hypoxic brain microvascular endothelial cells.In summary, exogenous VEGF-E prevents hypoxia-induced damage to cerebral microvascular endothelial cells by activating the PLCγ1/ERK and AKT pathways. This action inhibits the apoptosis pathway in hypoxic cerebral microvascular endothelial cells, thereby safeguarding the blood-brain barrier and the nervous system.

D-allose protects brain microvascular endothelial cells from hypoxic/reoxygenated injury by inhibiting endoplasmic reticulum stress

Ischemic stroke is an acute brain disease with a high mortality rate. Currently, the only effective method is to restore the blood supply. But the inflammation and oxidative stress induced by this approach can damage the integrity of the endothelial system, which hampers the patient's outcome. d-allose has the biological activity to protect against ischemia–reperfusion injury, however, the underlying mechanism remains unclear. Here, brain microvascular endothelial cells (RBMECs) were used as the study material to establish an IR-injury model. Cell viability of RBMECs was suppressed after hypoxia/reoxygenation (H/R) treatment and significantly increased after d-allose supplementation. RNAseq results showed 180 differentially expressed genes (DEGs) between the therapy group (H/R + Dal) and the model group (H/R), of which 151 DEGs were restored to control levels by d-allose. Enrichment analysis revealed that DEGs were mainly involved in protein processing in endoplasmic reticulum. 6 DEGs in the unfolded protein response (UPR) pathway were verified by qRT-PCR. All of them were significantly down-regulated by d-allose, indicating that endoplasmic reticulum stress (ERS) was relieved. In addition, d-allose significantly inhibited the phosphorylation level of eIF2α, a marker of ERS. The downstream molecules of Phosphorylation of eIF2α, Gadd45a and Chac1, which trigger cycle arrest and apoptosis, respectively, were also significantly inhibited by d-allose. Thus, we conclude that d-allose inhibits the UPR pathway, attenuates eIF2α phosphorylation and ERS, restores the cell cycle, inhibits apoptosis, and thus enhances endothelial cell tolerance to H/R injury.

Panax notoginseng Saponins Protect Brain Microvascular Endothelial Cells against Oxygen-Glucose Deprivation/Resupply-Induced Necroptosis via Suppression of RIP1-RIP3-MLKL Signaling Pathway.

Recently, necroptosis has emerged as one of the important mechanisms of ischemia stroke. Necroptosis can be rapidly activated in endothelial cells to cause vascular damage and neuroinflammation. Panax notoginseng saponins (PNS), an ingredient extracted from the root of Panax notoginseng (Burk.) F.H. Chen, was commonly used for ischemic stroke, while its molecular mechanism and targets have not been fully clarified. Our study aimed to clarify the anti-necroptosis effect of PNS by regulating RIP1-RIP3-MLKL signaling pathway in brain microvascular endothelial cells (BMECs) subjected to transient oxygen-glucose deprivation (OGD/resupply [R]). In vitro, the necroptosis model of rat BMECs was established by testing the effect of OGD/R in the presence of the pan-caspase inhibitor z-VAD-FMK. After administration of PNS and Nec-1, cell viability, cell death modality, the expression of RIP1-RIP3-MLKL pathway and mitochondrial membrane potential (Δψm) level were investigated in BMECs upon OGD/R injury. The results showed that PNS significantly enhanced cell viability of BMECs determined by CCK-8 analysis, and protected BMECs from necroptosis by Flow cytometry and TEM. In addition, PNS inhibited the phosphorylation of RIP1, RIP3, MLKL and the downstream expression of PGAM5 and Drp1, while similar results were observed in Nec-1 intervention. We further investigated whether PNS prevented the Δψm depolarization. Our current findings showed that PNS effectively reduced the occurrence of necroptosis in BMECs exposed to OGD/R by inhibition of the RIP1-RIP3-MLK signaling pathway and mitigation of mitochondrial damage. This study provided a novel insight of PNS application in clinics.

Hypoxia-induced blood-brain barrier dysfunction is prevented by pericyte-conditioned media via attenuated actomyosin contractility and claudin-5 stabilization.

The blood-brain barrier (BBB) regulates molecular and cellular entry from the cerebrovasculature into the surrounding brain parenchyma. Many diseases of the brain are associated with dysfunction of the BBB, where hypoxia is a common stressor. However, the contribution of hypoxia to BBB dysfunction is challenging to study due to the complexity of the brain microenvironment. In this study, we used a BBB model with brain microvascular endothelial cells and pericytes differentiated from iPSCs to investigate the effect of hypoxia on barrier function. We found that hypoxia-induced barrier dysfunction is dependent upon increased actomyosin contractility and is associated with increased fibronectin fibrillogenesis. We propose a role for actomyosin contractility in mediating hypoxia-induced barrier dysfunction through modulation of junctional claudin-5. Our findings suggest pericytes may protect brain microvascular endothelial cells from hypoxic stresses and that pericyte-derived factors could be candidates for treatment of pathological barrier-forming tissues.

Long noncoding RNA SNHG1 protects brain microvascular endothelial cells against oxygen-glucose deprivation/reoxygenation-induced injury by sponging miR-298 and upregulating SIK1 expression.

Growing evidence shows that long non-coding RNAs (lncRNAs) are widely involved in the progression of multiple diseases, including ischemic stroke. The aim of this study was to explore the function and underlying mechanism of lncRNAs small nucleolar RNA host gene 1 (SNHG1) in ischemic stroke. SNHG1 and salt-induced kinase 1 (SIK1) were upregulated in oxygen-glucose deprivation/reperfusion (OGD/R)-induced bEnd3 cells. SNHG1 downregulation promoted OGD/R-induced injury through decreasing cell proliferation and increasing apoptosis, which was reversed by upregulating SIK1 or downregulating miR-298. Moreover, SIK1 interference had similar functions with SNHG1 knockdown in OGD/R-treated bEnd3 cells. In addition, miR-298 was a direct target of SNHG1 and could specifically bind to SIK1. Furthermore, SNHG1 functioned as a molecular sponge of miR-298 to regulate SIK1 expression. SNHG1 knockdown enhanced OGD/R-induced injury in bEnd3 cells by regulating miR-298/SIK1 axis, which might provide promising therapeutic target for treatment of ischemic stroke.

4-Methoxybenzylalcohol protects brain microvascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced injury via activation of the PI3K/AKT signaling pathway.

Damage to the blood-brain barrier (BBB) during the process of cerebral ischemic injury is a key factor that affects the treatment of this condition. The present study aimed to assess the potential effects of 4-methoxybenzyl alcohol (4-MA) on brain microvascular endothelial cells (bEnd.3) against oxygen-glucose deprivation/reperfusion (OGD/Rep) using an in vitro model that mimics in vivo ischemia/reperfusion injury. In addition, the present study aimed to explore whether this underlying mechanism was associated with the inhibition of pro-inflammatory factors and the activation status of the PI3K/Akt signaling pathway. bEnd.3 cells were subjected to OGD/Rep-induced injury before being treated with 4-MA, following which cell viability, lactate dehydrogenase (LDH) release and levels of nitric oxidase (NO) were detected by colorimetry, pro-inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6, were detected by ELISA. The expression levels of occluding and claudin-5were evaluated by immunofluorescence staining. The expression levels of AKT, phosphorylated (p)-Akt, endothelial nitric oxide synthase (eNOS) and p-eNOS were also measured by western blot analysis. After bEnd.3 cells were subjected to OGD/Rep-induced injury, cell viability and NO levels were significantly decreased, whilst LDH leakage and inflammatory factor (TNF-α, IL-1β and IL-6) levels were significantly increased. Treatment with 4-MA significantly ameliorated cell viability, LDH release and the levels of NO and pro-inflammatory factors TNF-α, IL-1β and IL-6 as a result of OGD/Rep. Furthermore, treatment with 4-MA upregulated the expression of occludin, claudin-5, Akt and eNOS, in addition to increasing eNOS and AKT phosphorylation in bEnd.3 cells. These results suggest that 4-MA can alleviate OGD/Rep-induced injury in bEnd.3 cells by inhibiting inflammation and by activating the PI3K/AKT signaling pathway as a possible mechanism. Therefore, 4-MA can serve as a potential candidate for treating OGD/Rep-induced injury.

EGb761 Protects Brain Microvascular Endothelial Cells Against Oxygen-Glucose Deprivation-Induced Injury Through lncRNA Rmst/miR-150 Axis.

In the present study, we aimed to illustrate the roles and working mechanisms of long non-coding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (Rmst) and EGb761 in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). OGD exposure augmented the level of Rmst while reduced the expression of miR-150 in bEnd.3 cells. MiR-150 could directly bind to Rmst in bEnd.3 cells. Rmst silencing abrogated the inhibitory influences on the proliferation and migration and the promoting impact on the apoptosis of bEnd.3 cells caused by OGD exposure. Rmst overexpression intensified OGD-induced injury in bEnd.3 cells. OGD induced the injury of bEnd.3 cells through Rmst/miR-150 axis. EGb761 attenuated the damage in bEnd.3 cells induced by OGD through targeting Rmst/miR-150 axis. EGb761 might be an effective therapeutic agent to protect brain microvascular endothelial cells from hypoxia-ischemia induced injury.

LncRNA MALAT1 up-regulates VEGF-A and ANGPT2 to promote angiogenesis in brain microvascular endothelial cells against oxygen-glucose deprivation via targetting miR-145.

Stroke is one of the leading causes of death and long-term disability around the world. Angiogenesis is supposed to protect brain microvascular endothelial cells (BMECs) from oxidative and ischemic stress. Previous studies indicated that interaction between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miR-145 was involved in myocardial ischemia reperfusion, suggesting MALAT1 and miR-145 were also mediated with the progress of angiogenesis and cell migration in oxygen–glucose deprivation (OGD)-induced BMECs. The present study aimed to investigate the functional roles of MALAT1 in regulating miR-145 and its downstream pro-angiogenesis factors, vascular endothelial growth factor (VEGF)-A and Angiopoietin-2 (ANGPT2) during the progress of angiogenesis in OGD-induced BMECs. An in vitro OGD model was employed in mouse BMECs to mimic brain hypoxic and ischemic conditions; MTT was used to determine cell viability. qRT-PCR was used to determine the expression of long non-coding RNA (lncRNA)-MALAT1 and miR-145 under OGD conditions; in vitro tube formation assay was used to investigate angiogenic effect of MALAT1 and miR-145. The relationship between lncRNA-MALAT1/miR-145 and miR-145/VEGF-A/ANGPT2 was evaluated by qRT-PCR and Western blot, and direct binding was assessed using dual luciferase assay. Results showed that the levels of lncRNA-MALAT1 and miR-145 were up-regulated in OGD-induced BMECs. miR-145 functioned as an anti-angiogenic and pro-apoptotic factor in OGD treated BMECs via down-regulating VEGF-A and ANGPT2 directly. While lncRNA-MALAT1 enhanced the expressions of VEGF-A and ANGPT2 by targetting miR-145 to promote angiogenesis and proliferation of BMECs under OGD conditions. Our present study revealed the inhibitory functions of miR-145 on angiogenesis through direct targetting on VEGF-A and ANGPT2 for the first time and proved the protective role of lncRNA-MALAT1 for BMECs under OGD conditions through the direct regulation of miR-145.

Zhongfenggao Protects Brain Microvascular Endothelial Cells from Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury by Angiogenesis.

Zhongfenggao (ZFG) is prescribed for the treatment of cerebrovascular diseases in critical projects of the State Administration of Traditional Chinese Medicine. ZFG has been found to nourish qi, activate blood circulation, remove blood stasis, dredge collaterals, and strengthen the brain and mind. The present study investigated the effects of ZFG on oxygen-glucose deprivation-reoxygenation (OGD/R) induced injury to brain microvascular endothelial cells (BMECs), and the mechanisms underlying such effects. BMECs are essential target cells of ischemic stroke. In order to simulate ischemic-like conditions in vitro, BMECs were exposed to glucose deprivation and hypoxia for 2 h. Results indicate that ZFG may protect OGD/R-induced injury to BMECs by promoting angiogenesis. Further, we observed that ZFG significantly inhibited apoptosis induced by OGD/R injury. ZFG significantly promoted migration and microtubule formation in BMECs under OGD/R conditions. Additionally, ZFG increased levels of the vascular endothelial growth factor (VEGF) significantly and activated the Notch and Wnt signaling pathways. The results of the present study indicate that ZFG may display a protective effect against OGD/R-induced BMECs injury by promoting angiogenesis via Notch and Wnt signaling pathways. These results provide novel insights into the mechanisms underlying the therapeutic action of ZFG which shows promise as a potential drug candidate for treating cerebral ischemia-reperfusion.

MALAT1 lncRNA Induces Autophagy and Protects Brain Microvascular Endothelial Cells Against Oxygen–Glucose Deprivation by Binding to miR-200c-3p and Upregulating SIRT1 Expression

There is growing evidence that long noncoding RNAs (lncRNAs) play important roles in various biological processes. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is one of the most highly upregulated lncRNAs in cerebral ischemia. However, the molecular mechanism of MALAT1 during cerebral ischemia is still unclear. This experiment is intended to investigate the role of MALAT1 in cerebral ischemia and its relationship with autophagy. Oxygen–glucose deprivation (OGD) in brain microvascular endothelial cells (BMECs) was used to mimic ischemic-like conditions in vitro. Real-time PCR, MTT, LDH assay and western blot were used to evaluate the levels of MALAT1, miR-200c-3p, SIRT1, cell survival and proteins. We found that the expression of MALAT1 and LC3BII were upregulated and p62 was downregulated by OGD. Inhibition of MALAT1 attenuated the autophagy activation and promoted cell death. We further revealed that MALAT1 downregulated the expression of miR-200c-3p by directly binding to miR-200c-3p. Furthermore, miR-200c-3p inhibited the autophagy and survival in BMECs by binding to 3′UTR of SIRT1, whereas MALAT1 overturned the inhibitory effect of miR-200c-3p. In conclusion, our study illuminated a novel Malat1-miR-200c-3p-SIRT1 pathway in the regulation of autophagy, in which, MALAT1 activates autophagy and promotes cell survival by binding to miR-200c-3p and upregulating SIRT1 expression.

Hydroxysafflor yellow a protects brain microvascular endothelial cells against oxygen glucose deprivation/reoxygenation injury: Involvement of inhibiting autophagy via class I PI3K/Akt/mTOR signaling pathway

The present study aimed to test whether Hydroxysafflor yellow A (HSYA) protects the brain microvascular endothelial cells (BMECs) injury induced by oxygen glucose deprivation/reoxygenation (OGD/R) via the PI3K/Akt/mTOR autophagy signaling pathway. Primary rat BMECs were cultured and identified by the expression of factor VIII-related antigen before being exposed to OGD/R to imitate ischemia/reperfusion (I/R) damage in vitro. The protective effect of HSYA was evaluated by assessing (1) cellular morphologic and ultrastructural changes; (2) cell viability and cytotoxicity; (3) transendothelial electrical resistance (TEER) of monolayer BMECs; (4) cell apoptosis; (5) fluorescence intensity of LC3B; (6) LC3 mRNA expression; (7) protein expressions of LC3, Beclin-1, Zonula occludens-1 (ZO-1), phospho-Akt (p-Akt), Akt, phospho-mTOR (p-mTOR) and mTOR. It was found that HSYA (20, 40, and 80 μM) and 3-MA effectively reversed the cellular morphological and ultrastructural changes, increased cell survival, normalized the permeability of BMECs, and suppressed apoptosis induced by OGD/R (2 h OGD followed by 24 h reoxygenation). Concurrently, HSYA and 3-MA also inhibited OGD/R-induced autophagy evidenced by the decreased number of autophagosomes and down-regulated levels of LC3 and Beclin-1 proteins and mRNAs. HSYA (80 μM), in combination with 3-MA showed a synergistic effect. Mechanistic studies revealed that HSYA (80 μM) markedly increased the levels of p-Akt and p-mTOR proteins. Blockade of PI3K activity by ZSTK474 abolished its anti-autophagic and pro-survival effect and lowered both Akt and mTOR phosphorylation levels. Taken together, these results suggest that HSYA protects BMECs against OGD/R-induced injury by inhibiting autophagy via the Class I PI3K/Akt/mTOR signaling pathway.

KLF4 protects brain microvascular endothelial cells from ischemic stroke induced apoptosis by transcriptionally activating MALAT1

In this study, we investigated the expression profile and functional role of Kruppel-like family of transcription factor 4 (KLF4) in mouse cerebral microvascular endothelial cells (MECs) after focal cerebral ischemia and in cultured b.End3 cells after oxygen-glucose deprivation (OGD). Its downstream regulation was also studied. Our data showed that KLF4 was significantly upregulated in the cerebral MECs after transient ischemic insult and in cultured b.End3 cells after 24 h OGD exposure. In in vitro b.End3 cell model, KLF4 shRNA significantly increased OGD-induced activation of caspase-3 and also increased OGD-induced b.End3 cell death. KLF4 shRNA substantially enhanced OGD induced Bim and Bax expression at mRNA and protein levels and also aggravated OGD induced upregulation of E-selectin, MCP-1 and IL-6. Bioinformatic analysis suggested that MALAT1 promoter has a possible KLF4 binding site. In this study, we validated this possible binding site and also demonstrated that enforced KLF4 expression increased MALAT1 transcription. Functionally, knockdown of MALAT1 phenocopied the effect of KLF4 shRNA on enhancing OGD induced cell apoptosis and OGD induced upregulation of pro-apoptotic factors and pro-inflammatory cytokines. Based on these findings, we infer that MALAT1 is a transcriptional target of KLF4 in its protective role in cerebral MECs after ischemic insult.

Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury.

Fermented Chinese formula Shuan-Tong-Ling (STL), composed of fourteen medicinal herbs, was an experiential formula by Dr. Zhigang Mei for treating vascular encephalopathy, but the underlying mechanisms remained unknown. In this study, we aimed to investigate the protective effects of fermented STL on hydrogen peroxide- (H2O2-) induced injury in rat brain microvascular endothelial cells (BMECs) and the possible mechanisms. Cultured BMECs were treated with H2O2, STL, or nicotinamide (NAM, a SIRT1 inhibitor). Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to detect cell proliferation and senescence-associated beta-galactosidase (SA-β-gal) was used to examine cell senescence. Cell nuclei were observed by 4′,6-diamidino-2-phenylindole. Additionally, changes in reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were measured. Expression of SIRT1, p21, and PGC-1α was determined by western blot. Cell proliferation significantly increased with STL treatment in a dose-dependent manner. H2O2 treatment could intensify cell senescence and nuclei splitting or pyknosis. With STL treatment, the reduced ROS level was accompanied by increased SOD and GSH activity. Further assays showed upregulation of SIRT1 and PGC-1α and downregulation of p21 after STL treatment. The results revealed that STL could protect BMECs against oxidative stress injury at least partially through the SIRT1 pathway.

Inhibition of endothelin A receptor protects brain microvascular endothelial cells against hypoxia‑induced injury.

Endothelin-1 (ET-1)-induced cell damage is commonly involved in ischemia/hypoxia-associated diseases. PD155080 [sodium 2-benzo (1.3)dioxol-5-yl-3-benzyl-4-(4‑metho-xyphenyl)-4-oxobut-2-enoate] is a selective endothelin A receptor (ETAR) antagonist that inhibits ET-1‑induced cell damage. The aim of this study was to investigate the effects of PD155080 on hypoxia-induced rat brain microvascular endothelial cell (BMEC) injury. BMECs were isolated from the cerebral cortex of Wistar rats and cultured in an anoxia chamber, containing 95% N(2) and 5% CO(2) for 12 h. BMEC injury was assessed by determining cellular ultra-microstructural changes and cell viability by MTT assay, trypan blue (TB) staining and measuring the lactate dehydrogenase (LDH) levels. ET-1 mRNA expression was detected by in situ hybridization and reverse transcription PCR (RT-PCR); the ET-1 protein level was measured by radioimmunoassay. Following exposure to hypoxic conditions, the viability of the BMECs was markedly decreased and the ultrastructure of the BMECs was damaged, as demonstrated by chromatin margination, chromatin agglutination, plasma edema, the increased number of intracellular liposomes and vacuoles, mitochondrial swelling and the expansion of a rough surfaced endoplasmic reticulum. The levels of ET-1 and ET-1 mRNA expression in the BMECs were increased following exposure to hypoxic conditions. Of note, the administration of PD155080 greatly enhanced the viability of the BMECs and ameliorated hypoxia-induced cellular injury. PD155080 also inhibited hypoxia-induced ET-1 production by the BMECs. In conclusion, PD155080 exerts protective effects against hypoxia-induced BMEC injury.

Carnosine protects brain microvascular endothelial cells against rotenone‐induced oxidative stress injury through histamine H1 and H2 receptors in vitro

Although it is believed that carnosine has protective effects on various cell types, its effect on microvascular endothelial cells has not been well defined. In the present study, we investigated the protective effects of carnosine in microvascular endothelial cells using an in vitro rotenone-induced oxidative stress model. Mouse brain microvascular endothelial cells were exposed to 1μmol/L rotenone for 18h. In some experiments, carnosine (100nmol/L-1mmol/L) was added 30min prior to rotenone exposure. When used, histamine receptor antagonists (100nmol/L-10μmol/L) were added 15min before carnosine treatment. After rotenone exposure, apoptosis of microvascular cells was analysed by Hoechst 33342 staining, whereas mitochondrial membrane potential was assessed by JC-1 staining. Intracellular carnosine and histamine levels were determined using HPLC or ultra-HPLC. Over the range 1μmol/L-1mmol/L, carnosine concentration-dependently decreased the number of apoptotic cells after 18h exposure to rotenone. This effect was reversed by the histamine H1 receptor antagonists pyrilamine and diphenhydramine (1 and 10μmol/L) and the H2 receptor antagonists cimetidine (100nmol/L-10μmol/L) and zolatidine (10μmol/L). α-Fluoromethylhistidine (100μmol/L), a selective and irreversible inhibitor of histidine decarboxylase, also significantly inhibited the protective effects of carnosine. At 0.1mmol/L, carnosine restored the decrease in mitochondrial membrane potential after 6h exposure to 1μmol/L rotenone and this effect was also reversed by the H1 and H2 receptor antagonists. Moreover, intracellular carnosine levels increased as early as 1h after carnosine treatment, whereas intracellular histamine levels increased 18h after carnosine treatment. The results of the present study indicate that carnosine protects brain microvascular endothelial cells against rotenone-induced oxidative stress injury via histamine H1 and H2 receptors. The findings suggest that carnosine may be beneficial in the treatment of microvascular endothelial dysfunction induced by oxidative stress.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury.

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation -induced hypoxia inducible factor-1α expression. In this study, we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression. MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner. Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α. Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression; however, DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA. These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer’s disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H2O2-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-κB (NF-κB) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-κB activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.