Proliferation Of Brain Endothelial Cells Research Articles

Annexin A2 promotes angiogenesis after ischemic stroke via annexin A2 receptor – AKT/ERK pathways

Promoting angiogenesis to restore circulation to the ischemic tissue is still an important therapeutic target in stroke. Our group and others previously reported that the Ca2+-regulated, phospholipid-and membrane-binding protein-Annexin A2 (ANXA2) functions in cerebrovascular integrity and retinal neoangiogenesis. Here, we hypothesized that ANXA2 may regulate angiogenesis after stroke, ultimately improve neurological outcomes. Compared with wild type (WT) mice, the density of microvessels in brain and the number of new vessels sprouting from aortic ring were significantly increased in Anxa2 knock-in (Anxa2 KI) mice. After focal cerebral ischemia, proliferation of brain endothelial cells in Anxa2 KI mice was significantly elevated at 7 days post-stroke, which further improved behavioral recovery. To assess the pro-angiogenic mechanisms of ANXA2, we used brain endothelial cells cultures to investigate its effects on cell tube-numbers and migration. Recombinant ANXA2 increased tube-numbers and migration of brain endothelial cells either under normal condition or after oxygen glucose deprivation (OGD) injury. Co-immunoprecipitation experiments demonstrated that these protective effects of recombinant ANXA2 were regulated by interaction with ANXA2 receptor (A2R), a protein found in cancer cells that can interact with ANXA2 to promote cell migration and proliferation, and the ability of ANXA2-A2R to activate AKT/ERK pathways. Inhibition of AKT/ERK diminished recombinant ANXA2-induced angiogenesis in vitro. Taken together, our study indicates that ANXA2 might be involved in angiogenesis after ischemic stroke. Further investigation of ANXA2-A2R will provide a new therapeutic target for stroke.

The Paracrine Effect of Hypoxic and Normoxic Cancer Secretion on the Proliferation of Brain Endothelial Cells (bEnd.3).

Background: This study aimed to investigate the disruption of cell cycle phases of bEnd.3 cells exposed to cancer paracrine secretion. Cancer cells have been reported to use the secretion of paracrine factors to compromise the endothelial barrier to prepare for their passage into the parenchyma. As cancer cells are known to act differently under conditions of hypoxia, we investigated how conditional media (CM) derived from breast and glioblastoma cells incubated under conditions of normoxia and hypoxia would affect proliferation of brain endothelial cells (bEnd.3). Methods: Brain endothelial cells (bEnd.3) were cultivated with normoxic and hypoxic CM generated from breast cancer MCF7 cells and glioblastoma U-87 cells. Cell proliferation was evaluated using the trypan blue exclusion assay and phases of the cell cycle were evaluated using flow cytometry. Results: bEnd.3 proliferations was suppressed more aggressively with hypoxic CM after 72 and 96 h; cell cycle analysis showed that paracrine treatment tended to prevent BECs from entering the G2 phase, thus suppressing cell division. Conclusions: MCF7 and U-87 cells induce suppressed proliferation of BECs deferentially under hypoxia by blocking cell cycle progression to the G2 phase.

Abstract P2-20-06: Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells

Abstract Background Brain Metastasis in Breast Cancer (BMBC) is a significant contributor to mortality & presents a challenge to modern treatment of advanced breast cancer. HER2+ breast cancers have a predilection for metastasis to the brain & therefore the HER2 inhibitor Nerlynx could exhibit efficacy in treating BMBC. Previous work suggests that Nerlynx can pass through the Blood-Brain Barrier (BBB) & may alter it during treatment of BMBC. Methods The BBB was modelled using hCMEC-D3 cells. BMBC was modelled using HER2- MDA-MB-231 cells or HER2+ SK-BR-3 cells. To evaluate changes in protein expression, hCMEC-D3 cells were treated with 50 nM Nerlynx ± SK-BR-3-conditioned medium & analysed using the Kinexus microarray service. Functional changes in electrical resistance, permeability, proliferation & migration were measured using Trans-Endothelial Resistance (TEER) & Paracellular Permeability (PCP) assays. Changes in TJ protein location were assessed using immunofluorescence (IF). Results Protein microarray analysis compared to the control, co-treatment with 50 nM Nerlynx & SK-BR-3-CM was associated with increased expression of migration-associated proteins (ROCK1, +166%) & cell cycle regulatory components (Rb, +114%; CDK6, +159%; PLK1, +168%) as well as the guanine exchange factor VAV1 (+123%), intracellular kinases RSK1 (+110%) & PKCβ (+107%), & the HGF receptor MET (+74%). In contrast, decreased expression of the transcription factor STAT2 (-98%), the cell cycle regulators CDK2 (-85%) & CDK1 (-75%), focal adhesion component Paxillin 1 (-74%) & the RTK Kit (- 59%) are features of co-treatment compared to control. Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with an increase in the expression of Paxillin 1 (+809%), AKT1 (+458%) & MET (+449%), as well as CDK6 (+310%), PDK1 (+215%), the RTK DDR1 (+212%), lymphangiogenic receptor VEGFR3 (+203%) & Rb (+183%). Compared to treatment with SK-BR-3-CM alone, co-treatment with Nerlynx was associated with a decrease in the expression of STAT2 (-98%), IRS1 (-92%), ROCK2 (-84%), CSK (-73%), Kit (-71%), STAT3 (-68%), JAK2 (-67%) & VEGFR2 (-64%) & an increase in the expression of ILK1 (+554%), Kit (+448%), AKT1 (+337%), Rb (+261%) & VAV1 (+184%). However, compared to treatment with Nerlynx alone, co-treatment with SK-BR-3 CM was associated with a decrease in the expression of IRS1 (-99%), STAT2 (-98%), Paxillin 1 (-92%), IKKα (-78%), CDK1 (- 65%) FAK (-63%) & STAT3 (-62%). hCMEC-D3 co-treated with MDA-MB231-CM, 100 nM Nerlynx treatment exhibited increased PCP, after 60 min post-treatment & a similar effect was seen after 5 nM Nerlynx treatment at 120 min. In hCMEC-D3 monolayers co-treated with SK-BR-3- CM, 100 nM Nerlynx there was a decrease in PCP by 120 min post-treatment. Conversely, treatment with 5 nM or 50 nM Nerlynx led to an increase in PCP by 60 min or 180 min, respectively. Nerlynx treatment inhibited the proliferation of hCMEC-D3 cells in a dose-dependent manner with larger effects seen upon the addition of medium conditioned by MDA-MB-231 cells or SK-BR-3 cells. There was a concurrent change in distribution of TJ proteins as assessed using IF. Discussion Nerlynx inhibits the proliferation of brain endothelial cells but its role in altering barrier function is unclear. Nerlynx exerts a greater effect on brain endothelial cells in the presence of medium conditioned by either HER2+ or HER2- cells, suggesting it may be efficacious in the treatment of BMBC. The MET signalling pathway is implicated as a potential regulator of TJ-mediated BBB permeability in BMBC. Further work including BBB co-culture models, alternative brain endothelial lines & evaluation at the gene & protein level are warranted. Citation Format: Tracey Amanda Martin, Isaac Meyers, Emily Appadurai, Wen G Jiang. Comparative effect of Nerlynx & HER2+ positive & negative breast cancer cell lines on the barrier function of human brain endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-06.

Vascular expression of angiopoietin1, α5β1 integrin and tight junction proteins is tightly regulated during vascular remodeling in the post-ischemic brain

The post-stroke angiogenic response is accompanied by changes of tight junctions (TJs) of the blood–brain barrier (BBB). However, the precise dynamic change of TJ proteins (TJPs) in the different stages of stroke-induced vascular remodeling and the molecules mediating these processes have yet to be fully defined. To investigate the temporal relationship between changes in TJPs, the pro-angiogenic factor α5β1 integrin and the anti-permeability factor Ang1 in cerebral vessels following cerebral ischemic stroke, male C57Bl/6 mice were subject to 90min of ischemia by temporary occlusion of the middle cerebral artery followed by reperfusion and their brains analyzed 0, 1, 2, 4, 7 and 14days post-ischemia. Immunofluorescent studies demonstrated that in the ischemic penumbra, TJPs claudin-5 and ZO-1 levels decreased during the early stages of vascular remodeling, but then increased in the later stages. In contrast, within the ischemic core, TJPs levels decreased over the 14-day time-course, plateaued at day 4, and remained at low levels up to day 14. In the penumbra, Ang1 expression was induced, peaking at the same time point as α5β1 expression. Consistent with these findings, oxygen glucose deprivation/reperfusion induced expression of α5β1 and Ang1 on brain endothelial cell (BEC) in a similar manner in vitro, which correlated closely with BEC proliferation and increased expression of TJPs. Our results demonstrate that in the post-ischemic penumbra, a tight temporal correlation exists between the angiogenic markers α5β1 and Ang1 and the TJPs, suggesting a potential role for Ang1 and α5β1 in promoting BBB integrity following ischemic stroke.

Microglial activation state exerts a biphasic influence on brain endothelial cell proliferation by regulating the balance of TNF and TGF-β1

BackgroundStudies of cerebral ischemia and other neuroinflammatory states have demonstrated a strong association between new vessel formation and microglial recruitment and activation, raising the possibility that microglia may be involved in promoting angiogenesis. As endothelial cell proliferation is a fundamental early step in angiogenesis, the aim of this study was to test this hypothesis by examining the influence of microglial secreted factors on brain endothelial cell (BEC) proliferation using BrdU incorporation.MethodsPrimary cultures of mouse BEC, microglia and astrocytes were used in this study. Proliferation of BEC was examined by BrdU incorporation. ELISA was used to quantify TNF and TGF-β1 levels within cell culture supernatants.ResultsMicroglia regulated BEC proliferation in a biphasic manner; microglia conditioned medium (MG-CM) from resting microglia inhibited, while that from activated microglia promoted BEC proliferation. A screen of microglial cytokines revealed that BEC proliferation was inhibited by TGF-β1, but promoted by TNF. ELISA showed that TNF and TGF-β1 were both present in MG-CM, and that while TGF-β1 dominated in resting MG-CM, TNF levels were massively increased in activated MG-CM, shifting the balance in favor of TNF. Antibody-blocking studies revealed that the influence of MG-CM to inhibit or promote BEC proliferation was largely attributable to the cytokines TGF-β1 and TNF, respectively.ConclusionThis data suggests that microglial activation state might be an important determinant of cerebral angiogenesis; inhibiting BEC proliferation and neovascularization in the normal central nervous system (CNS), but stimulating the growth of new capillaries under neuroinflammatory conditions.

Abstract 1308A: Novel antiinvasive and antiangiogenic mechanisms of mTOR inhibition in glioblastoma

Abstract Two key properties of glioblastomas are vascular proliferation and invasion of tumor cells into the surrounding tissues, both limiting the efficacy of surgery and radiochemotherapy treatment regimens. Inhibition of mammalian target of rapamycin (mTOR) by Temsirolimus (CCI-779) only has modest single compound activity in recurrent glioma. Given the fact that postoperative radiochemotherapy is the standard of care in the first-line treatment of newly diagnosed glioblastoma, the first part of the presented project aims at analyzing combined mTOR/radiotherapy in the syngeneic, orthotopic VM/Dk/SMA-560 mouse glioma model. The combined treatment of CCI-779, a small-molecule inhibitor of the mTOR kinase complex 1 (mTORC1) approved for advanced renal cell carcinoma and mantle cell lymphoma, at 20 mg/KG from day 3 until day 17 and focal irradiation at 6 Gy on day 5 after tumor inocculation demonstrated remarkable antiangiogenic and antitumoral activity as well as prolonged survival of tumor bearing animals of 9 days, significant compared with irradiation- or CCI-779-treatment alone. Loss of phosphatase and tensin homologue on chromosome ten (PTEN), which is a common event in glioblastoma, results in activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling pathway, leading to neovascularisation, cell cycle progression and escape from apoptosis. So far, conflicting data on the sensitivity of PTEN wild-type (wt) versus mutant cells exist. Similarly, the relevance of the feedback activation of Akt by mTOR inhibition is debated. Here, analysis of PTEN on mRNA, promoter methylation as well as protein levels clearly demonstrates for cell lines as well as primary glioma cells that proliferation of PTEN wt cells is also sensitive to mTOR inhibition albeight at higher concentrations. We further demonstrate by differential effects on the mTOR complexes (mTORC) 1 and 2 by shRNA as compared to sole mTORC 1 inhibition by CCI-779 that feedback activation of Akt, which is more prominent in PTEN mutant than in wild-type cells, may in fact have additional therapeutic antiinvasive and angiogenic effects via inhibition of a G-protein-interacting protein and vascular endothelial growth factor receptor (VEGFR)-2, respectively. CCI-779 exerted marked anti-angiogenic effects both by reducing levels of VEGFR and by inhibiting radiation-enhanced proliferation of brain endothelial cells. Moreover, CCI-779 applied after radiosensibilization inhibited glioma invasiveness in a supra-additive way and reverted the proinvasive effect of sublethal irradiation alone. The results support the clinical evaluation of combined targeted mTOR inhibition with CCI-779 and radiotherapy in patients with newly diagnosed glioblastomas that is going to be conducted in the European Organization for Research and Treatment of Cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1308A.

Dutch and arctic mutant peptides of β amyloid 1–40 differentially affect the FGF-2 pathway in brain endothelium

Single point mutations of the amyloid precursor protein generate Aβ variants bearing amino acid substitutions at positions 21–23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Aβ on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Aβ structure, suggesting that the toxic effect of the Aβ assemblies goes beyond mere multimerization.

Brain microvascular endothelial cell proliferation with angiotensin stimulation and receptor blockade

Aim Hypertension is a major risk factor for stroke. The angiotensin (ANGII) AT1 receptor antagonist losartan (LOS) is a commonly prescribed hypertension medication. AT1 receptor activation stimulates angiogenesis in most tissues, yet chronic in vivo blockade of the AT1 receptor results in angiogenesis in the brain. Since this paradoxical response could be due to alterations in cerebral blood flow rather than direct actions of receptor blockade in the microvessels, this study tested the growth response of human brain microvascular endothelial cells (HBMEC) to ANGII receptor stimulation or blockade in vitro. Methods Cultured HBMEC were exposed to serum-free media (SFM) alone and in the presence of LOS (10 μM), ANGII (1 μM), or both for 24 hours in 96-well plates. Cell density was quantified by CyQUANT (Molecular Probes) cell proliferation assay. Results Average fluorescence increased in cells receiving LOS alone (46,358±2771), and was even greater with ANGII stimulation (59,582 ± 7400) compared to SFM (6,940±168). Results from combined LOS + ANGII stimulation (49,583 ±3197) were not different from LOS treatment alone. Conclusions These data suggest that, unlike in other tissues, AT1 receptor blockade results in brain endothelial cell proliferation. This supports our previous findings that AT1 receptor blockade induces cerebral angiogenesis. It is possible that chronic hypertension therapy using ARBs such as losartan might improve brain blood flow through angiogenesis, potentially protecting high-risk patients from stroke. Supported in part by Frontiers in Physiology Professional Development Fellowship, APS.