Endothelial cells modified by adenovirus vector containing nine copies hypoxia response elements and human vascular endothelial growth factor as the novel seed cells for bone tissue engineering

AbstractEndothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

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Human Chorionic Gonadotropin Induces Nestin Expression in Endothelial Cells of the Ovary via Vascular Endothelial Growth Factor Signaling

Endocrinology ◽

10.1210/en.2007-0774 ◽

2007 ◽

Vol 149 (1) ◽

pp. 253-260 ◽

Cited By ~ 10

Author(s):

Noriyuki Takahashi ◽

Masanori T. Itoh ◽

Bunpei Ishizuka

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Chorionic Gonadotropin ◽

Growth Factor Signaling ◽

Vascular Endothelial ◽

Nestin Expression ◽

Capillary Endothelial Cells ◽

Theca Interna ◽

Endothelial Growth Factor

The intermediate filament protein nestin was originally found to be expressed in neuronal progenitor cells, but recent studies have shown that other cell types, including endocrine and vascular endothelial cells, express nestin. In the present study, we examined the expression and localization of nestin in the ovaries of developing, peripubertal, and adult rats. RT-PCR and Western blot analyses revealed that nestin mRNA and proteins were expressed in adult rat ovaries. Immunohistochemical analyses using adult rat ovaries showed that nestin was mainly localized to capillary endothelial cells of theca interna in follicles with more than two layers of granulosa cells and that its expression increased with follicle growth. Ontogenetically, ovarian nestin expression started at the peripubertal period when the first gonadotropin surge occurs. To test the possibility that gonadotropins induce nestin expression, prepubertal (postnatal d 21) rats were sc injected with equine chorionic gonadotropin (eCG) and/or human chorionic gonadotropin (hCG). A single injection of hCG, but not eCG, was sufficient to induce nestin expression in follicles, mainly in capillary endothelial cells of theca interna. Furthermore, pretreatment with an inhibitor of vascular endothelial growth factor receptor prevented the induction of the nestin expression by hCG. These findings demonstrate that the endogenous LH surge induces nestin expression in capillary endothelial cells of theca interna via the vascular endothelial growth factor signaling pathway. Nestin may be involved in angiogenesis in growing follicles, which is followed by follicle maturation and subsequent ovulation.

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Pro-Angiogenic Effects of Resveratrol in Brain Endothelial Cells: Nitric Oxide-Mediated Regulation of Vascular Endothelial Growth Factor and Metalloproteinases

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2012.2 ◽

2012 ◽

Vol 32 (5) ◽

pp. 884-895 ◽

Cited By ~ 43

Author(s):

Fabricio Simão ◽

Aline S Pagnussat ◽

Ji Hae Seo ◽

Deepti Navaratna ◽

Wendy Leung ◽

...

Keyword(s):

Nitric Oxide ◽

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Signaling Pathways ◽

Tube Formation ◽

Mitogen Activated Protein Kinase ◽

Vascular Endothelial ◽

Cerebral Endothelial Cells ◽

Endothelial Growth Factor

Resveratrol may be a powerful way of protecting the brain against a wide variety of stress and injury. Recently, it has been proposed that resveratrol not only reduces brain injury but also promotes recovery after stroke. But the underlying mechanisms are unclear. Here, we tested the hypothesis that resveratrol promotes angiogenesis in cerebral endothelial cells and dissected the signaling pathways involved. Treatment of cerebral endothelial cells with resveratrol promoted proliferation, migration, and tube formation in Matrigel assays. Consistent with these pro-angiogenic responses, resveratrol altered endothelial morphology resulting in cytoskeletal rearrangements of β-catenin and VE-cadherin. These effects of resveratrol were accompanied by activation of phosphoinositide 3 kinase (PI3-K)/Akt and Mitogen-Activated Protein Kinase (MAPK)/ERK signaling pathways that led to endothelial nitric oxide synthase upregulation and increased nitric oxide (NO) levels. Subsequently, elevated NO signaling increased vascular endothelial growth factor and matrix metalloproteinase levels. Sequential blockade of these signaling steps prevented resveratrol-induced angiogenesis in cerebral endothelial cells. These findings provide a mechanistic basis for the potential use of resveratrol as a candidate therapy to promote angiogenesis and neurovascular recovery after stroke.

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Mid-luteal angiogenesis and function in the primate is dependent on vascular endothelial growth factor

Journal of Endocrinology ◽

10.1677/joe.0.1680409 ◽

2001 ◽

Vol 168 (3) ◽

pp. 409-416 ◽

Cited By ~ 50

Author(s):

SE Dickson ◽

R Bicknell ◽

HM Fraser

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Luteal Phase ◽

Smooth Muscle Actin ◽

Proliferation Index ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is essential for the angiogenesis required for the formation of the corpus luteum; however, its role in ongoing luteal angiogenesis and in the maintenance of the established vascular network is unknown. The aim of this study was to determine whether VEGF inhibition could intervene in ongoing luteal angiogenesis using immunoneutralisation of VEGF starting in the mid-luteal phase. In addition, the effects on endothelial cell survival and the recruitment of periendothelial support cells were examined. Treatment with a monoclonal antibody to VEGF, or mouse gamma globulin for control animals, commenced on day 7 after ovulation and continued for 3 days. Bromodeoxyuridine (BrdU), used to label proliferating cells to obtain a proliferation index, was administered one hour before collecting ovaries from control and treated animals. Ovarian sections were stained using antibodies to BrdU, the endothelial cell marker, CD31, the pericyte marker, alpha-smooth muscle actin, and 3' end DNA fragments as a marker for apoptosis. VEGF immunoneutralisation significantly suppressed endothelial cell proliferation and the area occupied by endothelial cells while increasing pericyte coverage and the incidence of endothelial cell apoptosis. Luteal function was markedly compromised by anti-VEGF treatment as judged by a 50% reduction in plasma progesterone concentration. It is concluded that ongoing angiogenesis in the mid-luteal phase is primarily driven by VEGF, and that a proportion of endothelial cells of the mid-luteal phase vasculature are dependent on VEGF support.

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