High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels

Banumathi, Elayappan; Haribalaganesh, Ravinarayanan; Babu, Sardar Sheik Pran; Kumar, NS; Gurunathan, Sangiliyandi

doi:10.1016/j.mvr.2008.12.005

Cited by 33 publications

(24 citation statements)

References 16 publications

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“…The presence of Willebrand factor is the most widely used criterion for identification of cells of endothelial origin. We found that von Willebrand factor was strongly positive in the cultures of PAECs, consistent with observations from other investigators [30,31,32,33]. However, PAECs did not express α-smooth muscle actin, whereas PASMCs expressed the protein.…”

Section: Discussionsupporting

confidence: 92%

“…Therefore, in our subsequent experiments, the cell phenotype, immunological properties and ultrastructural characteristics were used to characterize the isolated cells as vascular endothelial cells. The rat PAECs that we obtained exhibited characteristic morphological features, immunoreactivity to von Willebrand factor, CD31 and CD34 as well as unique ultrastructural characteristics that were consistent with observations by other investigators [26,29,30,31,32,33,34,36,41,42,43,44,45,46,47,48]. …”

Section: Discussionsupporting

confidence: 91%

“…At low density, PAECs were flat and polygonal and displayed a characteristic ovoid nucleus. As soon as the cells became confluent, the primary culture of PAECs, like other vascular endothelial cells described by other investigators [26,29,30], grew in a typical ‘cobblestone'-like pattern that is characteristic of endothelium. PASMCs, however, showed a spindle-shaped appearance at low density and grew in a ‘hill-and-valley'-like pattern at a high density that was completely different from that of PAECs.…”

Section: Discussionmentioning

confidence: 67%

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Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries

Peng¹,

Wen²,

Shi³

et al. 2013

J Vasc Res

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Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 67%

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Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries

Peng¹,

Wen²,

Shi³

et al. 2013

J Vasc Res

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“…The rats were fed ad libitum with Purina 5008 diet. Rats were anesthetized with a ketamine/xylasine cocktail [39,40]. Once full anesthesia was reached, the rats were euthanized via decapitation.…”

Section: Rat Retinal Explant Angiogenesis Assaymentioning

confidence: 99%

“…Once full anesthesia was reached, the rats were euthanized via decapitation. The retina was isolated [39,40] and placed in ice-cold EBM. Meanwhile, 200 lL of GFR-Matrigel was added to each well of a 48-well tissue culture plate and polymerized for 60 min at 37°C.…”

Section: Rat Retinal Explant Angiogenesis Assaymentioning

confidence: 99%

MG624, an α7-nAChR antagonist, inhibits angiogenesis via the Egr-1/FGF2 pathway

et al. 2011

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Small cell lung cancer (SCLC) demonstrates a strong etiological association with smoking. Although cigarette smoke is a mixture of about 4,000 compounds, nicotine is the addictive component of cigarette smoke. Several convergent studies have shown that nicotine promotes angiogenesis in lung cancers via the α7-nicotinic acetylcholine receptor (α7-nAChR) on endothelial cells. Therefore, we conjectured that α7-nAChR antagonists may attenuate nicotine-induced angiogenesis and be useful for the treatment of human SCLC. For the first time, our study explores the anti-angiogenic activity of MG624, a small-molecule α7-nAChR antagonist, in several experimental models of angiogenesis. We observed that MG624 potently suppressed the proliferation of primary human microvascular endothelial cells of the lung (HMEC-Ls). Furthermore, MG624 displayed robust anti-angiogenic activity in the Matrigel, rat aortic ring and rat retinal explant assays. The anti-angiogenic activity of MG624 was assessed by two in vivo models, namely the chicken chorioallantoic membrane model and the nude mice model. In both of these experimental models, MG624 inhibited angiogenesis of human SCLC tumors. Most importantly, the administration of MG624 was not associated with any toxic side effects, lethargy or discomfort in the mice. The anti-angiogenic activity of MG624 was mediated via the suppression of nicotine-induced FGF2 levels in HMEC-Ls. MG624 decreased nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduced the levels of Egr-1 on the FGF2 promoter. Consequently, this process decreased FGF2 levels and angiogenesis. Our findings suggest that the anti-angiogenic effects of MG624 could be useful in anti-angiogenic therapy of human SCLCs.

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Getting intimate with trypsin, the leading protease in proteomics

2013

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Nowadays, mass spectrometry‐based proteomics is carried out primarily in a bottom‐up fashion, with peptides obtained after proteolytic digest of a whole proteome lysate as the primary analytes instead of the proteins themselves. This experimental setup crucially relies on a protease to digest an abundant and complex protein mixture into a far more complex peptide mixture. Full knowledge of the working mechanism and specificity of the used proteases is therefore crucial, both for the digestion step itself as well as for the downstream identification and quantification of the (fragmentation) mass spectra acquired for the peptides in the mixture. Targeted protein analysis through selected reaction monitoring, a relative newcomer in the specific field of mass spectrometry‐based proteomics, even requires a priori understanding of protease behavior for the proteins of interest. Because of the rapidly increasing popularity of proteomics as an analytical tool in the life sciences, there is now a renewed demand for detailed knowledge on trypsin, the workhorse protease in proteomics. This review addresses this need and provides an overview on the structure and working mechanism of trypsin, followed by a critical analysis of its cleavage behavior, typically simply accepted to occur exclusively yet consistently after Arg and Lys, unless they are followed by a Pro. In this context, shortcomings in our ability to understand and predict the behavior of trypsin will be highlighted, along with the downstream implications. Furthermore, an analysis is carried out on the inherent shortcomings of trypsin with regard to whole proteome analysis, and alternative approaches will be presented that can alleviate these issues. Finally, some reflections on the future of trypsin as the workhorse protease in mass spectrometry‐based proteomics will be provided. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 32:453–465, 2013.

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High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels

Cited by 33 publications

References 16 publications

Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries

Development of a New Method for the Isolation and Culture of Pulmonary Arterial Endothelial Cells from Rat Pulmonary Arteries

MG624, an α7-nAChR antagonist, inhibits angiogenesis via the Egr-1/FGF2 pathway

Getting intimate with trypsin, the leading protease in proteomics

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