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In vivo endothelial gene regulation in diabetes.

Maresh JG, Shohet RV - Cardiovasc Diabetol (2008)

Bottom Line: RNA was isolated from >50,000 endothelial cells and subjected to oligo dT amplification prior to transcriptional analysis on microarrays displaying long oligonucleotides representing 32,000 murine transcripts.Five regulated transcripts were selected for analysis by real-time PCR.Up-regulation of glycam1, slc36a2, ces3, adipsin and adiponectin was confirmed by real-time PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Hawaii John A, Burns School of Medicine, Honolulu, USA. maresh@hawaii.edu

ABSTRACT

Background: An authentic survey of the transcript-level response of the diabetic endothelium in vivo is key to understanding diabetic cardiovascular complications such as accelerated atherosclerosis and endothelial dysfunction.

Methods: We used streptozotocin to induce a model of type I diabetes in transgenic mice that express green fluorescent protein under the control of an endothelial-specific promoter (Tie2-GFP) allowing rapid isolation of aortic endothelium. Three weeks after treatment, endothelial cells were isolated from animals with blood glucose > 350 mg/dl. Aortae from the root to the renal bifurcation were rapidly processed by mincing and proteolytic digestion followed by fluorescent activated cell sorting to yield endothelial cell populations of >95% purity. RNA was isolated from >50,000 endothelial cells and subjected to oligo dT amplification prior to transcriptional analysis on microarrays displaying long oligonucleotides representing 32,000 murine transcripts. Five regulated transcripts were selected for analysis by real-time PCR.

Results: Within replicate microarray experiments, 19 transcripts were apparently dysregulated by at least 70% within diabetic mice. Up-regulation of glycam1, slc36a2, ces3, adipsin and adiponectin was confirmed by real-time PCR.

Conclusion: By comprehensively examining cellular gene responses in vivo in a whole animal model of type I diabetes, we have identified novel regulation of key endothelial transcripts that likely contribute to the metabolic and pro-inflammatory responses that accompany diabetes.

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Comparison of real-time PCR and microarray results. Combined real-time PCR and microarray results of selected genes uncovered by the microarray analysis described in table 1. * indicates a p-value < .05, ** indicates a p-value < .005.
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Figure 2: Comparison of real-time PCR and microarray results. Combined real-time PCR and microarray results of selected genes uncovered by the microarray analysis described in table 1. * indicates a p-value < .05, ** indicates a p-value < .005.

Mentions: Microarray results have been placed in the GEO database under series record GSE9072. Transcripts within the aortic endothelium dysregulated by at least 70% in response to the type I diabetic model are shown in fig. 1. The average microarray-based fold change, reference sequence identity, and p-value are included in Table 2. Five transcripts displaying a high level of dysregulation in replicate array hybridizations were chosen for analysis by real-time PCR. As shown in fig. 2, there is concordance between the measurement of the diabetic response by microarray vs. real-time PCR.


In vivo endothelial gene regulation in diabetes.

Maresh JG, Shohet RV - Cardiovasc Diabetol (2008)

Comparison of real-time PCR and microarray results. Combined real-time PCR and microarray results of selected genes uncovered by the microarray analysis described in table 1. * indicates a p-value < .05, ** indicates a p-value < .005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365940&req=5

Figure 2: Comparison of real-time PCR and microarray results. Combined real-time PCR and microarray results of selected genes uncovered by the microarray analysis described in table 1. * indicates a p-value < .05, ** indicates a p-value < .005.
Mentions: Microarray results have been placed in the GEO database under series record GSE9072. Transcripts within the aortic endothelium dysregulated by at least 70% in response to the type I diabetic model are shown in fig. 1. The average microarray-based fold change, reference sequence identity, and p-value are included in Table 2. Five transcripts displaying a high level of dysregulation in replicate array hybridizations were chosen for analysis by real-time PCR. As shown in fig. 2, there is concordance between the measurement of the diabetic response by microarray vs. real-time PCR.

Bottom Line: RNA was isolated from >50,000 endothelial cells and subjected to oligo dT amplification prior to transcriptional analysis on microarrays displaying long oligonucleotides representing 32,000 murine transcripts.Five regulated transcripts were selected for analysis by real-time PCR.Up-regulation of glycam1, slc36a2, ces3, adipsin and adiponectin was confirmed by real-time PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Hawaii John A, Burns School of Medicine, Honolulu, USA. maresh@hawaii.edu

ABSTRACT

Background: An authentic survey of the transcript-level response of the diabetic endothelium in vivo is key to understanding diabetic cardiovascular complications such as accelerated atherosclerosis and endothelial dysfunction.

Methods: We used streptozotocin to induce a model of type I diabetes in transgenic mice that express green fluorescent protein under the control of an endothelial-specific promoter (Tie2-GFP) allowing rapid isolation of aortic endothelium. Three weeks after treatment, endothelial cells were isolated from animals with blood glucose > 350 mg/dl. Aortae from the root to the renal bifurcation were rapidly processed by mincing and proteolytic digestion followed by fluorescent activated cell sorting to yield endothelial cell populations of >95% purity. RNA was isolated from >50,000 endothelial cells and subjected to oligo dT amplification prior to transcriptional analysis on microarrays displaying long oligonucleotides representing 32,000 murine transcripts. Five regulated transcripts were selected for analysis by real-time PCR.

Results: Within replicate microarray experiments, 19 transcripts were apparently dysregulated by at least 70% within diabetic mice. Up-regulation of glycam1, slc36a2, ces3, adipsin and adiponectin was confirmed by real-time PCR.

Conclusion: By comprehensively examining cellular gene responses in vivo in a whole animal model of type I diabetes, we have identified novel regulation of key endothelial transcripts that likely contribute to the metabolic and pro-inflammatory responses that accompany diabetes.

Show MeSH
Related in: MedlinePlus