Limits...
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.

Show MeSH

Related in: MedlinePlus

Adiponectin and glycam 1 expression in whole aorta vs. aortic endothelium. Diabetes-induced changes in mRNA abundance of adiponectin and glycam 1 in whole aorta vs. aortic endothelium, measured by real-time PCR. * indicates a p-value < .05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2365940&req=5

Figure 3: Adiponectin and glycam 1 expression in whole aorta vs. aortic endothelium. Diabetes-induced changes in mRNA abundance of adiponectin and glycam 1 in whole aorta vs. aortic endothelium, measured by real-time PCR. * indicates a p-value < .05.

Mentions: Dysregulation of glycam1 and adiponectin was confirmed in a third independent experiment in which an alternative amplification system, the SPIA system, was employed to amplify RNA directly into cDNA. This endothelial regulation, shown in fig. 3, was determined using real-time PCR and is in agreement with the microarray and real-time analysis shown in fig. 2. Also shown in fig. 3, a similar analysis of whole aortic tissue from diabetic mice reveals much less dysregulation. The smaller response of Glycam 1 and adiponectin within diabetic aorta vs. the FACS-sorted product most likely reflects the relatively low proportion of endothelial cells in whole aorta.


In vivo endothelial gene regulation in diabetes.

Maresh JG, Shohet RV - Cardiovasc Diabetol (2008)

Adiponectin and glycam 1 expression in whole aorta vs. aortic endothelium. Diabetes-induced changes in mRNA abundance of adiponectin and glycam 1 in whole aorta vs. aortic endothelium, measured by real-time PCR. * indicates a p-value < .05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Adiponectin and glycam 1 expression in whole aorta vs. aortic endothelium. Diabetes-induced changes in mRNA abundance of adiponectin and glycam 1 in whole aorta vs. aortic endothelium, measured by real-time PCR. * indicates a p-value < .05.
Mentions: Dysregulation of glycam1 and adiponectin was confirmed in a third independent experiment in which an alternative amplification system, the SPIA system, was employed to amplify RNA directly into cDNA. This endothelial regulation, shown in fig. 3, was determined using real-time PCR and is in agreement with the microarray and real-time analysis shown in fig. 2. Also shown in fig. 3, a similar analysis of whole aortic tissue from diabetic mice reveals much less dysregulation. The smaller response of Glycam 1 and adiponectin within diabetic aorta vs. the FACS-sorted product most likely reflects the relatively low proportion of endothelial cells in whole aorta.

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