Mouse models and techniques for the isolation of the diabetic endothelium.

Understanding the molecular mechanisms underlying diabetic endothelial dysfunction is necessary in order to improve the cardiovascular health of diabetic patients. Previously, we described an in vivo, murine model of insulin resistance induced by feeding a high-fat diet (HFD) whereby the endothelium may be isolated by fluorescence-activated cell sorting (FACS) based on Tie2-GFP expression and cell-surface staining. Here, we apply this model to two new strains of mice, ScN/Tie2-GFP and ApoE(-/-)/Tie2-GFP, and describe their metabolic responses and endothelial isolation. ScN/Tie2-GFP mice, which lack a functional toll-like receptor 4 (TLR4), display lower fasting glucose and insulin levels and improved glucose tolerance compared to Tie2-GFP mice, suggesting that TLR4 deficiency decreases susceptibility to the development of insulin resistance. ApoE(-/-)/Tie2-GFP mice display elevated glucose and cholesterol levels versus Tie2-GFP mice. Endothelial isolation by FACS achieves a pure population of endothelial cells that retain GFP fluorescence and endothelial functions. Transcriptional analysis of the aortic and muscle endothelium isolated from ApoE(-/-)/Tie2-GFP mice reveals a reduced endothelial response to HFD compared to Tie2-GFP mice, perhaps resulting from preexisting endothelial dysfunction in the hypercholesterolemic state. These mouse models and endothelial isolation techniques are valuable for assessing diabetic endothelial dysfunction and vascular responses in vivo.

Transcriptional analysis of the endothelial response to diabetes reveals a role for galectin-3.

To characterize the endothelial dysfunction associated with Type II diabetes, we surveyed transcriptional responses in the vascular endothelia of mice receiving a diabetogenic, high-fat diet. Tie2-GFP mice were fed a diet containing 60% fat calories (HFD); controls were littermates fed normal chow. Following 4, 6, and 8 wk, aortic and leg muscle tissues were enzymatically dispersed, and endothelial cells were obtained by fluorescence-activated cell sorting. Relative mRNA abundance in HFD vs. control endothelia was measured with long-oligo microarrays; highly dysregulated genes were confirmed by real-time PCR and protein quantification. HFD mice were hyperglycemic by 2 wk and displayed vascular insulin resistance and decreased glucose tolerance by 5 and 6 wk, respectively. Endothelial transcripts upregulated by HFD included galectin-3 (Lgals3), 5-lipoxygenase-activating protein, and chemokine ligands 8 and 9. Increased LGALS3 protein was detected in muscle endothelium by immunohistology accompanied by elevated LGALS3 in the serum of HFD mice. Our comprehensive analysis of the endothelial transcriptional response in a model of Type II diabetes reveals novel regulation of transcripts with roles in inflammation, insulin sensitivity, oxidative stress, and atherosclerosis. Increased endothelial expression and elevated humoral levels of LGALS3 supports a role for this molecule in the vascular response to diabetes, and its potential as a direct biomarker for the inflammatory state in diabetes.

Hypercholesterolemia potentiates aortic endothelial response to inhaled diesel exhaust.

BACKGROUND: Inhalation of diesel exhaust induces vascular effects including impaired endothelial function and increased atherosclerosis. OBJECTIVE: To examine the in vivo effects of subchronic diesel exhaust exposure on endothelial cell transcriptional responses in the presence of hypercholesterolemia. METHODS: ApoE (-/-) and ApoE (+/+) mice inhaled diesel exhaust diluted to particulate matter levels of 300 or 1000 µg/m³ vs. filtered air. After 30 days, endothelial cells were harvested from dispersed aortic cells by fluorescent-activated cell sorting (FACS). Relative mRNA abundance was evaluated by microarray analysis to measure strain-specific transcriptional responses in mice exposed to dilute diesel exhaust vs. filtered air. RESULTS: Forty-nine transcripts were significantly dysregulated by >2.8-fold in the endothelium of ApoE (-/-) mice receiving diesel exhaust at 300 or 1000 µg/m³. These included transcripts with roles in plasminogen activation, endothelial permeability, inflammation, genomic stability, and atherosclerosis; similar responses were not observed in ApoE (+/+) mice. CONCLUSIONS: The potentiation of diesel exhaust-related endothelial gene regulation by hypercholesterolemia helps to explain air pollution-induced vascular effects in animals and humans. The observed regulated transcripts implicate pathways important in the acceleration of atherosclerosis by air pollution.

In vivo endothelial gene regulation in diabetes.

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.

Tobacco smoke dysregulates endothelial vasoregulatory transcripts in vivo.

We hypothesized that human smoking and its deleterious effects on endothelial function can be modeled by exposure of mice to tobacco smoke, and further that these changes would be reflected in gene regulation in vascular endothelium. We used for these studies a mouse strain that expresses green fluorescent protein under the control of an endothelial-specific promoter, Tie-2. Mice were exposed to sidestream smoke from reference cigarettes at 34 mg total suspended particulates/m3. After exposure for 5 days/wk for 1 and 6 wk, aortas were pooled from treatment and control groups. Endothelial cells were rapidly isolated by collagenase treatment followed by fluorescent activated cell sorting to yield populations of >95% purity. RNA isolated from >500 endothelial cells was amplified and analyzed on deeply representative long oligo microarrays. Transcripts dysregulated by >2.5-fold were confirmed by real-time PCR and selected proteins by immunofluorescent localization. In the endothelial cells, the observed more than threefold upregulation of complement factor H (Cfh), calcitonin receptor-like (Calcr1), and soluble epoxide hydrolase (Epxh2) may play a role in hypertensive responses of the vasculature to smoking. We have identified gene regulation in vivo in vascular endothelium that potentially underlies hypertensive responses to tobacco smoke.

In vivo transcriptional response of cardiac endothelium to lipopolysaccharide.

OBJECTIVE: Vascular endothelial cells must integrate stimuli from multiple sources, including plasma, leukocytes, and neighboring components of the vessel. These stimuli are difficult to recapitulate in vitro. We have developed a method to examine the in vivo regulation of gene expression in endothelial cells and have applied it to a model of sepsis. METHODS AND RESULTS: We used fluorescent-activated cell sorting to isolate highly purified endothelial cells from the hearts of transgenic mice that express green fluorescent protein driven by the endothelial-specific promoter Tie2. We treated these mice with intraperitoneal lipopolysaccharide and identified those genes within cardiac endothelium that were >3-fold dysregulated 4 and 24 hours later by microarray analysis. These findings were confirmed by real-time polymerase chain reaction and compared with in vitro regulation in a murine endothelial cell line. CONCLUSIONS: The in vivo regulation was distinct and, in general, more robust than that seen in vitro. We identified endothelial-expressed genes not previously recognized to be regulated in response to lipopolysaccharide. This approach provides insight into the cardiac-specific responses of the endothelium that contribute to the specific responses of the heart to sepsis, and can be generalized to the exploration of endothelial responses in any organ.