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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2011 Nov 10;32(2):317–324. doi: 10.1161/ATVBAHA.111.234856

Inhibitor of Differentiation-3 mediates high fat diet-induced visceral fat expansion

Alexis Cutchins 1,2, Daniel B Harmon 1,5, Jennifer L Kirby 1,3, Amanda C Doran 1,4, Stephanie N Oldham 1, Marcus Skaflen 1, Alexander L Klibanov 1,2, Nahum Meller 1,6, Susanna R Keller 3, James Garmey 1,2, Coleen A McNamara 1,2,4
PMCID: PMC3262109  NIHMSID: NIHMS342157  PMID: 22075252

Abstract

Objective

Inhibitor of differentiation-3 (Id3) has been implicated in promoting angiogenesis, a key determinant of high fat diet (HFD)-induced visceral adiposity. Yet the role of Id3 in high fat diet (HFD)-induced angiogenesis and visceral adipose expansion is unknown.

Methods and Results

Id3−/− mice demonstrated a significant attenuation of HFD-induced visceral fat depot expansion compared to WT littermate controls. Importantly, unlike other Id proteins, loss of Id3 did not affect adipose depot size in young mice fed chow diet or differentiation of adipocytes in vitro or in vivo. Contrast enhanced ultrasound revealed a significant attenuation of visceral fat microvascular blood volume in HFD-fed mice null for Id3 compared to WT controls. HFD induced Id3 and VEGFA expression in the visceral stromal vascular fraction (SVF) and Id3−/− mice had significantly lower levels of VEGFA protein in visceral adipose tissue compared to WT. Furthermore, HFD-induced VEGFA expression in visceral adipose tissue was completely abolished by loss of Id3. Consistent with this effect, Id3 abolished E12-mediated repression of VEGFA promoter activity.

Conclusions

Results identify Id3 as an important regulator of HFD-induced visceral adipose VEGFA expression, microvascular blood volume, and depot expansion. Inhibition of Id3 may have potential as a therapeutic strategy to limit visceral adiposity.

Keywords: obesity, visceral adiposity, helix-loop-helix factors, Id3, VEGFA

Obesity, an important contributor to atherosclerosis and diabetes1–3, is increasing in an epidemic manner. Yet, not all obese individuals are at the same risk of developing these diseases. Individuals with central fat accumulation, or visceral obesity, are at higher risk4–7. As such, understanding the molecular and cellular mechanisms mediating visceral adiposity may have important implications for future therapies to limit morbidity and mortality due to obesity.

Angiogenesis is tightly linked with adipogenesis8. New blood vessel formation contributes to adipose tissue growth by delivering nutrients, growth factors, progenitor and inflammatory cells. Recent evidence suggests that modulation of adipose tissue angiogenesis may be a novel therapeutic option for treating obesity and preventing obesity-related morbidity.9

The helix-loop-helix (HLH) factors, Id1 and Id3 regulate tumor angiogenesis10, yet their role in adipose angiogenesis and diet-induced obesity are unknown. The inhibitor of differentiation or inhibitor of DNA binding (Id) proteins (Id1, Id2, Id3 and Id4), belong to the family of HLH transcription regulators. Id proteins lack a DNA-binding domain but act as dominant negative inhibitors of gene regulation by associating with the broadly expressed E proteins and preventing them from forming homo- or heterodimers with other bHLH factors and binding DNA11. Id proteins are negative regulators of cell differentiation and play key roles in the regulation of lineage commitment, cell fate decisions, and in the timing of differentiation12. Ids 1–4 are all expressed in cultured preadipocytes13,14 and in adult adipose tissue in vivo15, yet expression in response to differentiation media or high fat feeding suggests unique roles for each of the Id proteins in the regulation of adipocyte differentiation and adipose depot development13–15. Recent studies provide evidence that Id2 and Id4 promote adipocyte differentiation and adipose development. Mice null for Id2 have smaller inguinal and intrascapular adipose depot sizes at four days of age15, while data from Id4 null mice demonstrate smaller epididymal and brown depots than their litter mate controls at birth and on chow diet13. Previous studies reveal conflicting data on the impact of Id3 overexpression on adipocyte differentiation of cultured preadipocytes14, 16. In vivo studies demonstrate that Apoe−/− mice null for Id3 have increased adiponectin protein in visceral adipose and in serum16, however the effect of Id3 on the development of adiposity is unknown.

The present study is the first to demonstrate that mice null for Id3 have a significant attenuation of high fat diet (HFD)-induced obesity. Furthermore, the effect of loss of Id3 on adiposity was more marked in the visceral depots. Consistent with these findings, Id3 was more abundantly expressed in visceral compared to subcutaneous fat, and Id3 expression was induced by HFD in visceral but not subcutaneous adipose tissue. Mice null for Id3 had no differences in adipocyte differentiation markers or enzymes involved in lipid metabolism when fed either a chow or HFD. Instead, mice null for Id3 had significantly reduced visceral depot VEGFA expression and blood volume compared to C57BL/6 (WT) controls. Consistent with the paradigm of Id3 function, the HLH factor E12 significantly repressed VEGFA promoter activation; an effect antagonized by expression of Id3. Collectively, these results provide evidence that, in mice fed a HFD, Id3 promotes VEGFA expression, microvascular blood volume and depot expansion in visceral adipose tissue, suggesting a potential new target to limit visceral adiposity.

Research Design and Methods

Animals

Id3−/− mice used in these studies were on a pure C57BL/6 background (confirmed by microsatellite testing). Male Id3−/− and C57BL/6 (WT) litter-mate controls were used for all experiments. Epididymal, subcutaneous and retroperitoneal fat depots were harvested for analysis according to the technique of Hausman et al17. Details of the materials and methods used for the evaluation of body composition by DEXA, measurement of serum parameters, isolation of adipocytes, quantitative PCR, Western blotting, promoter-reporter assays and metabolic cage measurements are provided in the Online Data Supplement.

Statistics

Details are provided in the online data supplement.

Results

Id3 expression is induced by high fat feeding in visceral but not in subcutaneous fat

C57BL/6 (WT) male mice were fed either a chow or HFD containing 60% kcal from fat for four weeks starting at 3–4 weeks of age. After four weeks, there was a significant increase in both visceral (Figure 1A) and subcutaneous (SC) (Figure 1C) adipose depots with HFD, although the effect on the visceral depot was more marked. Western blot analysis revealed a clear HFD-induced increase in Id3 protein expression in the visceral, but not SC depots (Figures 1B and D), suggesting a preferential role for Id3 in HFD-induced visceral adiposity. Evaluation of Id3 mRNA and protein levels in the two depots from the same animals fed four weeks of HFD revealed 2–3 fold higher Id3 expression in the visceral depot at both the mRNA and protein levels (Figures 1E and 1F). To determine if the HFD-induced changes in Id3 expression were due to increased Id3 in the stromal vascular fraction (SVF) or adipocytes, Western blot analysis for Id3 protein was performed on both fractions. Notably, HFD induced a two-fold increase in Id3 protein in the SVF but had no effect on Id3 expression in isolated visceral adipocytes (Figure 1G and H).

Figure 1. HFD induces Id3 expression in the stromal-vascular fraction (SVF) of visceral adipose tissue.

Figure 1

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Four week old C57BL/6 mice were fed either chow or HFD for 4 weeks. A.) Average visceral (epididymal) depot weights (n=9–10 mice for each group). (B.) Id3 protein expression in the visceral depot (n=3 in each group). (C.) Average SC depot weights. (D.) Id3 protein expression in the subcutaneous depot (n=5 in each group). For the Western blots, the histograms represents the fold increase of Id3 expression on HFD compared to chow. (E.) Quantitative PCR of total RNA harvested from whole adipose tissue (n=5 in each group). Id3 was normalized to the corresponding cyclophilin signal. (F.) Id3 protein expression in whole visceral and subcutaneous tissue of WT mice (n=5 in each group). The histograms represents the fold increase of Id3 expression in the visceral depot (V) compared to subcutaneous (SC). For Western blot analyses, equivalent amounts of protein were loaded into each well and equal loading was confirmed by Amido Black stain. Visceral adipose tissue of four week old C57BL/6 mice fed either chow or HFD for 4 weeks was separated into stromal vascular fraction (SVF) and isolated adipocytes. (G.) Id3 protein expression in the SVF (n=4 from each group), normalized to tubulin. (H.) Id3 protein expression in isolated visceral adipocytes (n=5 in each group, normalized to tubulin).

Mice null for Id3 have reduced visceral adiposity in response to HFD

To evaluate the consequences of loss of Id3 on adiposity in vivo, we fed Id3−/− and WT littermates either chow or HFD. Mice null for Id3 had reduced HFD-induced weight gain (Figure 2A) and percent body fat (Figure 2B), but no difference in lean body mass (Figure 2C) compared to littermate controls. Body length was not different between genotypes. However, Id3−/− mice had a smaller waist circumference compared to controls (Supplemental Figures IA and B and representative photo, C). In contrast, there were no differences in the weight of Id3−/− mice when compared to their WT littermate controls at baseline or with chow diet feeding (Figure 2A). Collectively, these results suggest that Id3 may be important in regulating processes specifically involved in HFD-induced visceral adipose tissue accumulation.

Figure 2. Increased adiposity in response to a HFD is attenuated in Id3−/− mice.

Figure 2

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(A.) Four week old male Id3−/− and C57BL/6 (WT) littermates were fed either chow or a HFD for 20 weeks. (A.) Monthly body weights (n=9–10). (B.) Monthly dual energy X-ray absorptiomery (DEXA) tests. Values are represented as percent total body fat as derived from the weight of each animal at the time of DEXA (n=9–10). (C.) Lean body mass of WT and Id3−/− mice as calculated by DEXA measurements after 16 weeks of HFD (n=9–10). (D, E.) Weight of visceral (epididymal in D and retroperitoneal in E) and (F.) subcutaneous adipose depots of WT and Id3−/− mice fed either chow or a HFD for 20 weeks. Each circle represents one mouse with the line representing the average depot weight. *Represents p<0.05. (n.s. indicates a p-value of ≥ 0.05.).

Examination of specific adipose tissue depot weights after 20 weeks of HFD demonstrated significantly smaller visceral (epididymal and retroperitoneal) depot weights in mice null for Id3 (Figure 2D and E). In contrast, while there was a trend to smaller SC depots in Id3−/− mice fed 20 weeks of HFD, there was no significant difference in SC (Figure 2F) or brown fat (data not shown) depot weights between genotypes fed chow or HFD

Id3−/− mice have smaller visceral adipocytes in response to HFD compared to WT controls

To determine if the decreased visceral fat pad size in mice null for Id3 could be secondary to reduced visceral adipocyte size, H&E staining was performed on the epididymal depots of WT and Id3−/− mice fed HFD for 20 weeks. The adipocytes visualized in the Id3−/− epididymal tissue were smaller than the WT adipocytes and there were more adipocytes counted per high power field (Figures 3A, B and C). Although previous work has suggested a role for Id3 in adipocyte differentiation in vitro14, histological analysis revealed a similar appearance of the adipocytes. Further, Western blot analysis of differentiation markers and quantitative PCR for proteins involved in lipid metabolism confirmed no difference in expression between the visceral adipose tissue of WT and Id3−/− mice (Supplemental Figure II and III). In fact, in vitro analysis of MEFs as well as stromal vascular cells isolated from WT and Id3−/− visceral adipose tissue demonstrated no difference in oil-red-O staining after differentiation with dexamethasone, IBMX and insulin (Supplemental Figure IV). Consistent with the findings of smaller visceral adipocytes, Id3−/− mice had significantly lower insulin levels compared to WT controls. Glucose levels measured at the same time and circulating lipid levels were equivalent between the two groups (Supplemental Figure VA, B, and C). Evaluation of metabolic parameters in mice fed five weeks of chow showed no difference in food intake, total activity or oxygen consumption between WT and Id3 null mice (data not shown). In contrast, Id3 null mice fed five weeks of HFD had significantly higher average and resting oxygen consumption (Supplemental Table II). In addition, Id3−/−mice had a trend toward lower serum free fatty acid (FFA) levels compared to WT controls (1886 ± 217 vs. 2472 ± 197 μM, P = 0.076, n = 5–6 mice per group).

Figure 3. Id3−/− mice fed a HFD have smaller visceral adipocytes.

Figure 3

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Four week old male Id3−/− and C57BL/6 (WT) littermate controls were fed HFD for 20 weeks. Three sections from epididymal adipose depots, one hundred microns apart were evaluated for cell number per high powered field (HPF) (A.) and for cell size (B.). (C.) Representative images are shown for WT and Id3−/− adipose tissue sections.

Id3-dependent visceral depot expansion and regulation of microvascular blood volume occurs at early time points

Differences in metabolism of Id3−/− and WT mice fed five weeks of HFD and the increase in Id3 expression in visceral adipose tissue after four weeks of HFD (Figure 1) suggested that the effect of loss of Id3 on visceral adipose depot size may occur sooner than 20 weeks and be mediated by early responses of the adipose depots to HFD. Indeed, similar to mice fed a HFD for 20 weeks, mice fed four weeks of HFD displayed significantly reduced visceral adipose depots: epididymal (Figure 4A) and retroperitoneal (data not shown). While, as expected, HFD induced an increase in SC depot weight, there was no difference between genotypes in SC depot weights after HFD. The SC depot was slightly greater in the Id3−/− mice fed chow diet (Figure 4B). Together, data suggests that loss of Id3 attenuates the early development of diet-induced obesity through mechanism(s) independent of adipocyte differentiation.

Figure 4. The effect of Id3 on HFD-induced visceral adiposity occurs within the first month of feeding and is associated with reduced microvascular blood volume.

Figure 4

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Four week old male C57BL/6 (WT) and Id3−/− littermates were fed either chow or a HFD for 4 weeks. (A.) Weight of visceral (epididymal) and (B.) subcutaneous adipose depots. Each circle represents one mouse with the line representing the average depot weight. (C.) Contrast-enhanced ultrasound (CEU) was performed on epididymal adipose depots of WT and Id3−/− mice. Shown here are representative images (left) and quantification (right) of adipose tissue microbubble accumulation.

Given the established role of angiogenesis in modulating visceral adiposity and the role of Id3 in regulating tumor angiogenesis, we sought to determine if mice null for Id3 had reduced visceral depot microvasculature by performing contrast enhanced ultrasound (CEU) of the visceral depot. CEU has been shown to be an efficient and accurate noninvasive imaging modality to assess angiogenesis in mice18. Results demonstrated that compared to WT, Id3−/− mice fed 4 weeks of HFD had significantly lower visceral adipose tissue microbubble accumulation, a measure of microvascular blood volume as an index of microvascular density (Figure 4C, representative picture with quantitation). Consistent with a role for Id3 in regulating microvascular density, co-immunostaining of visceral adipose tissue from WT mice fed HFD with anti-CD31 and anti-Id3 antibodies revealed CD-31 positive vessels co-expressing Id3 (Supplemental Figure VI).

Id3−/− mice have reduced expression of VEGFA in visceral fat

To determine a potential mechanism whereby Id3 may regulate angiogenesis in visceral adipose, we evaluated the expression of the potent angiogenic factor, VEGFA, in visceral adipose tissue of WT and Id3−/−mice. Results demonstrated that chow fed mice null for Id3 had significantly less VEGFA protein expressed in visceral adipose tissue (Figure 5A). HFD significantly induced VEGFA expression in visceral adipose of WT but not of Id3−/−mice. In contrast, VEGFA expression was much lower in subcutaneous adipose tissue and was not affected by loss of Id3, even in mice challenged with a HFD (Figure 5B). Consistent with these data suggesting a visceral depot specific effect of Id3 on HFD-induced VEGFA expression, VEGFA ELISAs confirmed lower local VEGFA protein concentration in visceral adipose tissue from HFD fed Id3−/− mice compared to WT controls. This was not due to a global effect of loss of Id3 in the whole animal on VEGFA expression as there were no differences in circulating VEGFA levels between genotypes (Figure 5C). Furthermore, consistent with increased Id3 expression in the SVF in response to HFD, VEGFA is also increased in the SVF after HFD (Figure 5D).

Figure 5. Epididymal fat pads from Id3−/− mice have less VEGFA protein expression compared to WT controls.

Figure 5

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Four week old male Id3−/− and C57BL/6 (WT) littermates were fed either chow or a HFD for 4 weeks. (A.) Western blot of VEGFA protein expression in visceral fat from WT and Id3−/− mice. (B.) Quantitative histogram of Western blots from visceral and subcutaneous adipose from WT and Id3−/− mice (n=2–3 for each group). (VEGFA is expressed as fold increase over WT chow fed and normalized to tubulin). (C.) ELISA measurements for VEGFA concentration from Id3−/−and WT control mice fed HFD for 4 (serum) or 7 (epididymal adipose lysates) weeks. (n = 5–6 mice for each genotype were assayed in duplicate). (D.) Quantitative histogram of Western blots of visceral stromal vascular fraction (SVF) from WT mice fed Chow or HFD for 4 weeks. VEGFA levels are expressed as fold increase over chow fed mice and normalized to tubulin. Representative Western blot results are provided in the inset.

The VEGFA promoter contains several E-boxes, CANNTG consensus binding sites for bHLH factors19. To more fully elucidate the molecular mechanism whereby Id3 may promote VEGFA expression, we transiently co-transfected a 5.2 kb VEGFA promoter-luciferase reporter construct (Figure 6A) with expression plasmids encoding Id3 and the Id3-binding partner E12 into NIH 3T3 cells. Results demonstrated a significant repression of VEGFA promoter activation by co-transfected E12; an effect significantly antagonized by co-transfection of increasing amounts of Id3 (Figure 6B).

Figure 6. The bHLH protein E12 inhibits expression of the VEGFA Promoter; an effect antagonized by Id3.

Figure 6

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(A.) The VEGFA promoter contains numerous E box sequences (CANNTG) where bHLH transcription factors such as E12 can bind and regulate transcription. (B.) NIH 3T3 cells were transfected with 0.05 μg of the 5.2 Kb VEGFA-promoter-luciferase reporter together with specified expression vectors (EV = empty vector, quantities transfected are in μg). Luciferase activity is normalized to protein levels. Data are the result of three separate experiments of duplicate samples.

Discussion

Many genes that regulate embryonic development are re-expressed in the adult animal in disease states, such as cancer and cardiovascular disease20–22. These genes make particularly appealing targets of therapy, as they are generally not expressed in normal adult tissues where modulation of expression could lead to untoward effects. Id3 is expressed early in embryonic development, with expression declining as the embryo matures23,24. Previous studies have implicated Id3 in cancer and cardiovascular disease10,25, however the present study provides the first evidence implicating Id3 in visceral adipose depot expansion in response to HFD. While loss of Id3 did not alter visceral depot sizes at baseline, it did result in a significant attenuation of HFD and age-induced visceral depot expansion. This suggests that Id3, independent of its developmental role, functions in the adult animal to modulate the response to HFD leading to visceral adiposity.

Decreased visceral adiposity of Id3 null mice on HFD was associated with lower serum insulin levels and higher metabolic rates. This is consistent with published reports demonstrating an inverse relationship between visceral adipose tissue mass and whole body insulin sensitivity and metabolic rates1–6. In Id3 null mice, the smaller visceral adipose tissue depots contain smaller adipocytes, which have been associated with reduced basal lipolysis26. Consistent with decreased adipocyte lipolysis, Id3 null mice showed a trend toward lower FFA levels under HFD conditions. Note that less fat storage in adipose tissue in Id3 null mice does not lead to increased circulating lipid levels and, as indicated by improved insulin sensitivity, increased lipid deposition in other tissues like liver and skeletal muscle. Instead, FFAs may be burned at an increased rate to cover increased energy expenditure. These observations are consistent with a hypothesis that Id3 regulates adipose angiogenesis and consequently adipose tissue expansion and whole body metabolism. Indeed, when angiogenesis was inhibited pharmacologically in mouse models of obesity smaller adipose tissue mass, improved whole body metabolism, and increased use of fatty acids as energy substrates were observed27. However, we cannot rule out the possibility that the metabolic phenotype of our Id3 null mice is due to changes in other tissues that play roles in the regulation of insulin sensitivity and energy homeostasis such as skeletal muscle, liver and brain. The answer to this question will need to await the availability of mice with cell type-specific deletion of Id3.

In addition to a unique role in visceral versus subcutaneous adipose tissue, our data also demonstrate a unique role for Id3 in adipose tissue biology relative to other Id proteins. Both Id2 and Id4 have been implicated in promoting differentiation of adipocytes in vivo and in vitro13, 15. Overexpression of Id2 increased expression of PPARγ, aP2, C/EBPa, and adiponectin and promoted lipid accumulation in 3T3-L1 preadipocytes in response to an adipocyte differentiation cocktail. Conversely, compared to wildtype, MEFs null for Id2 had decreased expression of PPARγ, aP2 and adiponectin when treated with adipocyte differentiation media. Moreover, Id2−/− mice had decreased intrascapular and inguinal adipose tissue weights at birth15. Similarly, mice lacking Id4 had decreased fat mass compared to control mice even on chow diet and MEFs null for Id4 had decreased levels of similar markers of adipocyte differentiation13. The role of Id3 in adipocyte differentiation has been less clear. Moldes et al. demonstrated that Id1, Id2 and Id3 mRNA levels, abundant in multiplying 3T3-F422A cells, drop significantly when the cells are induced to differentiate. Coupled with data demonstrating that over-expression of Id3 resulted in decreased glycerophosphate dehydrogenase activity, a marker of adipocyte differentiation, the authors concluded that Id3 prevented differentiation of preadipose cells14. In contrast, our laboratory recently demonstrated that overexpression of Id3 did not alter the expression of the adipocyte differentiation marker, GLUT-4, in OP9 or 3T3-L1 cell lines treated with six and ten days of adipocyte differentiation medium respectively16, although the effect of Id3 on growth or the differentiated phenotype as measured by oil-red-O staining of these cells was not examined. The present study demonstrated no difference in oil-red-O staining of WT and Id3−/− MEFs or adipose SVF cells treated with differentiation media. Instead, the present study provides the first in vivo data linking Id3 with adipose tissue development and vascularization in response to HFD. Although both the visceral depot and adipocyte size were smaller in the Id3−/− mice compared to WT controls, there was no difference in the appearance of the mature adipocytes and Western blot analysis of adipose tissue from WT and Id3−/− mice revealed no differences in aP2, PEPCK or CEBP-α protein levels (Figure 2S). In addition, there were no differences in mRNA expression of adipocyte differentiation markers between WT and Id3−/− visceral adipocytes (data not shown). Results provide evidence that, in contrast to Id2 and Id4, loss of Id3 does not affect adipocyte differentiation or adipose depot size at baseline, but rather attenuates visceral adipose depot expansion in response to HFD.

Our results, demonstrating no differences in adipocyte differentiation markers or proteins involved in lipid metabolism (Figure 3S) in adipose tissue from wildtype compared to Id3 null mice, led us to investigate alternate reasons for decreased visceral adiposity in the Id3 null mice. Angiogenesis is necessary for adipose tissue expansion and studies have shown that inhibition of angiogenesis inhibits that growth27. It has also been proposed that visceral fat secretes pro-angiogenic factors, suggesting visceral fat is better adapted for rapid expansion28. In addition to promoting adipocyte hypertrophy, adipose tissue angiogenesis allows for inflammatory cell infiltration29. Increased levels of lymphocytes are visible in murine visceral adipose tissue after just three weeks of high-fat feeding30. Continued high-fat feeding leads to enhanced CD8+ T cell infiltration31 and promotes a CD4+ TH1 bias32, both of which help recruit M1 macrophages to visceral adipose tissue – a key event in the development of insulin resistance3, 33. Macrophages34 and mast cells35 have also been shown to further enhance adipose tissue angiogenesis in the context of diet-induced obesity, suggesting a positive feedback loop that contributes to the systemic metabolic abnormalities observed in obese individuals. Id proteins have been established as necessary for tumor angiogenesis. Data from this study demonstrates not only that mice null for Id3 have less microvascular blood volume in their epididymal fat depots but also that Id3 regulates visceral adipose VEGFA expression at the protein and promoter level. VEGFA has been implicated as an important mediator of angiogenesis36–38, and serum VEGFA levels are significantly increased in obese compared to lean humans and mice 39.

Taken together, results suggest that loss of Id3 attenuates visceral fat expansion by inhibiting HFD-induced visceral fat VEGFA expression and increased capillary density. Future studies using postmortem histology and other techniques to assess visceral adipose angiogenesis are needed to confirm and extend these findings. Nonetheless, as inhibition of Id3 has been proposed as a strategy to limit angiogenesis in vivo40 and inhibition of adipose angiogenesis is a current target to attenuate obesity9, results hold promise to lead to novel approaches to limit visceral adiposity.

Supplementary Material

1

Acknowledgments

We acknowledge Toni Barbera and Kathryn Corbin in the Diabetes Center Animal Characterization Core at the University of Virginia (NIH DK063609) for their assistance. We also thank Pat D’Amore for providing us with the VEGF promoter-reporter construct.

Sources of funding:

This work was supported by NIH grants 1R01HL62522, R01 HL096447 and 5P01HL055798 (C.A.M.), NIH Training Grant 5T32 HL007355-29 (to A.C. and J.L.K.), American Heart Association Pre-Doctoral Fellowship (A.C.D.).

Footnotes

Disclosures:

None.

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