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. 2011 Dec;96(12):E1990-8.
doi: 10.1210/jc.2011-1567. Epub 2011 Oct 12.

Adipose tissue extracellular matrix and vascular abnormalities in obesity and insulin resistance

Michael Spencer  1 , Resat Unal, Beibei Zhu, Neda Rasouli, Robert E McGehee Jr, Charlotte A Peterson, Philip A Kern
Affiliations

Adipose tissue extracellular matrix and vascular abnormalities in obesity and insulin resistance

Michael Spencer et al. J Clin Endocrinol Metab. 2011 Dec.

Abstract

Context: Insulin resistance is associated with inflammation, fibrosis, and hypoxia in adipose tissue.

Objective: This study was intended to better characterize the extracellular matrix (ECM) and vascularity of insulin-resistant adipose tissue.

Design: Adipose expression of collagens, elastin, and angiogenic factors was assessed using real-time RT-PCR and immunohistochemistry (IHC) in abdominal sc adipose tissue. Adipocyte-macrophage coculture experiments examined the effects of polarized macrophages on adipose ECM gene expression, and the effects of collagens were measured in an angiogenesis assay.

Participants and setting: A total of 74 nondiabetic subjects participated at a University Clinical Research Center.

Interventions: Interventions included baseline adipose biopsy and measurement of insulin sensitivity.

Main outcome measures: Outcome measures included characterization of vascularity and ECM in adipose tissue.

Results: CD31 (an endothelial marker) mRNA showed no significant correlation with body mass index or insulin sensitivity. In a subgroup of 17 subjects (nine obese, eight lean), CD31-positive capillary number in obese was decreased by 58%, whereas larger vessels were increased by 70%, accounting for the lack of change in CD31 expression with obesity. Using IHC, obese (compared with lean) subjects had decreased elastin and increased collagen V expression, and adipocytes cocultured with M2 macrophages had reduced elastin and increased collagen V expression. In obese subjects, collagen V was colocalized with large blood vessels, and the addition of collagen V to an angiogenesis assay inhibited endothelial budding.

Conclusions: The adipose tissue from obese/insulin-resistant subjects has fewer capillaries and more large vessels as compared with lean subjects. The ECM of adipose tissue may play an important role in regulating the expandability as well as angiogenesis of adipose tissue.

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Figures

Fig. 1.
Fig. 1.
Identification of blood vessels in lean and obese adipose tissue. Obese (A–D) and lean (E) adipose tissue samples were immunoreacted with antibodies to CD31 (B, green) to identify endothelial cells and ASMA (A, red) to delineate the vessel wall, overlaid in D. C, The bright-field overlay of the same image. In the merged images from a representative obese (D) and lean (E) subject, larger blood vessels (arrowheads) are recognizable because they accumulate both ASMA and CD31. Capillaries are small and display only endothelial cell staining (arrows). Magnification, ×200; bar, 125 μm. F, Images from obese and lean subjects were scored for the number of capillaries and larger vessels, as described in Subjects and Methods, and normalized to cross-sectional area. Vessels are defined as structures that stained with CD31 and ASMA, and capillaries stained for only CD31. G, The areas containing the capillaries and vessels were categorized as fibrotic and nonfibrotic. *, P < 0.05 vs. lean.
Fig. 2.
Fig. 2.
Elastin expression in adipose tissue. Adipose elastin was visualized with a histochemical stain, as described in Subjects and Methods. Representative fibrotic (A) and nonfibrotic (C) areas of a lean subject are compared with fibrotic (B) and nonfibrotic (D) areas from an obese subject. Elastin was present in fibrotic areas and to a lesser extent in nonfibrotic areas of lean subjects. Obese subjects exhibited much less elastin staining. Magnification, ×200; bar, 125 μm. E, The amount of elastin was quantified as described in Subjects and Methods, and total elastin in lean and obese subjects is shown, expressed as a percentage of total area. *, P < 0.05.
Fig. 3.
Fig. 3.
Collagen V localizes to fibrotic areas and large vessels. The immunohistochemical staining pattern of collagens IV (A) and V (B) are shown. Collagen IV, a basement membrane protein, was most abundant in blood vessels (arrowheads) and surrounding adipocytes (arrow) and was seldom found in fibrotic areas (asterisk) within the adipose tissue section. In contrast, collagen V was very abundant in fibrotic areas (asterisk) and surrounding blood vessels (arrowheads). Magnification, ×200; bar, 125 μm. C, Quantification of collagen V staining in lean and obese (BMI > 27 kg/m2) subjects. D, Relationship between collagen V mRNA abundance and SI. *, P < 0.05.
Fig. 4.
Fig. 4.
Collagen V localization in large vessels in obese subjects. Adipose tissue sections from lean (A–C) and obese (D–F) subjects were immunoreacted with antibodies against ASMA (B and E, green) and collagen V (C and F, red) to localize collagen V to blood vessels in merged images (A and D). Vessels from lean subjects exhibited very little collagen V accumulation. Vessels from obese subjects contained significantly more collagen V. In most vessels in obese subjects, collagen V could be seen to extend from the endothelial basement membrane to the outer edges of the vessel. Magnification, ×200; bar, 125 μm.
Fig. 5.
Fig. 5.
Macrophages alter elastin and collagen V gene expression in adipocytes. As described in Subjects and Methods, a coculture system was used to determine the effects of macrophage coculture on adipocyte ECM expression. THP-1 monocytes were differentiated into M1, M2a, or M2c polarized macrophages and cocultured with mature adipocytes for 24 h. Gene expression of collagen V (A) and elastin (B) was quantified by real-time RT-PCR in adipocytes alone or after culture with the polarized macrophages, as indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. adipocytes alone.
Fig. 6.
Fig. 6.
Collagen V inhibits angiogenesis. HUVEC were layered onto an ECM, as described in Subjects and Methods, and medium was then added in the presence of increasing concentrations of the respective collagens. A–C, Micrographs show the extent of endothelial cell branching after 10 h incubation alone (A) or with the addition of collagen I (B) and collagen V (C) at 10 μg/ml. Magnification, ×200; bar, 125 μm. D, Quantification of the branch points from the addition of increasing concentrations of collagen V. E, Collagen I, III, and V were added to the ECM at a concentration of 10 μg/ml, followed by incubation for 10 h and counting of branch points. *, P < 0.05 vs. control; ***, P < 0.01 vs. control.
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