Role of hypertrophic adipocytes, collagen VI, and CD38 in adipose tissue fibrosis in obesity

Figures

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  Fig. 1. Fibrosis in human adipose tissue. A: Light and electron microscopy. Sirius Red staining (left and middle panels): representative images of fibrosis in visceral (omental) fat. In the right panel pericellular fibrosis is shown by high-resolution scanning electron microscopy (HRSEM, see Methods); scale bars: 50 μm (left), 15 μm (middle) and 7 μm (right). B: Fibrosis is significantly increased only in omental fat when compared with lean controls. C: Perilipin1 (PLIN1) immunohistochemistry. PLIN1-negative adipocytes (*) located near PLIN1-immunoreactive adipocytes are visible in a visceral biopsy from a patient with obesity; scale bar 40 μm. D: The percentage of PLIN1-negative parenchymal areas was higher in both subcutaneous and omental fat from patients with obesity. E: Correlation between PLIN1-negative parenchymal areas of visceral and subcutaneous fat in each patient. F: Correlation between the percentage of PLIN1-negative parenchymal areas and total fibrosis in subcutaneous and omental fat. Box-and-whisker plots with median and min-max values (B and D) represent the data distribution. Statistical significance was assessed by two-way ANOVA followed by the Benjamini– Yekutieli…

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  Fig. 2. Transmission (TEM) and high-resolution scanning (HRSM) electron microscopy of human visceral (omental) fat. A: TEM image from a lean control showing sparse collagen fibrils in close contact with the external adipocyte surface (arrows). B: TEM image from a representative biopsy from a patient with obesity showing abundant collagen fibrils tightly associated with the adipocyte surface. C: Representative image of a hypertrophic adipocyte (Ad) from obese fat showing microfibrils (F; possibly tropocollagen) apparently sprouting directly from the cell surface. COL: collagen fibrils. D: High-magnification transverse section of collagen fibrils shown in B, including enlarged dense fibrils (black arrows) compared with normal-like fibrils (white arrow). E: Longitudinal section of collagen fibrils showing altered fibril morphology (black arrow) compared with normal-like fibrils (white arrow). Scale bars: 1.2 μm in A and B, 280 nm in C, 100 nm in D and E. F: Size of collagen fibrils in lean, with obesity and fibrosis > 5% and Ullrich patients (see also 2.8 section of Results). G: Representative HRSEM images of omental fat from a lean patient (G) and a patient with obesity and fibrosis > 5%…

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  Fig. 3. Expression of collagen genes and inflammatory markers. A: In vitro gene expression of COL1A1, COL3A1, and COL6A3 in a human adipocyte cell line (hMADS) and in human dermal fibroblasts (NhDF). Mature adipocytes express COL1A1 at levels comparable to those of fibroblasts, but higher levels of COL3A1 and COL6A3 than NhDF cells. Hypertrophic adipocytes (HA), compared with mature adipocytes, express higher levels of COL1A1 (ns) and lower levels of COL6A3. B: Light microscopy of differentiated hMADS. Addition of fatty acids to the medium induced hypertrophy of lipid droplets (LDs), mimicking the phenotype of hypertrophic obese adipocytes (HA) (left and middle panels). The mean size of LDs was significantly increased in differentiated hypertrophic hMADS cells (right panel). Scale bar: 10 μm. C: Quantitative data showing that CD68-immunoreactive macrophages are more abundant in both subcutaneous and visceral (omental) fat from patients with obesity. D: Positive correlations between the extent of fibrosis and the density of CD68-immunoreactive macrophages in fibrotic areas were observed in both visceral and subcutaneous fat. E: Increased expression of type I (COL1A1) and type III (COL3A1)…

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  Fig. 4. Congenital mutations in human COL6 genes in vivo appear to promote fibrillar collagen production by adipocytes. A: Representative light-microscopy images with Sirius Red staining of fat biopsies from the Ullrich case (left panel) and the Bethlem cases (middle and right panels). Details of the COL6 gene mutations are indicated. Abundant lobular and pericellular fibrosis is evident in all cases. Scale bars: 80 μm (left panel), 45 μm (middle panel), and 50 μm (right panel). The graph summarizes the percentage of fibrosis detected in subcutaneous fat from the patients studied. B: Representative transmission electron microscopy (TEM) image from the Ullrich case showing abundant fibrillar collagen in close contact with a mature adipocyte (see also Suppl. Figure 6). Some mature collagen fibrils (red arrows) are visible within the cytoplasm (left panel). The lumen of the rough endoplasmic reticulum appears dilated (yellow arrows). The right panels show transverse sections of collagen fibrils. In the upper right panel, some collagen fibrils appear enlarged. The lower right panel shows a higher-magnification view of the boxed area in right upper panel. Enlarged fibrils (white arrows, some…

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  Fig. 5. CD 38 immunohistochemistry and in vivo and in vitro data. A: Light microscopy. Representative images from lean (LEAN) and obese (OBESE) visceral fat. Most lean adipocytes were negative. Rare positive adipocytes showed a granular-like positivity (arrow). Weakly positive macrophages were also found in lean fat. Fibrous tissue was negative in both lean and obese fat. Intense granular-like positivity was observed in adipocytes from patients with obesity. Macrophages were intensely positive both when located in interstitial parenchymal tissue and when forming CLS in obese fat. Scale bars: 3 μm in all upper panels; 30 μm in the lower left and right panels; 10 μm in the upper middle panels (macrophages), 30 μm in the lower middle panel (CLS). B: A positive correlation was found between fibrosis and CD38 gene expression. C: CD38 gene expression in the human adipocyte cell line hMADS under standard conditions and after the addition of fatty acids to the culture medium to obtain hypertrophic adipocytes (see also Fig. 3). Aligned dot blot with mean ± S.E.M. (C) represents the data distribution. Statistical significance was assessed by unpaired two-tailed Student’s t-test (C). ***p < 0.001.

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  Fig. 6. Gene expression in visceral (omental) fat from patients with obesity in our case series. Correlations suggesting a role for myofibroblasts (ASMA with a positive tendency for Activin A) and peptidase D (PEPD) in the pathogenesis of visceral fat fibrosis were found (A, B and F). The data did not support a role for preadipocytes (PDGFRA and PREF1) or ENPP2 (C, D and E).