In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue (original) (raw)
In vivo imaging system showing enhanced leukocyte-EC-platelet interaction and perturbed blood flow in obese visceral fat pads. To visually analyze the microcirculation and cellular dynamics within adipose tissue in living mice, we developed in vivo imaging techniques (Figure 1) by modifying the methods that have previously been applied to other tissues (5, 6). The fat pads of anesthetized mice were observed through small dermal windows, without being exteriorized. Using a spinning-disk confocal microscope and a charge-coupled device camera, we visualized obese adipose tissue and assessed its cellular dynamics in living mice.
Schematic diagram of the confocal microscope for real-time in vivo imaging. To visualize cell dynamics in vivo, fluorescent dyes were injected into the tail veins of anesthetized mice. A small incision was then made for an observation window, and the mouse was set on a heating pad to maintain body temperature. An inverted microscope equipped for Nipkow–spinning disk confocal laser microscopy, which enabled scanning at up to 1,000 frames/s, was used to visualize the tissue. See Methods for details.
Blood cell dynamics were visualized by injecting FITC-dextran (Figure 2, A–I, and Supplemental Movies 1–6; supplemental material available online with this article; doi:10.1172/JCI33328DS1), and leukocytes were identified by nuclear staining with acridine orange (Figure 2, K, L, N, and O, and Supplemental Movies 7 and 8). Single-platelet kinetics were also visualized by injecting fluorescent anti-CD41 antibody (Supplemental Movie 9). We first compared epididymal adipose tissue in ob/ob mice with that in lean control ob/+ mice. In ob/+ mice, capillary blood flow within epididymal adipose tissue was continuous, and leukocytes interacting with the vessel walls were rarely observed (Figure 2, D and G, and Supplemental Movies 1 and 4). But there was a striking difference in the obese epididymal adipose tissue of 12-week-old ob/ob mice: leukocytes rolling on (ob/+, 1.85 ± 0.58 leukocytes/mm; ob/ob, 10.30 ± 2.67; n = 5, total 50 vessels; P < 0.01) or firmly adhering to the vessel walls (ob/+, 1.03 ± 0.45 leukocytes/mm; ob/ob, 12.84 ± 2.93; n = 5, total 50 vessels; P < 0.01) were frequently observed in postcapillary venules, which were identified based on vessel diameter and slower flow velocity than that seen in arterioles (Figure 2, K–P, and Supplemental Movies 7 and 8). Interestingly, firmly adhering leukocytes were often observed on vascular walls adjacent to CLSs (Figure 2O), which, as reported previously, consist of aggregated macrophages (3). In addition to leukocytes, adhesion of platelets to vessel walls also increased in postcapillary venules, and firm leukocyte adhesion was always coupled with platelet aggregation (Figure 2, E and N, and Supplemental Movies 2 and 8).
Abnormal leukocyte-EC-platelet interactions in obese adipose tissue revealed by intravital fluorescence microscopy. One-shot (A–C and O) and time-lapse images (D–I, K, L, and N) obtained by intravital fluorescence microscopy at the postcapillary venule (D–F) and capillary (G–I) levels in adipose tissue from ob/+, IgG-treated ob/ob, and anti–ICAM-1–treated ob/ob mice as indicated as well as untreated ob/ob mice (K, L, N, O, and Q). Time-lapse images were reconstructed in red-green-blue order from 3 sequential images obtained at 30-ms intervals from Supplemental Movies 1–6 (D–I, respectively). Following FITC-dextran injection (MW 150,000), blood cells were negatively visualized in cyan-magenta-yellow order. Erythrocyte, leukocyte, and platelet cell dynamics were visualized at high spatiotemporal resolutions. Blood cell type was determined from the cell size. Where indicated, anti–ICAM-1, anti–E-selectin (Esel), anti–P-selectin (Psel), and normal rat IgG was injected intravenously prior to observation. Note the firmly adherent leukocytes and platelets on the vascular wall and the low hematocrit within the postcapillary venules of ob/ob mice (E). (J) Relationship between blood flow velocity and vascular diameter in adipose tissue from ob/+ (blue), IgG-treated ob/ob (red), and anti–ICAM-1–treated ob/ob (green) mice. Blood flow velocity at the capillary level (<8 μm) was significantly slower in ob/ob than ob/+ mice, but was increased to ob/+ levels by anti–ICAM-1 treatment (n = 5, total 100 vessels/genotype). (K–P) Nuclear staining with acridine orange for specific visualization of leukocyte dynamics. Time-lapse images were reconstructed in red-green-blue order from 3 sequential images obtained at 90-ms intervals from Supplemental Movies 7 (L) and 8 (N), and enabled leukocyte rolling and firm adhesion were visualized. FITC-conjugated anti-CD41 antibody was used to visualize platelet dynamics (N). Number of rolling (M) and firmly attached (P) leukocytes per postcapillary venule length (leukocytes/mm; n = 5, total 50 vessels/genotype) and in epididymal adipose tissue from lean C57BL/6 mice fed normal chow (WT) and high-fat diet–induced obese mice (HFD). In ob/ob mice, the number of rolling/adherent leukocytes significantly increased in epididymal fat (EPI), but not in inguinal subcutaneous fat (SC) or quadriceps skeletal muscle (MUS). (Q and R) Vascular permeability of epididymal and subcutaneous adipose tissue analyzed using FITC-dextran (MW 4,000). Fluorescence intensities within the vessel (R2) and stromal space (R1) were measured, and R1/R2 ratio was used to assess the level of extravasation of FITC-dextran. Permeability increased in obese epididymal adipose tissue from ob/ob and high-fat diet–fed mice, but was normalized by anti–ICAM-1 (n = 3, total 60 points from 20 fields/genotype). Scale bars: 100 μm (A–C and K), 50 μm (O), 10 μm (D–I, L, N, and Q). *P < 0.05.
In contrast to epididymal fat pads, the inguinal subcutaneous fat pads of ob/ob mice showed no significant increases in the levels of leukocyte rolling or adhesion in the microcirculation (Figure 2, M and P). In addition, leukocyte-EC interactions were also not augmented in the skeletal muscle quadriceps femoris in ob/ob mice. Thus in obesity, leukocyte-EC-platelet interactions are apparently selectively enhanced in visceral adipose tissue.
Further analysis of the blood flow within the epididymal adipose tissue revealed that while the blood flow in lean adipose tissue was largely continuous, even at the capillary level, the flow in obese epididymal adipose tissue was varied and often discontinuous (Figure 2, G and H, and Supplemental Movies 4 and 5). The passing leukocytes appeared to perturb the flow in obese adipose tissue (Figure 2H and Supplemental Movie 5), and leukocytes firmly adhering to vessel walls were often observed at sites of discontinuous blood flow, suggesting that the leukocyte-EC interactions retard flow (15, 16). As a result, the average blood flow in the relatively small capillaries (<8 μm) was significantly slower in obese adipose tissue than in lean adipose tissue (ob/+, 391 ± 21 μm/s; IgG-treated ob/ob, 187 ± 17 μm/s; n = 5, total 100 vessels; P < 0.01; Figure 2J). Moreover, lower local hematocrits and slower blood flow velocities were seen in venules distal to capillaries with adherent leukocytes and platelet aggregations (Figure 2E). Such perturbation of blood flow was not observed in subcutaneous adipose tissue in ob/ob mice, again indicating that leukocyte-EC interactions are selectively activated in visceral adipose tissue.
Our observation of local hypocirculation in obese epididymal adipose tissue is in agreement with previous reports (13) and implies local hypoxia. This was confirmed when we administered pimonidazole and observed increased formation of pimonidazole adducts in adipocytes and stromal cells in obese epididymal adipose tissue (Figure 3, A–D), which suggests that perturbation of the blood flow and the resultant hypoxia likely contribute to adipose tissue dysfunction in obesity. The hypoxic state of obese adipose tissue was supported by our finding there of upregulated expression of hypoxia-inducible factor–1α (HIF-1α; Supplemental Figure 1).
CLS formation in adipose tissue in ob/ob mice. Epididymal adipose tissue obtained from 12-week-old ob/+, IgG-treated ob/ob, and anti–ICAM-1–treated ob/ob mice were examined with tissue imaging. Anti–ICAM-1 and control antibody was administered intraperitoneally (100 μg/mouse) 3 times per week for 2 weeks prior to observation. (A–D) Analysis of hypoxia using pimonidazole. Pimonidazole adducts were immunostained, adipocytes were counterstained with BODIPY, and nuclei stained with Hoechst. Adipose tissue from ob/ob mice was in a more hypoxic state than that from ob/+ mice, and anti–ICAM-1 mitigated the hypoxia. Pimonidazole adducts were found in both adipocytes and other cell types, particularly macrophages within CLSs. (D) The hypoxic state was quantified based on fluorescence intensity per low-power field (n = 5, total 50 fields/genotype). (E–H) ICAM-1 staining showed that macrophages within CLSs strongly express ICAM-1 in IgG-treated ob/ob mice. (I–L) Macrophages within CLSs strongly express CCR2 in IgG-treated ob/ob mice, indicating macrophage activation within CLSs. (M–O) Immunohistochemical analysis of F4/80 staining showed increased CLS formation (arrows) in IgG-treated ob/ob mice. (P and Q) Number of CLSs (P) and macrophages (Q) per number of adipocytes (n = 5, total 1,000 adipocytes from 50 fields/genotype). Anti–ICAM-1 reduced CLS number. (R–T) Dead cell staining (YO-PRO1) of CLSs. Some CLSs contained dead adipocytes and macrophages that positively stained with YO-PRO1 (arrows). (U) CLSs with YO-PRO1-1+ cells relative to total CLS number (n = 5, total 50 fields/genotype). (V) Double staining with PI and YO-PRO1 was performed to discriminate apoptotic (YO-PRO1+PI+) and necrotic (YO-PRO1+PI–) cell death in CLSs from ob/ob mice. Nuclei were counterstained with Hoechst. Scale bars: 100 μm (A–C, E, F, H–J, L–O, and R–T), 10 μm (G, K, and V). *P < 0.05.
Enhanced expression of adhesion molecules in ECs and macrophages in obese visceral adipose tissue. To investigate the molecular mechanisms underlying the heightened leukocyte-EC interactions within obese adipose tissue, we focused on expression of adhesion molecules by initially examining their expression levels in stromal-vascular fractions (SVFs) from epididymal fat pads (Figure 4, A–C). Real-time PCR analyses showed several-fold increases in the levels of ICAM-1, E-selectin, and P-selectin expression in SVFs from obese mice compared with control mice. To determine which cell populations within the SVFs contributed to the increases, surface expression of adhesion molecules and cell type–specific markers were analyzed by flow cytometry with gating to macrophages (F4/80+; Figure 4, D–H) or ECs (CD31+; Figure 4, I–L). When gated to ECs (CD31+ cells), surface expression of P-selectin, E-selectin, and ICAM-1 was increased in obese adipose tissue, whereas VCAM-1 expression was unchanged. That P- and E-selectins are not expressed under normal physiological conditions but are synthesized and exported to the EC surface under inflammatory conditions (17) indicates that the ECs are activated within obese adipose tissue.
Enhanced surface expression of adhesion molecules in macrophages and ECs in obese adipose tissue. (A–C) Adhesion molecule expression examined by real-time PCR. mRNA expression of P- and E-selectin and ICAM-1 in the SVF of adipose tissue from ob/+ and ob/ob mice was analyzed (n = 5). (D–L) FACS analysis of the surface expression of adhesion molecules in F4/80+ macrophages (D–H) and CD31+ ECs (I–L) from ob/+ (gray line) and ob/ob (black line) mice. Expression of ICAM-1, L-selectin, and CD11c was upregulated in ob/ob compared with ob/+ macrophages. E-selectin, P-selectin, and ICAM-1 expression was upregulated in ob/ob ECs compared with ob/+ ECs. (M) Expression of ICAM-1 in F4/80+ cells was examined in local adipose blood. The y axis denotes cell count; the x axis denotes signal intensity. *P < 0.05.
We found that obesity also markedly affected expression of adhesion molecules on macrophages (F4/80+ cells; Figure 4, D–F). As reported previously, the number of macrophages (defined as F4/80+CD11b+ cells) within SVFs was increased in obese adipose tissue (ob/+, 11.7% ± 1.7%; ob/ob, 26.1% ± 1.2%; n = 5; P < 0.01). When gated to macrophages (F4/80+ cells), the levels of ICAM-1 and L-selectin expression were significantly increased, whereas expression of CD18 (integrin β2-chain, the ligand for ICAM-1) and CD162 (P-selectin ligand 1; PSGL1) was unchanged (Figure 4, G and H). Thus expression of adhesion molecules is clearly upregulated in both ECs and macrophages within obese adipose tissue.
Although ICAM-1 is expressed in leukocytes and ECs under normal conditions, it is upregulated by proinflammatory cytokines in atherosclerotic lesions (10). Robker et al. recently reported that expression of ICAM-1 protein is localized to ECs in lean adipose tissue (18), but to our knowledge its expression in obese adipose tissue has not previously been documented. Because our FACS analysis indicated that ICAM-1 expression was clearly upregulated in both ECs and macrophages (Figure 4, D and K), we further analyzed localization of ICAM-1 in adipose tissue using the confocal microscopy–based tissue imaging method that we recently developed (7). Immunohistochemical staining showed strong ICAM-1 and CCR2 expression in macrophages within CLSs in obese mice, suggesting M1 activated phenotypes (4), with stromal macrophages expressing lower levels of ICAM-1 (Figure 3, E–L).
Locally enhanced platelet dynamics in obese adipose tissue. That firmly adherent leukocytes were often found adjacent to CLSs (Figure 2O) suggests that the enhanced surface expression of adhesion molecules such as ICAM-1 likely promotes extravasation of macrophages into CLSs. We found that firm leukocyte adhesion was always coupled to platelet aggregation in obese epididymal fat pads (Figure 2, E and N, and Supplemental Movies 2 and 8). Indeed, platelet dynamics in postcapillary venules were markedly enhanced in obese adipose tissue, such that local aggregation and rolling of platelets on the vascular walls were significantly increased (ob/+, 0.43 ± 0.25 adhered platelets/mm; ob/ob, 1.59 ± 0.36 adhered platelets/mm; n = 5, total 50 vessels; P = 0.01; Supplemental Movie 2). Furthermore, approximately 30% of the aggregated platelets were found with adherent leukocytes. To examine the activation status of systemic platelets, surface P-selectin expression was examined using flow cytometry with systemic blood obtained by cardiac puncture (Figure 5A). P-selectin is stored in α-granules in resting platelets and expressed on the platelet surface only during and after platelet degranulation (19). We found no significant difference in surface P-selectin levels in platelets from the systemic circulations of lean and obese mice (Figure 5A). In contrast, platelets obtained from blood within adipose tissue showed markedly elevated surface expression of P-selectin, which is indicative of local platelet activation within obese adipose tissue (Figure 5B).
Increased platelet P-selectin expression and formation of monocyte-platelet conjugates in obese adipose tissue. (A and B) Platelet surface P-selectin expression in systemic circulation (A) and local adipose blood (B) from ob/+ (gray line) and ob/ob (black line) mice was examined to evaluate the activation status of platelets. Platelets were gated based on CD41 expression and side scatter. Platelet activation was indicated in obese adipose tissue blood, but not in systemic blood from ob/ob mice. The y axis denotes cell count; the x axis denotes signal intensity. (C) Quantification of monocyte-platelet conjugates in the systemic circulation and adipose tissue blood. Systemic and local blood obtained from adipose tissue was examined by FACS using F4/80 (macrophage) and CD41 (platelet) gates. The ratio of the F4/80+CD41+ cell (conjugates) to the total F4/80+ cell count (macrophages) is shown (n = 5 per genotype). *P < 0.05.
To further analyze platelet dynamics, we used flow cytometry to examine platelet-monocyte conjugate formation (Figure 5C). Degranulated platelets aggregate with monocytes, initially via the binding of platelet surface P-selectin to its PSGL1 counter-receptor on the surface of macrophages. Previous studies have shown that monocyte-platelet aggregates are highly sensitive indicators of platelet activation, particularly in vivo (20). Activated platelets have also been shown to interact with monocytes and the endothelium, depositing chemokines on their surfaces and promoting atherogenesis (21, 22). We counted monocyte-platelet aggregates in blood within adipose tissue using flow cytometry and found that the number of aggregates was markedly increased in obese adipose tissue. In addition, the number of circulating platelet-monocyte conjugates in the systemic circulation tended to be increased, but did not reach statistical significance, which is consistent with local activation of platelets within obese adipose tissue.
EC barrier dysfunction in obese visceral adipose tissue. Another hallmark of inflammatory responses in the microcirculation is EC barrier dysfunction (i.e., increased vascular permeability), which we evaluated based on the ratio of fluorescence intensities within the vessel and stromal space following injection of mice with small-sized FITC-dextran (MW 4,000) (23, 24). We found that the FITC-dextran was highly extravasated into the stromal space in obese epididymal adipose tissue in ob/ob mice, but not in control lean adipose tissue, indicating that vascular permeability was significantly increased in the former (Figure 2, Q and R). By contrast, we did not find increased vascular permeability in subcutaneous fat pads in ob/ob mice.
Enhanced leukocyte-EC-platelet dynamics and EC barrier dysfunction in diet-induced obesity. To determine whether the observed enhancement of cellular dynamics is a fundamental feature of obese visceral adipose tissue, we analyzed the cellular dynamics in high-fat diet–induced obese mice. Body weights were significantly higher in 12-week-old mice fed a high-fat diet for 4 weeks than in mice fed normal chow (high-fat diet, 32.0 ± 0.25 g; normal chow, 25.6 ± 0.25 g; n = 5; P < 0.01). The levels of leukocyte rolling and adhesion were significantly higher in epididymal adipose tissue in diet-induced obese mice than in control mice, although the increases were smaller than those observed in ob/ob mice (Figure 2, M and P). We also observed increased vascular permeability in epididymal adipose tissue of mice with high-fat diet–induced obesity (Figure 2R). The results presented thus far clearly indicate that inflammatory changes are taking place in the microcirculation selectively within visceral obese adipose tissue. ECs, macrophages, and platelets are all locally activated and interacting via adhesion molecules.
Inhibition of adhesion molecules mitigates visceral adipose tissue inflammation. Levels of ICAM-1 expression were markedly increased on both macrophages and ECs in obese adipose tissue, and particularly high levels of ICAM-1 expression were present in macrophages within CLSs (Figure 3, E and F, and Figure 4, D and K). We also confirmed the presence of greater numbers of F4/80 cells (monocytes) expressing ICAM-1 in local adipose tissue blood (Figure 4M). Based on these findings, we hypothesized that inhibition of ICAM-1–mediated cell-cell interactions might affect leukocyte-EC dynamics and inflammation in obese adipose tissue. To test that idea, we injected either anti–ICAM-1 neutralizing antibody or control normal IgG into ob/ob mice. At 30 min after injection, the numbers of rolling (IgG-treated ob/ob, 9.96 ± 2.53 leukocytes/mm; anti–ICAM-1 ob/ob, 6.18 ± 1.33 leukocytes/mm; n = 5, total 50 vessels; P = 0.03) and adherent (IgG-treated ob/ob, 11.56 ± 4.28 leukocytes/mm; anti–ICAM-1 ob/ob, 1.30 ± 0.71 leukocytes/mm; n = 5, total 50 vessels; P < 0.01) leukocytes were significantly diminished by anti–ICAM-1 antibody compared with normal IgG (Figure 2, M and P). Administration of normal IgG did not significantly alter the levels of leukocyte rolling or adhesion compared with untreated ob/ob mice (Figure 2, M and P). Anti–ICAM-1 treatment also mitigated the reduction in small capillary (<8 μm) blood flow (IgG-treated ob/ob, 187 ± 17 μm/s; anti–ICAM-1 ob/ob, 421 ± 21 μm/s; n = 5, total 100 vessels; P < 0.01, Figure 2J). In addition, 24 h after administration of the anti–ICAM-1 antibody, vascular permeability had declined to a level similar to that seen in lean controls (Figure 2R). Thus, inhibition of ICAM-1 suppresses leukocyte-EC dynamics and improves EC function and blood flow.
Because surface expression of E- and P-selectins was increased in ECs, we also analyzed the effects of neutralizing antibodies against them. We found that the numbers of rolling leukocytes were significantly diminished by the antibodies. Similarly, the numbers of adherent leukocytes tended to be reduced by both P- and E-selectin blockade, but the difference did not reach statistical significance (Figure 2, M and P).
The observed acute effects of an ICAM-1 neutralizing antibody on leukocyte-EC dynamics prompted us to test whether chronic administration of the antibody further inhibits macrophage infiltration into adipose tissues. We administered the anti–ICAM-1 antibody or normal IgG 3 times per week for 2 weeks, after which the numbers of infiltrating macrophages and CLSs were determined. The number of CLSs was significantly higher in ob/ob mice than ob/+ mice (ob/+, 0.25 ± 0.16 CLS/field; IgG-treated ob/ob, 5.54 ± 0.93 CLS/field; n = 5, total 50 fields; P < 0.01; Figure 3, M–Q), as previously reported (3, 7, 25, 26). Treatment with anti–ICAM-1 antibody reduced CLS formation (anti–ICAM-1 ob/ob, 2.36 ± 0.61; n = 5, total 50 fields; P < 0.01 vs. IgG-treated ob/ob; Figure 3P), and there was a corresponding reduction in the numbers of infiltrating macrophages (ob/+, 0.43 ± 0.03 macrophages/adipocyte; IgG-treated ob/ob, 1.31 ± 0.06 macrophages/adipocyte; anti–ICAM-1 ob/ob, 1.05 ± 0.07 macrophages/adipocyte; n = 5, total 1,000 adipocytes; P < 0.01; Figure 3Q).
We then determined whether CLSs contained dead adipocytes or macrophages. Previous studies have shown that adipocytes in CLSs undergo necrotic cell death (3). Only 26% of CLSs contained dead cells, including adipocytes and stromal cells (YO-PRO1+ cells), in anti–ICAM-1–treated mice, while 54% of CLSs did so in control ob/ob mice (Figure 3, R–U). To distinguish apoptotic from necrotic cell death, double labeling with YO-PRO1 and propidium iodide (PI) was performed, and the results obtained showed almost 40% of the dead cells within CLSs (YO-PRO1+) had undergone apoptotic cell death (YO-PRO1+PI+) in obese mice (Figure 3V).
The reduction in macrophage infiltration was confirmed using flow cytometry based on counts of F4/80+CD11b+ cells (IgG-treated ob/ob, 26.1% ± 1.2% macrophages in SVF; anti–ICAM-1 ob/ob, 20.9% ± 1.4% macrophages in SVF; n = 5; P < 0.01). Thus anti–ICAM-1 treatment not only inhibited leukocyte-EC interaction, it also mitigated macrophage infiltration into obese adipose tissue.