PPARγ is expressed in endothelium of normal and neoplastic tissues. (a) Immunofluorescent double staining for vWF and PPARγ demonstrates PPARγ expression in endothelium of normal skin. vWF-stained endothelial cells are shown in green, and PPARγ-positive cells are red. PPARγ-positive nuclei inside the endothelium appear yellow (arrows). Red cells are PPARγ-expressing mural cells. Scale bar, 20 μm. (b) In human glioblastoma tissue, PPARγ (red) is expressed in both tumor (arrowheads) and endothelial cells. Colocalization of red and green fluorescence indicates PPARγ in blood vessels (arrows). Scale bar, 20 μm. (c) Western blot analysis of PPARγ expression of cell lysates from isolated tumor endothelial cells from T241 fibrosarcoma (T-EC), and from cultured tumor cells including T241 fibrosarcoma (T241), rhabdomyosarcoma (RMS), liposarcoma (LS), glioblastoma (U87), and LLC. (d) Western blot analysis of PPARγ expression from isolated LLC tumor cells (LLC) and corresponding endothelial cells from LLC-GFP tumor (T-EC) and skin endothelial cells (N-EC). Levels of β-actin demonstrate protein loading. (e) Quantitation of autoradiographic signals. Values represent arbitrary densitometric units normalized for GAPDH signals. T-EC, tumor endothelium; N-EC, normal (skin) endothelium. (f) Western blot analysis of PPARγ protein showing higher expression in proliferating than in quiescent endothelium. Starv., starvation; Prolif., proliferation; Confl., confluent (contact inhibition). GAPDH levels are shown to demonstrate equal loading of protein in each lane.

TZDs activate endothelial PPARγ in a lactacystin-reversible manner. (a) Western blot analysis of PPARγ protein in proliferating HUVECs. PPARγ protein levels decrease in a dose-dependent manner with rosiglitazone (Rosi). Control, C3H/10T 1/2 (fully differentiated C3H cells), a positive control for PPARγ; –, HUVECs cultured in starvation media; +, HUVECs cultured in stimulation (growth) media. (b) PPARγ protein decreases with 5 μM (5T) and 10 μM (10T) troglitazone. In contrast, PPARα activator WY 14643 at 5 μM (5W) has no effect. 1R, 1 μM concentration of rosiglitazone. (c) PPARγ activation can be reversed by coadministration of 2.5 μM lactacystin. HUVECs treated with both rosiglitazone and lactacystin (lanes 3 and 5) show PPARγ expression comparable to that of HUVECs treated with stimulation medium alone (lane 1). Lactacystin alone has no effect on basal PPARγ levels (lane 6).

TZDs have direct and indirect antiangiogenic effects. (a) Percent proliferation of BCE cells is determined by comparing cells exposed to an angiogenic stimulus (bFGF) with those exposed to bFGF and rosiglitazone, relative to unstimulated cells (low serum), in a 72-hour proliferation assay. Percent proliferation of tumor cells is determined by comparing cells exposed to 10% FBS with those exposed to 10% FBS and rosiglitazone, relative to starvation conditions (0.5% FBS). In both cases, percent proliferation = 100 × (cells_{stimulated+TZD} – cells_{low serum})/(cells_stimulated – cells_{low serum}). Each point represents the mean ± SD for three wells. Representative experiments of three separate assays are shown. The difference in inhibition between day 3 and day 7 is a result of increased endothelial cell death on day 7 in unstimulated cells in starvation media (cells_{low serum}). (b) Percent of proliferation for endothelial and tumor cells treated for 7 days with rosiglitazone. (c) Levels of the phosphorylated form of eIF-2α protein upon treatment with rosiglitazone in HUVECs. NIH3T3 fibroblasts (3T3) serve as a positive control. (d) VEGF levels (expressed as percent decrease) in U87 and LLC cells after 6 days of treatment with rosiglitazone.

PPARγ ligands inhibit in vivo angiogenesis in the CAM and bFGF-induced corneal neovascularization. (a) A normal CAM implanted with a methylcellulose disk. (b) After a 48-hour exposure to rosiglitazone (2.5 μg), avascular zones free of capillaries and small vessels were observed around the disc (arrows point to a 3-mm avascular zone). Scale bar, 22.5 mm. (c) bFGF-induced neovascularization in control cornea on day 6. (d–f) Systemic treatment with rosiglitazone at 50 mg/kg/d (d), 100 mg/kg/d (e), and 400 mg/kg/d (f). (g) Area of inhibition (percent) by various doses of daily and twice-daily rosiglitazone administration. Inhibition was determined on day 6 by the formula 0.2 × π × neovessel length × clock hours of neovessels (n = 6 eyes per group; experiment was performed three times). Lines are fitted to the data assuming a Gaussian distribution.

Systemic therapy with PPARγ ligands inhibits primary tumor growth. After tumors were 100–200 mm³ in size, rosiglitazone treatment (100 mg/kg/d) was initiated (day 0). On the last day of treatment, the statistical difference between control (Ctr; blue) and treated (pink) groups was determined by Student’s t test. (a) Glioblastoma (U87) (P < 0.01). (b) LLC (P < 0.001). (c) Liposarcoma (LS) (P < 0.001). (d) Rhabdomyosarcoma (RMS) (P < 0.001).

PPARγ ligand therapy reduces vessel density and endothelial cell proliferation. (a, blue inset) Representative control and treated glioblastoma tumors on treatment day 40 are shown. Scale bar, 1 cm. (a–d, insets) Hematoxylin-and-eosin staining of tumors from control and rosiglitazone-treated mice show no signs of cytologic differentiation. In both liposarcoma and rhabdomyosarcoma, increased necrosis (n) was observed in treated tumors. Scale bar, 200 μm. (a–d) In each panel the x axis represents vessel density defined as the percentage of vessel area (CD31)/tumor area in each tumor field. The y axis indicates the percent of analyzed fields with the given vessel density. Bars further to the right represent fields with higher microvessel density. Upon rosiglitazone treatment, a significant decrease in vessel density was observed for all tumors as indicated by left-shifting of all histograms (P < 0.001). For example, in rosiglitazone-treated glioblastoma, 4% vessel density is present in 28% of all counted fields. Total fields scored per tumor (Ctr:Rosi): U87, 124:178; LLC, 163:131; LS, 186:178; RMS, 314:229. (e) Endothelial cell (EC) proliferation in rosiglitazone-treated and control U87 and LLC tumors as determined by immunofluorescent double staining (MECA-32 and PCNA). U87, P < 0.001; LLC, P < 0.05. Total fields scored per tumor (Ctr:Rosi): LLC, 36:26; U87, 34:33.

Systemic therapy with PPARγ ligands prevents metastatic invasion after removal of primary LLC. (a) Visual comparison of mouse lungs on treatment day 17 with vehicle (Ctr) or rosiglitazone (R50, 50 mg/kg/d; R100, 100 mg/kg/d). Scale bar, 1 cm. (b) Rosiglitazone (R50 and R100) prevents metastasis, as represented by a significant decrease in lung weight, which correlates with tumor burden. R400, 400 mg/kg/d. NL, normal lung weight. (c) Hematoxylin-and-eosin staining showing individual LLC tumor cells inside blood vessels (black arrowheads) in lungs of rosiglitazone-treated mice. Scale bar, 50 μm. (d and e, insets) Typical patterns of centrosomal abnormalities in control LLC metastasis, as demonstrated by immunofluorescence staining for pericentrin (green). (d and e) In rosiglitazone-treated mouse lungs, immunofluorescence double staining for pericentrin and PECAM (endothelial marker) demonstrates LLC tumor cells inside blood vessels. Abnormal centrosomes of tumor cells stained with pericentrin are green (white arrowheads), and endothelium is red. The asterisk shows red blood cells (d, red). Hoechst staining of nuclei is blue. Scale bar, 5 μm. (f and g) Effect of rosiglitazone treatment on MMP and TIMP activity. Representative gelatin zymograms demonstrate that the activity of the predominant gelatinase detected in HUVECs appears to be unaffected by rosiglitazone treatment (f). In contrast, TIMP bioactivity assays demonstrate an increase in TIMP activity up to approximately 1.0 μM rosiglitazone (g).