The PPAR-γ Agonist Pioglitazone Modulates Proliferation and Migration in HUVEC, HAOSMC and Human Arteriovenous Fistula-Derived Cells

Figures

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  Figure 1. PPAR-γ expression in AVF tissues and cells. Immunohistochemical analysis of PPAR-γ in (a,b) normal AVF T0 and (c,d) failed AVF T1 veins. PPAR-γ protein was identified in AVF T0 tissues, localizing mainly in SMCs within the tunica media. In AVF T1 SMCs positive to PPAR-γ were detected in the tunica media of normal tissue surrounding the IH lesion but not directly involved in it (black arrows), whereas the IH characterized by the typical signs of intimal occlusion (black rectangle) did not express PPAR-γ. Mouse brown fat tissue was used as a positive control. (e) Quantification of PPAR-γ expressed as percentage of positive areas calculated on random fields captured from 10× images, by application of ImageJ (version 1.53) color deconvolution tool. (f) Expression of PPAR-γ mRNA in AVF cells (AVFCs T0 and AVFCs T1) analyzed by Real Time PCR. Results are shown as fold changes in AVFCs T1 relative to AVFCs T0. Each experiment was executed in triplicate (n = 3). Statistical analysis was performed by unpaired t test; * p = 0.0327; *** p = 0.0002. Abbreviations: AVF, arteriovenous fistula; AVFCs, arteriovenous fistula cells; n, number of values; PPAR-γ,…

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  Figure 2. Pioglitazone modulates HUVEC proliferation and migration. (a) Analysis of HUVEC proliferation performed through crystal violet stain and expressed as absorbance values read with a Spark multimodal microplate reader at 595 nm. Results are reported as normalized to control with vehicle (DMSO) at 24 h. (b) Analysis of HUVEC migration was performed through wound scratch assay by using an Incucyte S3 instrument. Representative images of wound healing taken at 0 h (initial wound process), 6 h, and 12 h, acquired with a 10× objective lens. Yellow: initial scratch wound area; pink: HUVEC migrated into the scratch area. 10× magnification. Measurement of migration parameters were (c) wound width, expressed in micrometers (μm) and (d) wound confluence, expressed as a percentage (%). Each experiment was executed in triplicate (n = 3). Statistical analysis was performed by two-way ordinary Anova test, followed by Tukey’s multiple comparisons tests; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GW, GW9662; HUVEC, Human Umbilical Vein Endothelial Cells; n, number of values; OD, optical density; PIO, pioglitazone.

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  Figure 3. Pioglitazone modulates HAOSMC proliferation and migration. (a) Analysis of HAOSMC proliferation performed through crystal violet stain and expressed as absorbance values red by Spark multimodal microplate reader at 595 nm. Results are reported as normalized to control with vehicle DMSO at 24 h. (b) Analysis of HUVEC migration performed through wound scratch assay by using Incucyte S3 instrument. Representative images of wound healings taken at 10× objective lens 0 h (initial wound process), 15 h, and 24 h. Yellow: initial scratch wound area; pink: HAOSMC migrated into the scratch area. 10× magnification. Measurement of migration parameters (c) wound width, expressed in micrometers (μm) and (d) wound confluence, expressed as percentage (%). Each experiment was executed in triplicate (n = 3). Statistical analysis was performed by two-way ordinary Anova test, followed by Tukey’s multiple comparisons tests; * p < 0.05; ** p < 0.01. Abbreviations: OD, optical density; GW, GW9662; HAOSMC, human aortic smooth muscle cells; n, number of values; PIO, pioglitazone.

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  Figure 4. AVFCs T1 isolated from failed AVF exhibit a more proliferative and migratory phenotype than AVFCs T0. (a) AVFCs T0 and AVFCs T1 were adherent cells with fibroblast-like morphology (20× magnification), and (b) exhibited an immunophenotype negative to CD34 and positive to CD44 and α-SMA. Scale bar: 50 μm, 20× magnification. (c) AVFCs T1 displayed an increased proliferative curve, as shown by crystal violet stain of cells cultured until three days. Data are reported as mean ± standard deviation. (d) Representative immunofluorescence of ki-67 expression in AVFCs T0 and AVFCs T1, and relative quantification measured as ratio of ki-67 positive cell number on total cells. Green: ki-67, blue: DAPI. Scale bar: 25 μm, 40× magnification. Real-time PCR analysis of remodeling and migration genes (e) SLUG, (f) MMP-9, and (g) VIMENTIN. Results are expressed as fold changes, and a statistical analysis was performed between AVFCs T0 and AVFCs T1. Each experiment was executed in triplicate (n = 3). Statistical analysis was performed by two-way Anova in (c) and unpaired Student t-test in (d–g). * p < 0.05; ** p < 0.01; **** p < 0.0001. Abbreviations: AVFCs, arteriovenous fistula cells; MMP-9,…

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  Figure 5. Pioglitazone determines proliferation decrease in AVFCs T0 and AVFCs T1. Analysis of AVFCs T0 (a,b) T1 proliferation evaluated by crystal violet (CV) stain and quantified in terms of absorbance by the Spark multimode microplate reader (Tecan) at 592 nm optical density (OD). Analysis of AVFCs T0 (c) and T1 (d) metabolic activity detected by MTT assay and quantified in terms of absorbance by the Spark multimode microplate reader (Tecan) at 570 nm OD. Number of values = 6. Data were normalized to untreated controls at 24 h, and analyzed by two-way Anova test with Tukey’s multiple comparisons tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Abbreviations: AVFCs, arteriovenous fistula cells; CTRL, control; CV, crystal violet; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl -2H-tetrazolium bromide; OD, optical density; PIO, pioglitazone.

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  Figure 6. Pioglitazone induces PPAR-γ expression and down-regulates migration/invasion genes in AVFCs T1. (a) PPAR-γ mRNA expression by real-time PCR and (b) representative Western Blot image (left) and densitometric analysis (right) of PPAR-γ protein expression in AVFCs T1 following treatment with pioglitazone at 10 μM for 48 h, corresponding at the experimental setting associated with growth cell decrease. (c) mRNA analysis of SLUG, MMP-9, and VIMENTIN genes in AVFCs T1 following PPAR-γ induction by pioglitazone. Real-time results are expressed as fold changes relative to untreated controls. All data are reported as mean ± SEM of at least three independent experiments (n = 3). Statistical analysis was performed by paired Student’s t-test relative to untreated controls. * p < 0.05; ** p < 0.01. Abbreviations: AVFCs, arteriovenous fistula cells; CTRL, control; GAPDH, glyceraldehyde 3-phospate dehydrogenase; n, number of values; PIO, pioglitazone; PPAR-γ, peroxisome-proliferator-associated-receptor-γ.

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  Figure 7. GW9662 reverses pioglitazone effects in AVFCs T1. (a) Analysis of cell proliferation in AVFCs T1 treated with pioglitazone (10 μM) alone or in combination with the PPAR-γ inhibitor GW9662 (5–10 μM). Absorbance values were analyzed by Spark multimode microplate reader (Tecan) at 592 nm OD and normalized to untreated control at 24 h. (b) Analysis of AVFCs T1 migration performed through wound scratch assay by using Incucyte S3 instrument. Representative images of wound healing were taken at 10× objective lens 0 h (initial wound process), 18 h and 24 h; yellow: initial scratch wound area, pink: AVFCs T1 migrated into the scratch area. Measurement of migration parameters (c) wound width, expressed in micrometers (μm) and (d) wound confluence, expressed as a percentage (%). Each experiment was executed in triplicate (n = 3). Statistical analysis was performed by two-way ordinary Anova test, followed by Tukey’s multiple comparisons tests; * p < 0.05; *** p < 0.001; **** p < 0.0001. Abbreviations: AVFCs, arteriovenous fistula cells; CTRL, control; GW, GW9662; n, number of values; OD, optical density; PIO, pioglitazone.