Macrophage phenotype and function are dependent upon the composition and biomechanics of the local cardiac tissue microenvironment

Figures

• 

  Figure 1. Young and aged cardiac extracellular matrix can be decellularized while retaining matrix protein content. (A) Picrosirius histological staining of collagen deposition in cardiac tissue samples isolated from mice of 1 month, 12 months, and 23 months of age. (B) Quantification of the percent area of positive picrosirius red expression per field of view for tissue samples from 1 month, 12 month, and 23 month old mice. (C) Alcian blue staining of sulfated proteoglycans in cardiac tissue samples isolated from mice of 1 month, 12 months, and 23 months of age. (D) Hydroxyproline mass percentage and (E) glycosaminoglycan mass percentage for both young and aged native as well as young and aged decellularized cardiac samples. (F) 4’,6-diamidino-2-phentylindole (DAPI) nuclear staining of native and decellularized tissue samples (G) Quantification of dsDNA content isolated from young and aged either native or decellularized cardiac tissue samples. Values shown represent mean with 25%/75%. Error bares represent standard deviation. N=4-5; * p<0.05; Scale bars (A, C) = 1mm; Scale bar (F) = 100um. Independent samples t-test used to assess significant trends in (D, E). One-way ANOVA with post-hoc…

• 

  Figure 2. Macrophage morphology is dependent upon the stiffness of the culture substrate. (A) Naïve bone-marrow derived macrophage culture on 8kPA gel, 32kPA gel, or tissue culture plastic (TCP). Macrophages cultured on gels of lower stiffnesses tended to exhibit more round morphologies with few to no filopodial extensions (black arrows, 8kPA, 32kPA gel groups). Conversely, macrophages cultured on gels of increased stiffness, such as on tissue culture plastic exhibited more spread morphologies often with several filopodial extensions (white arrows, TCP). (B) Cell area was found to be significantly increased for macrophages cultured on tissue culture plastic as compared to cells cultured on gels of 8kPA or 32kPA stiffness. At least 70 cells were counted per field of view for 2-3 independent replicates. (C) Cells cultured on tissue culture plastic demonstrated a greater percentage of cells exhibiting filopodia per field of view. At least 70 cells were counted per field of view for 2-3 independent replicates. (D) Naïve bone marrow-derived macrophages cultured on 32kPA gel coated with either young (8-16wk) or aged (20-24mo), decellularized cardiac tissue displayed comparable cell morphologies.…

• 

  Figure 3. Naïve macrophage response to pro-inflammatory M1 signaling cues is proportional to the stiffness of the culture substrate. (A) Nitrite concentration in cell culture media supernatants from cells cultured on increasingly stiff substrates (n=32). (B) Nitrite concentration in cell culture media supernatants as a function of substrate stiffness and cECM coating age (n=16). (C) Representative immunolabeling of inducible nitric oxide synthase (iNOS) and cell nuclei (DAPI) in Th1 cytokine treated macrophages cultured on 8 kPA gels, 64 kPA gels, or tissue culture plastic each coated with either young or aged cECM. Representative image brightness and contrast adjusted to reduce background and highlight sample differences. TCP= tissue culture plastic. Scale bars = 100um. Data reported represents mean values with boxes representing 25%/75% of mean. Error bars represent standard deviation. ANOVA with Tukey HSD post-hoc analysis. * p<0.05, ** p<0.001.

• 

  Figure 4. Naïve macrophage response to anti-inflammatory M2 signaling cues is attenuated when cells are cultured on stiff culture substrates. (A) Final urea concentrations in cell lysate following arginase activity assay for naïve macrophages cultured on differentially stiffened substrates (n=8-12). (B) Urea concentrations in cell lysates following arginase activity assay as a function of substrate stiffness and cECM coating age (n=4). (C) Representative immunolabeling of Arginase-1 (ARG) and cell nuclei (DAPI) in Th2 cytokine treated macrophages cultured on 8 kPA gels, 64 kPA gels, or tissue culture plastic each coated with either young or aged cECM. Image contrast adjusted to reduce background fluorescence. Scale bars = 100um. Data reported represents mean values with boxes representing 25%/75% of mean. Error bars represent standard deviation. ANOVA with Tukey HSD post-hoc analysis. * p<0.05, ** p<0.001.

• 

  Figure 5. Differentially aged decellularized cECM promotes altered gene expression patterns in macrophage populations. (A) Fold change qRT-PCR results of pro- (Nos2, Tnfa) and anti-inflammatory (Tgfb, Arg) gene expression in RNA isolated from whole heart tissue lysates isolated from either young (1 month), moderately aged (8 months) or advanced age (18-21 month) C57/Bl6 mice (n=3-5). (B) Heatmap of qRT-PCR results for pro- and anti-inflammatory gene expression in macrophages isolated from young mice (8-16 week) cultured on tissue culture plastic coated with either young(Y, 8-16 week) or aged(A, 18-21 month) cECM with either no supplemental cytokine treatment (M0, YM0, AM0), Th1 cytokine treatment (M1, YM1, AM1), or Th2 cytokine treatment (M2, YM2, AM2) (n=4). Significant effects are noted below heatmap in boxplots with data reported as mean values with boxes representing 25%/75% of mean. Error bars represent standard deviation. Significance was assessed using a Kruskal-Wallis 1-way ANOVA with post-hoc pairwise comparisons. * p<0.05.