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

Cardiac extracellular matrix is effectively decellularized

To assess the impact of cardiac extracellular matrix (cECM) age on naïve macrophage responses, it was necessary to decellularize native cardiac tissue to provide a cell free scaffold which preserved matrix protein content. A skeletal muscle decellularization protocol from Wolf et al [27] with additional blood clot dissociation steps was chosen to accomplish this goal. Successful decellularization of native tissue is characterized by retention of bioactive matrix proteins while sufficiently removing DNA content and cellular antigens from the native tissue, as these factors will induce confounding immune responses in macrophages [28]. Picrosirius red staining (Figure 1A) of collagen coupled with Alcian blue staining (Figure 1C) of sulfated proteoglycans demonstrated a qualitative increase in collagen and glycosaminoglycan content with increasing age in native cardiac tissues samples. Semi-quantitative analysis of the percent positive area per field of view for picrosirius red stained cardiac sections also demonstrated a mean increase in percent positive staining area with increased sample donor age (Figure 1B). Following decellularization, samples were found to be comprised of a significantly increased mass percentage of hydroxyproline, indicative of the increased contribution of collagen content to the overall mass of the decellularized cECM scaffold (Figure 1D) following removal of the cellular content. Conversely, glycosaminoglycan mass percentage was found to significantly decrease following decellularization (Figure 1E).

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 Tukey’s test used to assess significant trends in (G).

The chosen decellularization protocol was found to have a significant impact on tissue nuclear content. Nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) qualitatively demonstrated a decrease in nuclear content between the native and decellularized groups (Figure 1F). Quantitatively, PicoGreen double stranded DNA quantification assay results demonstrated a significant decrease in mean double stranded DNA content between native and decellularized samples (Figure 1G). Taken together, these assay results demonstrate a qualitative and quantitative removal of nuclear content with minimal loss of matrix protein content.

Substrate stiffness differentially impacts macrophage morphology

In order to assess the contribution of biomechanics as a predictor of macrophage morphological features, naïve bone marrow-derived macrophages isolated from young (8-16 week) mice were cultured on either 8kPA gel, 32kPA gel, or tissue culture plastic coated with decellularized cardiac extracellular matrix (cECM). Differing cell morphology was observed in response to increased culture substrate stiffness, with tissue culture plastic promoting more spread cell morphologies (Figure 2A white arrows), significantly greater cellular area (Figure 2B), and a greater percentage of cells per field of view containing filopodial extensions (Figure 2C).

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. 32kPA brightfield image from 2A reproduced in 2D for qualitative comparison of the different experimental cECM coatings. TCP= tissue culture plastic. Scale bars = 100um. Data reported represents mean values with boxes representing 25%/75% of mean. Error bars represent standard deviation. Black triangles signify data point outliers. ANOVA with Tukey HSD post-hoc analysis. * p<0.05, ** p<0.001.

In comparison, it was observed that less stiff gel culture groups tended to promote more round macrophage morphologies(Figure 2A, white arrows), significantly decreased cellular adhesion area (Figure 2B), and a decreased percentage of cells per field of view displaying filopodia as compared to macrophages cultured on tissue culture plastic (Figure 2C). In order to assess if the age of the mouse from which the cECM coating was derived affected macrophage morphology, macrophages were seeded onto 32kPA gels coated with decellularized cECM isolated from young or aged mice. It was noted that cell morphology did not appear substantially different between young or aged cECM coated gels, suggesting that substrate stiffness may be a greater predictor of macrophage morphological features than the age of the mice from which the decellularized cECM was isolated (Figure 2D).

Stiffer substrates promote pro-inflammatory phenotype and function in naïve macrophage populations

In order to study the ability of naïve macrophages to polarize to M1 phenotypes in response to Th1 cytokine signaling cues when cultured in conditions varying in stiffness and cECM coating, macrophages isolated from young (8-16 week) mice were seeded onto cECM-coated gels ranging in stiffness from 2kPA to 64kPA or onto tissue culture plastic, and were subsequently polarized with canonical Th1 cytokine interferon gamma (IFN-γ) and lipopolysaccharide (LPS). Pro-inflammatory polarization and function was assessed through media nitrite quantification (Figure 3A, 3B) and immunolabeling for macrophage protein inducible nitric oxide synthase (iNOS) (Figure 3C).

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.

In order to assess the significance of the observed differences, a two-way ANOVA for the independent variables gel stiffness and cECM coating age and dependent variable nitrite media supernatant concentration was performed. ANOVA results indicated a significant effect for both predictors gel stiffness (p<0.001, n=32) and cECM coating age (p<0.001, n= 92) with a significant interaction between variables noted as well (p<0.001, n=16). Tukey HSD post-hoc testing for multiple comparisons was used to assess significance between macrophage media nitrite concentrations between gels of different stiffnesses. A significant effect (p<0.001) was observed between all substrates of differing stiffnesses, save for between 8 kPA and 16 kPA (p=.348, mean diff=.2141μM) as well as between 32 kPA and 64 kPA (p=1.000, mean diff.=-.0174μM) groups. In order to assess the significance of interaction between gel stiffness and young or aged cECM gel coatings, independent samples t-tests were performed between equivalent stiffness gels coated with either young or aged decellularized cECM for each substrate stiffness tested. After correcting for multiple comparisons (Bonferroni correction – p<0.05/6=p<0.0083), a significant difference in nitrite media concentration was found between cECM coatings derived from young or aged mice for macrophages cultured on 16kPA (p=0.006, mean diff.=.3293μM), 32kPA (p<0.001, mean diff.=-.5547μM), and tissue culture plastic(TCP) (p<0.001, mean diff.=.8927μM). Qualitative immunolabeling for inducible nitric oxide synthase in fixed macrophage cultures appears to support quantified trends in macrophage media supernatant. These results indicate that expression of M1-associated protein inducible nitric oxide synthase (iNOS) and section of oxidant nitric oxide in response to Th1 stimuli is dependent upon both the stiffness of the culture substrate as well as upon the age of the host from which the decellular cECM coating was derived.

Substrate stiffness inversely correlates with arginase activity and expression

In order to study the ability of naïve macrophages to polarize to M2 phenotypes in response to Th2 cytokine interleukin-4 (IL-4) when cultured in conditions varying in stiffness and cECM coating, macrophages isolated from young (8-16 week) mice were seeded onto cECM-coated gels ranging in stiffness from 2kPA to 64kPA or onto tissue culture plastic, and were subsequently polarized with canonical Th2 cytokine interleukin-4 (IL-4). Alternative macrophage polarization was then assessed through quantification of M2 macrophage function and phenotype based on expression of arginase-1, which is upregulated in murine M2 macrophages [1, 2]. Arginase function was quantified through quantification of urea production, as urea is a downstream enzymatic product of arginase (Figure 4A, 4B). Arginase-1 protein expression was assessed by immunolabeling for arginase-1 (ARG) (Figure 4C).

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.

Urea concentration was found to exhibit variability between differentially stiffened gel macrophage cultures, which were all found to be greater than tissue culture plastic macrophage cultures. In order to assess the significance of the observed differences, a two-way ANOVA for variables gel stiffness and cECM coating age and dependent variable urea concentration was performed. ANOVA results indicated a significant effect for both predictors gel stiffness (p < 0.001, n = 8-12) and animal age from which the cECM coating was derived (p = 0.001, n = 26) with a significant interaction between variables noted as well (p = 0.001, n = 4). Tukey HSD post-hoc testing for multiple comparisons was used to assess significance between culture substrates of different stiffnesses. A significant effect (p<0.001) was observed between tissue culture plastic and all gel substrate groups. Additionally, significant differences were observed between all gel groups (p < 0.001) except for between 2 kPA and 16 kPA (p = 0.237, mean diff. = 0.5572μM) as well as between 8 kPA and 32 kPA (p=0.105, mean diff.=-0.6590μM) gels. In order to assess the significance of interaction between gel stiffness and the age of animal from which the cECM was derived, independent samples t-tests between equivalent stiffness gels coated with cECM derived from either young (8-16 week) or aged (18-21 month) mice were performed for each substrate stiffness tested. After correcting for multiple comparisons (Bonferroni correction – p < 0.05/6 = p < 0.0083), a significant effect was only observed between macrophages cultured on tissue culture plastic (TCP) coated with cECM derived from either young or aged donor cardiac tissue (p < 0.001, mean diff. = 1.670μM). Qualitative immunolabeling for macrophage Arginase-1 in fixed macrophage cultures appeared variable in expression. These results suggest that M2 polarization in response to canonical Th2 cytokine IL-4 appears to remain functional across culture groups of physiological stiffness (2kPA to 64kPA), however a significant attenuation in function was noted at supraphysiological stiffnesses, such as in the case of naïve macrophage culture on tissue culture plastic.

cECM isolated from differentially aged animals induces altered gene expression in naïve macrophage populations

In order to understand how pro- and anti-inflammatory gene expression changes in aging cardiovascular tissue, RNA was isolated from whole cardiac tissue homogenates isolated from either young (1 week), moderately aged (8 months), or advanced aged (18-21 months) mice. Relative gene expression was quantified and expressed as a fold change in pro- or anti-inflammatory gene expression normalized to housekeeping control gene Gapdh (Figure 5A).

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.

In native cardiac tissue, it was observed that expression of both pro- and anti-inflammatory cytokines increased expression levels with increasing age (Figure 5A). A significant difference was noted between expression levels of pro-inflammatory Nos2 expression in RNA isolated from 8 month and 18-21 month old cardiac samples as compared to 1 month cardiac RNA isolates. A significant increase in expression of Tgfb was also observed in 18-21 month cardiac RNA samples relative to 1 month RNA samples.

In order to better understand if these observed differences in native tissue gene expression levels may be a consequence of age-dependent alterations in the cECM, macrophages were seeded onto tissue culture plastic with young or aged cECM, followed by either no treatment (Y→M0, A→M0), treatment with Th1 cytokine IFN-γ and LPS (Y→M1, A→M1), or treatment with Th2 cytokine IL-4 (Y→M2, A→M2). Differential gene expression determined by normalizing to control M0, M1, or M2 macrophage cell gene expression patterns was chosen to highlight alterations in macrophage polarization dynamics induced by differential cECM treatments relative to baseline expression following polarization. It was observed that young cECM treatment in general induced greater fold changes in expression levels of both pro- and anti-inflammatory factors as compared to aged cECM treatment in macrophage groups with no cytokine treatments (Figure 5B). For macrophages treated with Th1 cytokines (M1, Y→M1, A→M1), the trends in gene expression levels noted for the M0 condition were largely maintained, with young cECM tending to induce greater levels of both pro- and anti-inflammatory gene expression levels as compared to aged cECM. It was noted that young cECM induced a relatively large induction of anti-inflammatory gene Arg (220 vs 90). For the M2 polarization condition, treatment with aged cECM promoted a large increase in expression of pro-inflammatory Nos2 relative to control M2 macrophage gene expression. A decrease in expression of anti-inflammatory factors arginase (Arg) and superoxide dismutase (Sod3) was observed in macrophages treated with aged cECM relative to M2 control macrophage gene expression. A significant effect was noted in the M0 treatment condition for anti-inflammatory genes Arg and Sod3. Young decellularized cECM was found to promote significantly greater mean expression of Sod3 relative to both uncoated TCP and aged cECM coated conditions (Figure 5B). Additionally, young cECM treatment was found to induce a significant increase in macrophage Arg expression relative to macrophages cultured on aged cECM-coated tissue culture plastic (Figure 5B). In the M1 condition, a significant increase was also noted between macrophages cultured on young cECM-coated tissue culture plastic relative to macrophages cultured on uncoated tissue culture plastic (Figure 5B). These results suggest that macrophage culture in the presence of young cardiac extracellular matrix without polarizing cytokine cues tends to promote anti-inflammatory gene expression relative to culture on aged cECM-coated tissue culture plastic. In the presence of Th1 or Th2 cytokine cues, young cECM tends to promote enhanced expression of appropriate canonical M1 or M2 macrophage genes while aged cECM culture tended to attenuate macrophage gene expression levels in response to cytokine cues.

Animal care and use

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Young wild type C57BL/6 mice were obtained from Jackson Labs (Bar Harbor, ME) and bred in a pathogen-free rodent facility for use in the described experiments. Eighteen to twenty-two month old C57BL/6 mice were obtained from the National Institute of Aging (NIA) aged rodent colony (Charles River, Wilmington, MA). Animals were housed according to the University of Pittsburgh guidelines in a pathogen free facility with 12 hr / 12 hr light to dark cycle with food and water ad libitum. Mice utilized for experiments were sacrificed by carbon dioxide inhalation followed by cervical dislocation, in accordance with Institutional Animal Care and Use Committee guidelines.

Decellularization of cardiac tissue

The decellularization protocol used in the present study is based on a skeletal muscle decellularization protocol first described by Wolf et al. with several modifications [27]. Briefly, young (8-16 week old) and aged (20-24mo) cardiac tissue was isolated from C57BL/6 mice (Jackson laboratories, NIA), cleaned to remove any connective tissue or vasculature removed during isolation, and washed with 1x PBS three times to wash blood content out of cardiac tissue. Samples were then minced and incubated in streptokinase (Sigma, St. Louis, MO) at 1000 IU/mL overnight at room temperature to remove any additional clotted blood from cardiac tissue. Samples were washed twice in 1x PBS. Cardiac tissue samples were then placed in a 2:1 v/v chloroform:methanol solution and were shaken on an orbital shaker at 300 rpm for 2 hours. Cardiac tissue then went through a graded series of ethanol (100, 90, 70, 50, 0) for 30 minutes each under constant shaking at 300 rpm. Samples were then incubated at 37° C in 0.2% Trypsin / 0.2% EDTA solution for 2 hours. Samples were washed with deionized water followed by 2x PBS for 30 minutes each at room temperature under constant agitation at 300rpm. Samples were then shaken at 300rpm with 2% (w/v) sodium deoxycholate (Sigma, St. Louis, MO) for 5 hours at room temperature, washed with deionized water for 30 minutes, washed with 2x PBS for 30 minutes, and shaken in 2% sodium deoxycholate overnight for 14-16 hours. Samples were subsequently shaken at 300rpm in 1% Triton X-100 (Fisher, Hampton, NH) for 1 hour followed by a 30 minute wash in deionized water. Samples were then shaken at 300 rpm in a 0.1% (w/v) peracetic acid (Sigma, St. Louis, MO) / 4% (v/v) ethanol solution for 2 hours to sterilize the decellularized cardiac tissue. Decellularized tissue samples were then washed twice in 1x PBS and deionized water for 30 minutes per wash. Decellularized cardiac tissue was then frozen in Type I water and lyophilized. Lyophilized cECM was then split for either DNA quantification and biochemistry quality control assays or hydrogel coating and cell treatment.

cECM DNA isolation

Lyophilized decellularized cardiac tissue samples were digested in proteinase K digest buffer (0.5% proteinase K (Thermo, Waltham, MA) in 10mM Tris-HCl, 100mM NaCl, 25mM EDTA, and 0.5% sodium dodecyl sulfate made up in Type I water) at a concentration of 50 mg cECM/mL proteinase K digest buffer at 50° C under agitation for 24 hours or until no visible tissue fragments remain. After full sample digestion, an equal volume of a 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol was added to each sample. Samples were mixed by inversion and were then centrifuged at 10,000g for 10 minutes at 4° C. Following centrifugation, the aqueous fraction of the solution containing any DNA present in the sample was carefully removed from the top of the solution. Care was taken to avoid the uptake of any precipitate formed at the aqueous/organic phase interface. Fractionation and aqueous phase collection was repeated until no visible precipitate formed at the aqueous/organic phase interface of the solution. A 1/10^th sample volume of 3M sodium acetate and 2x sample volume of 100% ethanol was then added to the aqueous phase and sample tube was mixed by inversion several times. Samples were then placed in dry ice until fully frozen, after which samples were centrifuged at 10,000g at 4° C for 10 minutes. Any DNA isolated from decellularized cECM samples will pellet out during centrifugation. Supernatant was carefully decanted off sample tubes and DNA isolates were washed with 70% ethanol and centrifuged at 10,000g for 10 minutes at 4° C. Ethanol wash supernatant was decanted and samples were spun again at 10,000g for 1 minute at 4° C. Any residual ethanol was pipetted out with care not to disturb the DNA pellet.

Doubled stranded DNA quantification

PicoGreen double stranded DNA (dsDNA) quantification kit (Thermo, Waltham, MA) was used for quantification of dsDNA isolated from native and decellularized cardiac samples. dsDNA standard solution was prepared from a 2,000 ng/mL stock and serially diluted in TE buffer. Isolated DNA samples were resuspended in 1mL of 1X TE buffer for 5 minutes at room temperature under gentle agitation. Samples were then serially diluted in 1X TE buffer. One hundred microliters of each sample and dsDNA standard was plated on a 96 well plate. One hundred microliters of Quant-iT PicoGreen reagent was added to each standard or sample and was incubated in the dark at room temperature for 5 minutes, after which time fluorescence emission was measured using a Synergy HTX plate reader (BioTek, Winooski, VT) using an excitation wavelength of 480 nm, an emission wavelength of 520 nm, and a cutoff wavelength of 515 nm. dsDNA concentration for each sample was calculated from the equation derived from the linear regression of dsDNA standard fluorescence emission vs. standard dsDNA concentration. The sample dilution whose reading was closest to the center of the standard curve was used for calculating sample dsDNA content, and calculated dsDNA content was multiplied by the dilution factor to determine accurate concentrations.

Tissue histology

Native cardiac tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned by the McGowan Institute for Regenerative Medicine Histology Core. Tissue sections were deparaffinized by xylene washes, followed by a graded series of ethanol washes, followed finally by tap water wash. Tissue sections were then stained with either picrosirius red to visualize collagen or alcian blue for sulfated proteoglycan visualization. Brightfield imaging of stained sections was performed with Nikon Eclipse50i (Nikon, Melville, NY). Tissue section images were stitched together to form composite images using Photoshop (Adobe, San Jose, CA). Semi-quantitative analysis of the percent area of positive staining in picrosirius red images was performed using NIH FIJI biological imagining analysis software (National Institutes of Health, Bethesda, MD).

Papain digest

Lyophilized decellularized cardiac extracellular matrix samples were massed to obtain dry weight prior to digestion. The papain digest solution was prepared by adding 0.125 mg papain / mL (Sigma, St. Louis, MO) and 0.10M L-cysteine (Sigma, St. Louis, MO) to phosphate-buffered EDTA (PBE) buffer (0.1 M Na₂HPO₄, 0.01 M Na₂EDTA, pH 6.5). Samples were digested in 1 mL of papain digestion solution and incubated at 60C for 12-16 hours or until samples were fully digested. Following complete digestion, samples would be split for either hydroxyproline quantification or glycosaminoglycan quantification.

Hydroxyproline assay

A 500 μg/mL stock solution of hydroxyproline (Sigma, St. Louis, MO) was prepared in PBE buffer and serially diluted to create assay standards. Fifty microliters of each sample and standard was added to a screw-top microcentrifuge tube. Fifty microliters of 2M NaOH was added to each tube and tops were secured. Samples and standards were incubated at 110° C for at least 18 hours. Tubes were then centrifuged and 10.5 μL of Type I water was added to each sample. One hundred microliters of 0.01M CuSO₄, 100 μL of 2.5M NaOH, and 100 μL of 6% H₂O₂ were added sequentially to each tube and tubes were vortexed well after each addition. After 6% H₂O₂ addition, tubes were incubated at room temperature until samples and standards turned clear or blue in color. Samples and standards were vortexed well and incubated at 80° C for 5 minutes. Tubes were briefly centrifuged after which tubes were placed in the -80° C freezer for 5 minutes. Four hundred microliters of 3N H₂SO₄ was then added to each tube. Samples and standards were allowed to cool to room temperature. Two hundred microliters of a 5% dimethylaminobenzaldehyde (Sigma, St. Louis, MO) in propanol solution was added to each tube and tubes were incubated in heat block at 70° C for 15 minutes. Standards and samples were vortexed well and 200 μL of each was plated in triplicate in a 96 well plate. The absorbance at 540nm for each standard or sample well was measured using a Synergy HTX plate reader (BioTek, Winooski, VT). Experimental sample hydroxyproline content was calculated using the equation derived from the linear regression of standards’ absorbances values.

Glycosaminoglycan assay

A 500 μg/mL standard of chondroitin-6-sulfate (Sigma, St. Louis, MO) was prepared in PBE buffer (0.1 M Na₂HPO₄, 0.01 M Na₂EDTA, pH 6.5). The 500 μg/mL standard was then serially diluted in PBE buffer to generate subsequent standards for curve fitting. Fifty microliters of each standard and papain-digested sample was pipetted into a 96 well plate in triplicate. Dimethylmethylene blue (DMMB) solution was prepared by dissolving 1 mg of 1,9-dimethylmethylene blue zinc chloride double salt (Sigma, St. Louis, MO) in 0.313 mL of 100% ethanol with 0.148 g NaCl and 0.19 g glycine added and then diluted up to 50 mL in Type I water. Two hundred fifty microliters of DMMB solution was added to each standard or sample well. Standard and sample absorbance at 525 nm was measured using a Synergy HTX plate reader (BioTek, Winooski, VT). Sample glycosaminoglycan content was calculated using the equation derived from the quadratic regression of standards’ absorbance values.

PDMS gel coating

Decellularized young and aged cardiac extracellular matrix was pepsin-digested at a concentration of 10 mg/mL for 48 hours. Young and aged cECM digests were then diluted down to a concentration of 1 mg/mL with 1X PBS, neutralized to 6 pH, and incubated with sterilized, functionalized polydimethylsiloxane gels of either 2 kPA (Advanced Biomatrix, San Diego, CA), 8 kPA (Advanced Biomatrix, San Diego, CA), 16 kPA (Advanced Biomatrix, San Diego, CA), 32 kPA (Advanced Biomatrix, San Diego, CA), or 64 kPA (Advanced Biomatrix, San Diego, CA) for 1 hour at room temperature. Following incubation, cECM digests were aspirated off of gels and gels were washed three times with 1X PBS. The final wash in 1X PBS was left on the gel to prevent any gel drying per manufacturer’s instructions prior to cell seeding.

Macrophage isolation, culture, and seeding

The tibia and femur were isolated from young (8-16 week) C57BL/6 mice (Jackson, Bar Harbor, ME) and all muscle and connective tissue was separated from the bone. Clean bones were then washed in macrophage culture media (Dubelco’s modified eagle medium, 10% fetal bovine serum, 1% HEPES, 1% Pen/Strep, 2% non-essential amino acids, and 10% L929 fibroblast culture supernatant) to remove any adherent tissue or debris. Tibias and femurs were then transferred to a sterile hood where the epiphysis of each bone was removed. 10mL of sterile macrophage culture media was then flushed through the diaphysis of each bone to collect murine bone marrow. Care was taken to ensure that all marrow was flushed from femur and tibia prior to discarding the bones. Bone marrow-media suspensions were pipetted up and down 5x to break up any cell clumps within the marrow isolated and then passed through a 100um cell strainer to remove any residual debris and further break up any cell clumps. Marrow suspensions were then centrifuged at 1500rpm for 5min, after which the culture media was decanted off and cells were resuspended in fresh macrophage culture media. Cells were counted and plated at a density of 1x10⁶ cells/mL in petri dishes with approximately 1.5x10⁷ cells per petri dish. After 7 days of culture, adherent cells were lifted from the plates by incubating in accutase (Fisher, Waltham, MA) for 5 min, after which cells were washed off the petri dishes, counted, and re-plated onto either the cECM coated hydrogels or onto cECM coated tissue culture plastic at a density of 1x10⁶ cells/mL in macrophage culture medium. After 24 hours, cells received either no treatment, treatment with Th1 cytokines IFN-g (20 ng/mL, Peprotech) and LPS (100 ng/mL, Sigma), or treatment with Th2 cytokine IL-4 (20 ng/mL, Peprotech) for 24 hours. Following cytokine treatment for 24 hours, media was collected from experimental wells to measure nitrite production. Cells in remaining wells were split and either fixed in 2% paraformaldehyde (Fisher Scientific) for 30 minutes for immunofluorescent staining or lysed for arginase activity assays.

Macrophage morphological imaging and quantification

To identify differences in macrophage morphological features induced by culture on substrates of differing stiffness or by substrate coating with cECM isolated from young or aged individuals, brightfield images of fixed cells were acquired with a 20x objective on a ZEISS Axio Observer microscope (ZEISS, Oberkochen, Germany). Cell area measurements were quantified with NIH FIJI biological imagining analysis software (National Institutes of Health, Bethesda, MD). Additionally, the percentage of cells exhibiting filopodial extensions were counted per image, with at least 75 cells being analyzed per field of view.

Macrophage immunofluorescence

After fixation, cells were washed three times for 5 minutes each with 1X PBS and stored in 1X PBS at 4C until ready for immunofluorescence staining. Cells were blocked in donkey blocking buffer (5% donkey serum (Fisher Scientific), 1% bovine serum albumin (Sigma), 0.1% Tween-20-Triton X-100 (Fisher Scientific)) for 1 hour at room temperature. Following block, cells were stained with primary antibody, either rabbit anti-iNOS (ab3523, Abcam, Cambridge, UK) diluted 1:100 in donkey blocking buffer or goat anti-liver arginase (ab91279, Abcam) diluted 1:50 in donkey blocking buffer, for 14-16 hours in the dark at 4° C. Cells were then washed three times with 1X PBS for five minutes per wash. Cells incubated with rabbit anti-iNOS primary were incubated with donkey anti-rabbit Alexa Fluor 594 secondary antibody (ab150064, Abcam) diluted 1:200 in donkey blocking buffer. Cell incubated with goat anti-liver arginase were incubated with donkey anti-goat Alexa Fluor 594 (ab150136, abcam) diluted 1:200 in donkey blocking buffer. Cells were incubated with secondary antibody for 1 hour at room temperature in the dark. Cells were washed three times with 1X PBS for five minutes per wash. Cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Biolegend 422801, San Diego, CA) for 1 minute in the dark at room temperature. Cells were washed five times for 5 minutes per wash in 1X PBS. Cells were stored in PBS in the dark at 4° C until ready for imaging. Immunofluorescence images were captured using a FLoid cell imaging station (Thermo Scientific, Waltham, MA). Acquisition settings were set at: blue laser intensity=30%, red laser intensity=50%, optical zoom=0%.

Nitric oxide quantification

Standards were made from a 0.1M NaNO₂ stock solution mixed with 1 mL of macrophage media which was subsequently serially diluted. 50uL of both standards as well as media collected from experimental groups wells was plated in triplicate in a 96 well plate. Fifty microliters of sulfanilamide (Sigma, St. Louis, MO) was added to each well and plates were incubated in the dark for 10 min at room temperature. Fifty microliters of N-(1-Naphthyl)ethylenediamine (Sigma, St. Louis, MO) was pipetted onto each well and plates were incubated in the dark for 10 min at room temperature. Following N-(1-Naphthyl)ethylenediamine treatment, cells were transferred to Synergy HTX plate reader (BioTek, Winooski, VT) and sample absorbance at 540 nm was measured for each sample and standard. Sample nitrite concentration were calculated from the linear equation derived from the linear regression of serially diluted nitrite standards.

Arginase activity quantification

Cells were lysed in lysis buffer composed of 0.1% Triton X-100 and 1% 100X Halt Protease Inhibitor (Themo Scientific, Waltham, MA) in Type I water. 50uL of lysis buffer was added per well to ensure sufficient protein concentrations. Cells were incubated with lysis buffer for 15min at room temperature. Following lysis, 25 μL of cell lysate was pipetted into a new 96 well plate and 25 μL of arginase activation solution (10 mM MnCl₂ in 50 mM Tris buffer, pH 7.5) was added to each sample. Samples were then covered and incubated at 55° C for 10 minutes. Samples were removed from the incubator and 50 μL of L-arginine (Sigma, St. Louis, MO) substrate solution was added to each well, after which samples were incubated for 2 hours at 37° C. To determine relative arginase activity, it is necessary to measure the concentration of urea in each well following arginase activation and incubation with substrate solution. To accomplish this, urea standards were made up at 100 mg/mL in lysis buffer and serially diluted to create standard curve. To account for dilutions of experimental samples during sample preparation, 75 μL of Type I water was added to each standard. Following sample and standard preparation, 20 μL of each was pipetted into a new 96 well plate and 200 μL of urea detection solution was added to both standard and sample wells. Plates were incubated for 10 min at room temperature. Plate absorbance at 430 nm was measured using a Synergy HTX plate reader (BioTek, Winooski, VT). Urea concentrations were calculated from the equation derived from the linear regression of urea standards’ absorbance values.

Cardiac tissue RNA isolation

Cardiac tissue was isolated from 1 month, 8 month, and 18-21 month old C57BL/6 mice and placed in RNAlater. If RNA was not immediately isolated, samples were frozen at -80C until RNA was to be isolated. RNA was isolated from cardiac tissue using the Qiagen RNeasy Mini Kit using the recommended protocol for tissue RNA isolation (Qiagen, Hilden, Germany). Tissue samples were disrupted and homogenized in Buffer RLT. Tissue lysate was centrifuged for 3 minutes at full speed. Lysate supernatant was carefully pipetted off and transferred to a new microcentrifuge tube. 1 volume of 70% ethanol was added to supernatant and mixed well. 600uL of sample was then transferred to an RNeasy spin column placed in a 2mL collection tube. The spin column lid was closed and samples were centrifuged at 10000 rpm for 15 seconds. The flow through in the collection tube was discarded. The remaining 600uL of sample was transferred to the spin column and samples were centrifuged at 10000 rpm for 15 seconds, with flow through subsequently being discarded. 700uL of RW1 was then transferred to the spin column and samples were centrifuged at 10000 rpm for 15 seconds. 500uL of Buffer RPE was then added to the spin column and samples were centrifuged at 10000 rpm for 15 seconds. Flow through was discarded. 500uL of Buffer RPE was added to spin columns and samples were centrifuged at 10000 rpm for 2 minutes. Spin columns were then removed from the collection tubes and collection tube and flow through was discarded. Samples were placed into a new collection tube and centrifuged at full speed for 1 minute to remove any residual buffer from samples. Spin columns were removed from collection tubes following centrifugation and placed into a new 1.5mL collection tube. 50μL of RNAse-free water was added to each spin column and samples were centrifuged for 1 minute at 10000 rpm to elute RNA isolate from samples. Flow through was then pipetted from the collection tube back onto spin column and samples were centrifuged for 1 minute at 10000 rpm to elute any residual RNA bound to the spin column. Sample RNA concentration and purity were determined using a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA). Only samples with A260/80 ratios within 2.0 ± 0.10 were considered sufficiently purified for subsequent cDNA synthesis and qRT-PCR.

Macrophage RNA isolation

Primary murine bone marrow-derived macrophages were isolated from C57BL/6 mice as previously described. Cells were cultured and treated as previously described, after which cell RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) protocol for cell monolayer RNA isolation. Briefly, cells were lysed by pipetting 600 μL of Buffer RLT in each well and using a sterile cell scraper to disrupt the cell monolayer. Six hundred microliters of 70% ethanol was added to each lysate and mixed well by pipetting. Seven hundred microliters of sample volume was then transferred to an RNeasy spin column placed in a 2 mL microcentrifuge collection tube and centrifuged for 15 seconds at 8000g. Flow through in the collection tube was discarded and remaining volume of sample lysate was transferred to the spin column and was centrifuged for 15 seconds at 8000g. Flow through in collection tube was discarded. Seven hundred microliters of Buffer RW1 was added to RNeasy spin columns and was centrifuged for 15 seconds at 8000g after which any flow through was discarded. Then, 500 μL of Buffer RPE was added to the RNeasy column and centrifuged for 15 seconds at 8000g. The flow through was discarded and 500 μL of Buffer RPE was added again to each spin column. Samples were centrifuged for 2 minutes at 8000g to wash column and ensure all residual ethanol is removed. Flow through was discarded and RNeasy spin columns were placed in a new 1.5 mL collection tube. Thirty microliters of RNAse-free water was pipetted directly onto the spin column and centrifuged for 1 minute at 8000g to elute RNA on spin column. Eluate in collection tube was then pipetted back onto column and centrifuged for 1 minute at 8000g to elute any residual RNA and concentrate sample. Sample RNA concentration and purity were determined using a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA). Only samples with A260/80 ratios within 2.0 ± 0.10 were considered sufficiently purified for subsequent cDNA synthesis and qRT-PCR.

Macrophage and cardiac RNA qRT-PCR

Sample RNA was mixed well by pipetting prior to cDNA synthesis. Total mass of RNA utilized for cDNA synthesis reaction was normalized to least concentrated RNA sample in order to ensure consistent cDNA synthesis across samples. Samples which were more concentrated were diluted with appropriate volume of RNAse-free water to normalize RNA mass used in cDNA reaction. Nine microliters of RNA or RNA diluted in RNAse-free water were added to microcentrifuge tubes. One microliter of 20X RT Enzyme Mix and 10 μL of 2X RT Buffer Mix from High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) was added to each tube. Sample tubes were prepared on ice. When ready for cDNA synthesis, sample tubes were placed in Veriti thermocycler (Applied Biosystems, Foster City, CA). cDNA reaction was catalyzed by thermocycle of 37° C for 60 minutes followed by 95° C for 5min and final rest temperature of 4° C. cDNA was stored at -20° C until ready for qRT-PCR. For the qRT-PCR reaction, 1 μL of sample cDNA was added to 8uL of RNAse-free H₂O, 10uL of TaqMan gene expression master mix (Applied Biosystems, Foster City, CA), and 1 μL of TaqMan primer in a MicroAmp Optical 96-well reaction plate (Applied Biosystems Ref. 43067, Foster City, CA) on ice. Plate wells were sealed with MicroAmp Optical Adhesive Film (Applied Biosystems, Foster City, CA) and centrifuged at 300g for 4 minutes at 4° C. qRT-PCR reaction was catalyzed and Ct values were obtained using a QuantStudio 3 RT-PCR system (Applied Biosystems, Foster City, CA). Relative gene expression was determined by using the double delta C_t method of gene expression relative to housekeeping control gene, GAPDH. Primers chosen for gene expression quantification were previously reported targets of downstream Th1 and Th2 signaling pathways in macrophages: Nos2 (Mm00440502_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Tnfa (Mm00443258_m1), Tgfb (Mm01178820_m1), Sod3 (Mm01213380_s1), Arg (Mm00475988_m1) (Thermo Scientific).

Statistics

All statistical analysis was performed using IBM SPSS statistical analysis package (IBM, Armonk, NY). A two-way ANOVA was used to assess the significance of predictor variables gel stiffness and age of cardiac extracellular matrix for dependent variables of either media nitrite concentration or urea concentration in Figures 3, 4, respectively. Tukey’s post-hoc testing was performed to assess the significance of inter-group differences. In the any case where a significant interaction term was identified from two-way ANOVA results, comparisons of interest were defined a priori and independent samples t-tests were leveraged to assess significance of variable interactions defined a priori against p-value adjusted with Bonferroni correction to account for multiple comparisons. For qRT-PCR experimental data which exhibited non-normal distributions, a Kruskal-Wallis one-way ANOVA was used to assess the significance of the predictor variable age of cardiac extracellular matrix coating. Post hoc comparisons were then performed pairwise to further elucidate significant difference between groups.