Urokinase Plasminogen Activator Induces Pro-Fibrotic/M2 Phenotype in Murine Cardiac Macrophages

<div><p>Objective</p><p>Inflammation and fibrosis are intertwined in multiple disease processes. We have previously found that over-expression of urokinase plasminogen activator in macrophages induces spontaneous macrophage accumulation and fibrosis specific to the heart in mice. Understanding the relationship between inflammation and fibrosis in the heart is critical to developing therapies for diverse myocardial diseases. Therefore, we sought to determine if uPA induces changes in macrophage function that promote cardiac collagen accumulation.</p> <p>Methods and Results</p><p>We analyzed the effect of the uPA transgene on expression of pro-inflammatory (M1) and pro-fibrotic (M2) genes and proteins in hearts and isolated macrophages of uPA overexpressing mice. We found that although there was elevation of the pro-inflammatory cytokine IL-6 in hearts of transgenic mice, IL-6 is not a major effector of uPA induced cardiac fibrosis. However, uPA expressing bone marrow-derived macrophages are polarized to express M2 genes in response to IL-4 stimulation, and these M2 genes are upregulated in uPA expressing macrophages following migration to the heart. In addition, while uPA expressing macrophages express a transcriptional profile that is seen in tumor–associated macrophages, these macrophages promote collagen expression in cardiac but not embryonic fibroblasts.</p> <p>Conclusions</p><p>Urokinase plasminogen activator induces an M2/profibrotic phenotype in macrophages that is fully expressed after migration of macrophages into the heart. Understanding the mechanisms by which uPA modulates macrophage function may reveal insights into diverse pathologic processes.</p> </div

Cardiac IL-6 is elevated but does not mediate uPA-induced cardiac fibrosis.

<p><b>A.</b> IL-6 concentrations in explant culture media from hearts of SR-uPA^+/0 and NTG mice. N  = 5 mice per genotype. <b>B.</b> Collagen content of hearts from SR-uPA^+/0 and NTG littermates at 15 weeks of age. Solid circles represent mice heterozygous for IL-6; open circles are mice homozygous (+/+ or −/−) for IL-6. Bars are medians. N  = 3–5 mice per heterozygous or homozygous genotype. <b>C.</b> Macrophage accumulation in hearts of SR-uPA^+/0 and NTG littermates at 7 weeks of age. Solid circles represent mice heterozygous for IL-6; open circles are mice homozygous (+/+ or −/−) for IL-6. Bars are medians. N  = 3–5 mice per heterozygous or homozygous genotype. Representative heart sections stained with picrosirius red of <b>D.</b> NTG mice, <b>E.</b> SR-uPA^+/0<i>il6</i>^+/+ and <b>F.</b> SR-uPA^+/0<i>il6</i>^−/− mice. Bars represent 100 µm.</p

Transcriptome analysis of SR-uPA^+/0 versus NTG macrophages.

<p>Interaction networks in which key molecules are transcriptionally differentially regulated in SR-uPA^+/0 vs. NTG macrophages. Ingenuity Pathways Analysis (IPA) was used to make the network diagrams. In each network diagram, nodes represent specific molecules and transcriptional changes in SR-uPA^+/0 vs. nontransgenic macrophages are indicated with blue or red circles (red = upregulated in SR-uPA^+/0 vs. nontransgenic macrophages, green = downregulated).</p

SR-uPA^+/0 macrophages are polarized to adopt M2 phenotype.

<p><b>A.</b> Expression of M2 genes measured by <i>qrtPCR</i> in SR-uPA^+/0 and NTG macrophages. Columns represent the mean of arbitrary <i>C</i> units normalized to <i>Gapdh</i>, error bars are S.D., n = 5 mice per genotype. <b>B.</b> Increase in expression of markers of M2 activation in response to IL-4 (10 ng/ml) in SR-uPA^+/0 and NTG bone marrow derived macrophages. Columns represent means and error bars represent S.E.M. <i>P</i> values are calculated by Mann-Whitney as data were non-parametric, n = 5–6 mice per genotype. <b>C.</b> Production of TNF-alpha protein after serial treatment with IL-4 followed by LPS in SR-uPA^+/0 and NTG macrophages. Circles represent the mean of duplicate samples from individual mice, n  = 3 mice per genotype. <b>D.</b> Fold increase in expression of TNF-alpha mRNA after treatment with LPS or LPS+IL-4 in SR-uPA^+/0 and NTG macrophages, n  = 4 mice per genotype. <b>E.</b> Arginase activity in conditioned media from SR-uPA^+/0 and NTG macrophages. N = 4 mice per genotype <b>F.</b> Immunoblot for Arg1 in protein extract from SR-uPA^+/0 and NTG macrophages.</p

SR-uPA^+/0 macrophages adopt full M2 phenotype in the heart.

<p>Picrosirius red stain for collagen in hearts from <b>A.</b> 5 weeks old, <b>B.</b> 7 weeks old, and <b>C.</b> 11 weeks old SR-uPA^+/0 mice. Bars represent 100 µm. <b>D.</b> Fold increase in expression of <i>Arg1</i>, <i>Ym1</i> and <i>Fizz1</i> mRNA in CD-45+ cell fractions from hearts of SR-uPA^+/0 mice at time-points of increasing fibrosis. Star (*) indicates significant <i>P</i> values versus 4–5 week time-point. N  = 4–7 mice per time-point. <b>E.</b> Comparison of <i>Arg1</i> expression from bone marrow macrophages, cardiac macrophages (CD45+) and cardiac CD45 negative fraction (CD45–). Pound (#) indicates significant <i>P</i> values versus other cell types, n  = 6–7 mice per condition. <b>F.</b> Arginase activity in cardiac explant conditioned media from SR-uPA^+/0 and NTG hearts, n  = 7–8 mice per genotype.</p

Pro-fibrotic effect of SR-uPA^+/0 macrophages is limited to cardiac fibroblasts.

<p>Fold increase in expression in <i>Col1a1</i> mRNA in response to conditioned media from SR-uPA^+/0 in comparison to NTG macrophages in <b>A.</b> isolated cardiac fibroblasts <b>B.</b> NIH-3T3 cells. Error bars represent S.D. N  = 4–6 mice per genotype. <b>C. </b><i>Col1a1</i> expression (arbitrary <i>C</i> units normalized to <i>Gapdh</i>) in cardiac fibroblasts treated with conditioned media from SR-uPA^+/0 macrophages treated with BEC or vehicle, N  = 4 mice per treatment, error bars represent S.D.</p