Role of inflammation in right ventricular damage and repair following experimental pulmonary embolism in rats

Abstract

Right ventricular (RV) dysfunction is associated with poor clinical outcome following pulmonary embolism (PE). Previous studies in our laboratory show that influx of neutrophils contributes to acute RV damage seen in an 18 h rat model of PE. The present study describes the further progression of inflammation over 6 weeks and compares the neutrophil and monocyte responses. The RV outflow tract became white in colour by day 1 with influx of neutrophils (tissue myeloperoxidase activity increased 17-fold) and mononuclear cells with characteristics of M1 phenotype (high in Ccl20, Cxcl10, CcR2, MHCII, DNA microarray analysis). Matrix metalloproteinase activities were increased and tissue was thinned to produce a translucent appearance in weeks 1 through 6 in 40% of hearts. RV contractile function was significantly reduced at 6 weeks of PE. In this later phase, there was accumulation of myofibroblasts, the presence of mononuclear cells with M2 characteristics (high in scavenger mannose receptors, macrophage galactose lectin 1, PDGFR1, PDGFRbeta), enrichment of the subendocardial region of the RV outflow tract with neovesels (alpha-smooth muscle immunohistochemistry) and deposition of collagen fibres (picrosirius red staining) beginning scar formation. Thus, while neutrophil response is associated with the early, acute inflammatory events, macrophage cells continue to be present during the proliferative phase and initial deposition of collagen in this model, changing from the M1 to the M2 phenotype. This suggests that the macrophage cell response is biphasic.

Figure 1

Pictures of hearts taken 6 weeks after vehicle treatment (a), on day 1 (b), week 3 (c) or week 6 (d) after PE. Arrows point to the right ventricular outflow tract in each panel. Bar = 3 mm.

Figure 2

Myeloperoxidase activity measured in right ventricular tissue vehicle treatment (day 1 or week 6) or after PE (day 1, day 4 and weeks 1, 2, 4 and 6). Values are mean ± SE, n = 8–10 hearts per group. Asterisks indicate a significant difference from other groups (*) or a significant difference from vehicle (**).

Figure 3

Gelatinase zymogram from right ventricular tissue isolated from animals on day 1 and week 6 after vehicle treatment or at the indicated times after PE. The right panel shows a separate experiment determining gelatinase activity on day 1 after PE and day 1 after PE in animals treated with anti-PMN antibody to reduce neutrophil influx into RV tissue. Extracts from 8 to 10 hearts were pooled for each group. Density absorbance values are graphed for each of the major bands of activity.

Figure 4

Representative sections taken from a 6-week vehicle heart and stained with CD68 immunohistochemistry for (a) monocytes, (b) picrosirius red for collagen or (c) α-SMA for smooth muscle or myofibroblasts. Arrows indicate the structures staining positive in each panel.

Figure 5

Histology of the right ventricular outflow tract seen with CD68 immunohistochemistry identifying monocyte/macrophage cells. (a, arrows) CD68+ cells are in clusters in the right ventricular outflow tract at day 1 after PE, (b, arrows) aggregated in regions of damage at day 4 after PE, (c, arrow) in the debrided, thinned outflow tract week 1 after PE and (d) in the loose connective tissue above the developing fibres seen on the endocardial side 6 weeks after PE. Sections are serial to those in Figures 6 and 7. Endocardial side is downward.

Figure 6

Histology of the right ventricular outflow tract seen with picrosirius red collagen stain. (a, arrows) Collagen fibres are seen between myocytes and at the epicardial area on day 1 after PE, (b, arrow) in the damaged region at day 4 after PE, (c, arrow) in the endocardial side of the debrided, thinned outflow tract week 1 after PE and (d) in the endocardial side 6 weeks after PE. Sections are serial to those in Figures 5 and 7. Endocardial side is downward.

Figure 7

Histology of the right ventricular outflow tract seen with α-SMA stain. (a, arrows) Stain is localized to blood vessels on day 1 after PE (b) and day 4 after PE. (c, black arrow) Stain is in blood vessels 1 week after PE and also in a diffuse stain pattern within the endocardial side. The insert comes from the area indicated by the white arrow, showing neovessels. (d, black arrow) Stain is observed in blood vessels at 6 weeks. The white arrow (d) indicates the region where the insert was taken to show neovessel formation. Sections are serial to those in Figures 5 and 6. Endocardial side is downward.

Figure 8

Differential expression of genes describing mononuclear cell M1 and M2 subtypes in right ventricles at (a) day 1 (b) and week 6 after PE in comparison with time-matched vehicle-treated tissue. Fold changes are statistically significant using t-test with Benjamini and Hochberg adjustment for false discovery rates (n = 5 arrays/group). Gene symbols are CC chemokine ligand 20 also known as MIP-3α (Ccl20), CXC chemokine ligand 10 also known as IP-10 (Cxcl10), CC chemokine receptor 2 (Ccr2), major histocompatibility complex II (Fcgr2b), scavenger mannose receptors (Mrc1), macrophage galactose specific lectin 1 (CD301, Mgl1), platelet-derived growth factor receptor 1 (PDGFR1) and platelet-derived growth factor receptor beta polypeptide (PDGFRβ).