The cardiac repair benefits of inflammation do not persist: evidence from mast cell implantation

Abstract

Multiple mechanisms contribute to progressive cardiac dysfunction after myocardial infarction (MI) and inflammation is an important mediator. Mast cells (MCs) trigger inflammation after MI by releasing bio-active factors that contribute to healing. c-Kit-deficient (Kit(W/W-v) ) mice have dysfunctional MCs and develop severe ventricular dilatation post-MI. We explored the role of MCs in post-MI repair. Mouse wild-type (WT) and Kit(W/W-v) MCs were obtained from bone marrow (BM). MC effects on fibroblasts were examined in vitro by proliferation and gel contraction assays. MCs were implanted into infarcted mouse hearts and their effects were evaluated using molecular, cellular and cardiac functional analyses. In contrast to WT, Kit(W/W-v) MC transplantation into Kit(W/W-v) mice did not improve cardiac function or scar size post-MI. Kit(W/W-v) MCs induced significantly reduced fibroblast proliferation and contraction compared to WT MCs. MC influence on fibroblast proliferation was Basic fibroblast growth factor (bFGF)-dependent and MC-induced fibroblast contractility functioned through transforming growth factor (TGF)-β. WT MCs transiently rescue cardiac function early post-MI, but the benefits of BM cell implantation lasted longer. MCs induced increased inflammation compared to the BM-injected mice, with increased neutrophil infiltration and infarct tumour necrosis factor-α (TNF-α) concentration. This augmented inflammation was followed by increased angiogenesis and myofibroblast formation and reduced scar size at early time-points. Similar to the functional data, these beneficial effects were transient, largely vanishing by day 28. Dysfunctional Kit(W/W-v) MCs were unable to rescue cardiac function post-MI. WT MC implantation transiently enhanced angiogenesis and cardiac function. These data suggest that increased inflammation is beneficial to cardiac repair, but these effects are not persistent.

Keywords: c-Kit-deficient mice; cell transplantation; inflammatory response; mast cells; myocardial infarction; myofibroblasts.

Figure 1

Impaired mast cells (MCs) are associated with deterioration of cardiac function in Kit ^W/W‐v mice after ischaemic injury. (A) Wild‐type (WT) and Kit ^W/W‐v mice underwent myocardial infarction and received isogenic (WT and Kit ^W/W‐v) MCs or medium alone. Per cent fractional shortening was evaluated by echocardiography before (day −3) and after (days 7 and 14) infarction (n = 5, *P < 0.05 for WT MCs versus WT medium‐injected; ##P < 0.01 for Kit ^W/W‐v MCs versus WT MCs). (B) Whole heart serial sections demonstrated increased scar size 14 days after implantation with isogenic MCs in Kit ^W/W‐v mice compared to WT recipients of WT MCs (n = 5, **P < 0.01 versus WT). (C) An MTT assay demonstrated that co‐culture with WT MCs increased WT fibroblast (FB) proliferation compared to co‐culture with Kit ^W/W‐v MCs (n = 3, ***P < 0.001 versus WT). (D) A gel contraction assay demonstrated co‐culture with WT MCs increased the contractile function of WT FBs, while co‐culture with Kit ^W/W‐v MCs did not (n = 3, ***P < 0.001 versus FBs). Gel contraction is expressed as the per cent shrinkage compared with a control gel without cells.

Figure 2

Functional mast cells (MCs) increased cardiac fibroblast (FB) proliferation through bFGF. (A) Co‐culture with WT MCs induced dose‐dependent increased proliferation of cardiac WT FBs by an MTT assay. Data are expressed as % proliferation compared to FBs alone (n = 8, P < 0.01 versus 1:0 FBs:MCs). (B) After 48 hrs co‐culture, MCs induced proliferation of FBs to a similar extent as stimulation with bFGF, and the effect was prevented by an FGF‐2 neutralizing antibody (n = 3, **P < 0.01 versus FBs alone). bFGF expression was increased by MC or bone marrow (BM) cell implantation in the infarcted (C) but not the non‐infarcted (D) myocardium at 3 and 7 days after implantation as assessed by ELISA (n = 6, **P < 0.01 versus medium‐injected control).

Figure 3

Functional mast cells (MCs) induced cardiac fibroblast‐ (FB‐) to‐myofibroblast conversion through TGF‐β. (A) Wild‐type MCs induced the contraction of WT FBs in a collagen gel contraction assay to a similar extent as exogenous TGF‐β, and the effect was prevented by a TGF‐β neutralizing antibody. Gel contraction is expressed as the per cent shrinkage compared with a control gel without cells (n = 3, *P < 0.01 versus FBs alone). TGF‐β expression was increased in (B) infarcted but not in (C) non‐infarcted regions of the myocardium after MC and bone marrow (BM) cell transplantation 3 and 7 days after myocardial infarction and cell implantation by ELISA (n = 6, *P < 0.05 and **P < 0.01 versus medium‐injected).

Figure 4

Mast cell (MC) transplantation transiently improved mouse cardiac function post‐myocardial infarction (MI). C57BL/6 mice underwent coronary ligation to induce MI, and wild‐type MCs, bone marrow (BM) cells or medium alone were injected into the myocardium. Sham‐operated mice underwent thoracotomy but no ligation. (A) All ligated groups exhibited a drastic decrease in fractional shortening at day 1 after MI assayed by echocardiography (n = 9). The MC and BM recipient groups both showed early improvement at day 7. These improvements were lost in the MC group but persisted up to day 28 in the BM group (**P < 0.01 for MCs versus medium alone; ##P < 0.01 for BM cells versus medium alone; §P < 0.05 for MCs versus BM). Pressure–volume analysis at day 7 and 28 after cell implantation was used to evaluate ejection fraction (B), end‐systolic and end‐diastolic volumes (C and D), and dP/dt max and dP/dt min (E and F, n = 9, **P < 0.01 versus medium alone). In all cases, the benefits of MC transplantation were apparent at day 7, but absent by day 28.

Figure 5

Functional mast cell (MC) implantation is associated with augmentation of the inflammatory response. Infiltration of neutrophils into the (A) infarct region was increased at day 3 post infarction for the MC‐implanted group and at day 7 for both the MC‐ and bone marrow (BM) cell‐implanted groups while (B) no differences were seen in the non‐infarct regions with or without cell transplantation, evaluated by flow cytometry (n = 3, **P < 0.01 versus medium alone). Infiltration of macrophages into the (C) infarct region was increased in cell‐implanted mice at both time‐points while the (D) non‐infarct region saw no increase, evaluated by flow cytometry against F4/80 (n = 3, **P < 0.01 versus medium alone). TNF‐α concentration in the (E) infarct increases by 3 days after MC implantation and by 7 days after BM cell implantation while (F) no such increase was seen in the non‐infarcted regions, assessed by ELISA (n = 6, **P < 0.01 versus medium alone).

Figure 6

The healing myocardium sequentially mobilizes two monocyte subsets. Infiltration of leucocyte into the infarct region (CD45⁺ cells A and B) was increased at day 3 and 7 post infarction for both the MC‐ and bone marrow (BM) cell‐implanted groups while no differences were seen in the medium alone group without cell transplantation, evaluated by immunofluorescent labelling (n = 3, *P < 0.05, **P < 0.01 versus medium alone). Infiltration of Ly‐6C^hi/CD11b⁺ monocytes peaked at 3 days post‐MI (C and E, n = 3, **P < 0.01 versus medium alone) whereas the Ly‐6C^lo/CD11b⁺ peaked at 7 days post‐MI (D and F, n = 3, *P < 0.05, **P < 0.01 versus medium alone) in both the MC and BM groups and was significantly higher in these groups compared to the medium‐injected control group. However, there was more mobilization of Ly‐6C^hi/CD11b⁺ monocytes in the MC group compared with the BM group at 3 and 7 days post‐MI (E, n = 3, **P < 0.01). On the other hand, there was more mobilization of Ly‐6C^lo/CD11b⁺ monocytes in the BM group compared with the MC group at 7 days post‐MI (F, n = 3, **P < 0.01); scale bar = 50 μm

Figure 7

Enhanced angiogenesis is the predominant contributor to improved cardiac function after cell implantation. (A) Capillary density in the border zone was increased at day 3, 7 and 28 after wild‐type mast cell (MC) or bone marrow (BM) cell implantation, quantified by isolectin staining (n = 5/group,*P < 0.05 and **P < 0.01 versus medium‐injected control, scale bar = 100 μm). (B) Whole heart serial sections at day 7 and 28 post‐cell implantation illustrate ventricular dilation in MC and medium injected controls at the later time‐point. Infarct scar area was quantified as a percentage of left ventricular (LV) area (n = 6/group, **P < 0.01 versus medium‐injected control). (C) α‐SMA staining for myofibroblast accumulation in the border zone post implantation shows increased myofibroblast accumulation in the MC injected hearts 7 days post transplantation compared to their BM‐injected counterparts. Quantification of the α‐SMA ⁺ area excluded blood vessel structures (n = 5/group, **P < 0.01 versus medium‐injected control, scale bar = 100 μm).