Hyaluronan enhances bone marrow cell therapy for myocardial repair after infarction

Abstract

Hyaluronan (HA) has been shown to play an important role during early heart development and promote angiogenesis under various physiological and pathological conditions. In recent years, stem cell therapy, which may reduce cardiomyocyte apoptosis, increase neovascularization, and prevent cardiac fibrosis, has emerged as a promising approach to treat myocardial infarction (MI). However, effective delivery of stem cells for cardiac therapy remains a major challenge. In this study, we tested whether transplanting a combination of HA and allogeneic bone marrow mononuclear cells (MNCs) promotes cell therapy efficacy and thus improves cardiac performance after MI in rats. We showed that HA provided a favorable microenvironment for cell adhesion, proliferation, and vascular differentiation in MNC culture. Following MI in rats, compared with the injection of HA alone or MNC alone, injection of both HA and MNCs significantly reduced inflammatory cell infiltration, cardiomyocyte apoptosis, and infarct size and also improved cell retention, angiogenesis, and arteriogenesis, and thus the overall cardiac performance. Ultimately, HA/MNC treatment improved vasculature engraftment of transplanted cells in the infarcted region. Together, our results indicate that combining the biocompatible material HA with bone marrow stem cells exerts a therapeutic effect on heart repair and may further provide potential treatment for ischemic diseases.

Figure 1

HA promotes the adhesion and proliferation of bone marrow mononuclear cells in culture. (a) Freshly isolated MNCs were cultured on dishes coated with fibronectin, gelatin or HA. The adhered cells were identified using nuclear staining with DAPI (white). (b) The quantification of the adhered cells in the different coating conditions. Data are presented as the mean ± SEM. n = 4 per group. **P < 0.01 (c) The identification of proliferating MNCs under different coating conditions using Ki67 immunostaining (green). Nuclei (blue). (d) The quantification of Ki67-positive cells under the different coating conditions. The data are presented as the mean ± SEM. n = 4 per group. **P < 0.01. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, Hyaluronan; MNC, mononuclear cell.

Figure 2

HA promotes MNC differentiation, paracrine genes expression, and survival. (a) Immunocytochemistry was performed to identify vascular cell differentiation from the MNCs after they were cultured under the different coating conditions for 4 days. Nuclei (blue); Flk-1 (green); vWF (red); SM22-α (red). (b) The quantification of MNC vascular differentiation under the different coating conditions. The data are presented as the mean ± SEM. n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001. Scar bar: 50 µm (c) The quantitative reverse-transcription PCR results showing the paracrine gene expression profiles of MNCs cultured under the different coating conditions during hypoxia. (d) Representative propidium iodine staining, which was analyzed using flow cytometry to identify doxorubicin-induced MNC apoptosis. (e) The quantification of doxorubicin-induced MNC apoptosis. The data are presented as the mean ± SEM. n = 4 per group. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; FGF-2, fibroblast growth factor-2; Flk-1, fetal liver kinase 1; HA, hyaluronan; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; MNC, mononuclear cell; PDGF-b, platelet-derived growth factor-b; SDF-1, stromal cell-derived factor-1; SM22-α, smooth muscle 22-α vWF, von Willebrand Factor.

Figure 3

HA/MNC injection reduces cardiomyocyte apoptosis following infarction. (a) Representative TUNEL staining (red) co-stained with troponin I (green) showing cardiomyocyte apoptosis in heart sections taken from different treatment groups 1 day post-MI. Scale bar: 100 µm for all panels. (b) Quantification of cardiomyocyte apoptosis. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 per group. CM, cardiomyocyte; DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, hyaluronan; MI, myocardial infarction; MNC, mononuclear cell; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4

HA/MNC injection decreases neutrophil and macrophage infiltration after infarction. (a) Representative CD15 staining of neutrophils at the border zone from each group 3 days post-MI. Scale bar: 20 µm for all panels. (b) The number of infiltrated neutrophils at the border zone from each group. Data are presented as mean ± SEM. ** P < 0.01, ***P < 0.001. n = 6 per group. (c) Representative CD68 (green) staining of macrophages at the border zone from each group at 3 days post-MI. Membrane (red). Scale bar: 50 µm for all panels. (d) Quantification of infiltrated macrophages in different experimental groups. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n = 8 per group. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, Hyaluronan; MI, myocardial infarction; MNC, mononuclear cell; WGA, wheat germ agglutinin.

Figure 5

HA/MNC injection improves heart performance and reduces scar formation post-MI. (a) Histograms depicting the left ventricle ejection fraction 1 day and 28 days post-MI in the sham and other experimental groups. *P < 0.05, ***P < 0.001. (b, c) The statistical analysis of the echocardiographic results of the left ventricle internal dimensions at diastole (LVIDd, b) and systole (LVIDs, c) in the sham and other treatment groups. The data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001. n = 8 per group. (d) The infarct size of Sections 3, 4, and 5 from the left ventricle regions is indicated in the artist's rendition and was determined using Masson's trichrome staining. (e) The statistical analysis of scar length in various groups. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n = 8 per group. HA, Hyaluronan; MI, myocardial infarction; MNC, mononuclear cell.

Figure 6

HA/MNC injection improves transplanted cell retention after MI. (a) Representative pictures of DiI⁺ MNC (red) in the area injected with cells alone (left panel) or with cells and HA (right panel). Nuclei are blue. (b) The cell retention rates as reflected by DiI⁺ cell counts. The data are presented as the mean ± SEM. *P < 0.05. n = 8 per group. Scale bar: 50 µm for all panels. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, Hyaluronan; MI, myocardial infarction; MNC, mononuclear cell.

Figure 7

HA/MNC injection increases capillary and arteriole densities post-MI. (a) Representative immunostaining of isolectin (green) for endothelial cells, overlapped with cardiomyocytes stained with cardiac troponin I (cTnI, red) at the peri-infarct and the infarct from search group. Nuclei are blue. Scale bar: 50 µm for all panels. (b–c) The quantification of capillary density at the peri-infarct (b) and infarct (c) areas. The data are presented as the mean ± SEM.*P <0.05. n = 8 per group. (d) Representative immunofluorescence staining of smooth muscle 22 (SM22)-α and cTnI at the peri-infarct and infarct areas from each group. Scale bar: 100 µm for all panels. (e–f) The quantification of arteriole density at the (e) peri-infarct areas (f) and infarct areas. The data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001. n = 8 per group. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, Hyaluronan; MI, myocardial infarction; MNC, mononuclear cell; SM22-α, smooth muscle 22-α.

Figure 8

Injection of HA/MNC enhances transplanted cell differentiation into vascular cells. (a, b) Representative images of red DiI fluorescence, overlapped with immunostaining of (a) vWF or (b) SM22-α in green. (c) Quantification of ratio of cells double positive with DiI and vWF, SM22-α or TnI. *P < 0.05. Scar bar: 50 µm. DAPI, 4′, 6-diamidino-2-phenylindole fluorescent dye; HA, Hyaluronan; MNC, mononuclear cell; SM22-α, smooth muscle 22-α vWF, von Willebrand Factor.