Keywords

Myocardial infarction; cardiac stem cells; timp 1; c kit; cd63; Catenin; proliferation; differentiation

Abstract

We previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances cardiac cell type differentiation in the infarcted myocardium. To prove this hypothesis, we isolated c-kit+ve cells from four weeks old C57BL/6 mice and cultured them in vitro in presence of ES conditioned media (ESCM), ES-TIMP-1-CM or TIMP-1. Our immunostaining data validate the existence of a novel CD63+ve/c-kit+ve cells. When treated with TIMP-1, these cells showed significantly (p < 0.05) increased proliferation and differentiation into cardiac myocytes, vascular smooth muscle cells, and endothelial cells. Western blot analysis revealed significantly (p < 0.05) increased expression of CD63, phosphorylated and total β-catenin proteins. Furthermore, our RT-PCR data showed increased cardiac gene expression (GATA-4, Mef2C, and Nkx-2.5) when compared to ESCM and control cells. Based on the in vitro findings, we investigated the effect of intramyocardial delivery of TIMP-1 on endogenous CD63+ve/c-kit+ve cells following myocardial infarction (MI). C57BL/6 and TIMP-1 KO mice underwent coronary artery ligation followed by intramyocardial delivery of 20μl of culture media (CC), ESCM, ES-TIMP-1-CM or TIMP-1. Subsequent immunohistochemistry analysis demonstrated the presence of a CD63+ve/c-kit+ve cell population within the peri-infarct area and confirmed intramyocardial delivery of ES-TIMP-1-CM or TIMP-1 significantly (p < 0.05) enhanced their proliferation. Percentage of CD63+ve/c-kit+ve cells was significantly (p < 0.05) lower in TIMP-1 KO mice compared to C57BL/6 animals. RT-PCR analysis revealed TIMP-1 KO animals expressed significantly less CD63 and TIMP-1 mRNAs compared to C57BL/6 mice. Activated CD63+ve/c-kit+ve cells were also able to differentiate into major cardiac cell types as previously shown in vitro. The differentiation potential of these cells was however higher in C57BL/6 mice compared to TIMP-1 KO mice. We also demonstrate that CD63+ve/c-kit+ve cells differentiation is regulated by CD63/β-catenin pathway in vivo. Additionally, we provide evidence that TIMP-1 protects the heart from adverse cardiac remodeling through inhibition of cardiac apoptosis and fibrosis leading to significantly (p < 0.05) improved contractile function. Collectively, our data show TIMP-1 plays a dual protective role in the MI heart. It activates a unique stem cell population, CD63+ve/c-kit+ve, which proliferates and differentiates into functional myocytes, smooth muscle cells and endothelial cells mediated through CD63/β-catenin pathway. TIMP-1 also protects the heart from adverse cardiac remodeling. Increased cardiac regeneration and inhibition of adverse cardiac remodeling consequently lead to restored cardiac function.

Notes

If this is your thesis or dissertation, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu

Graduation Date

2015

Semester

Summer

Advisor

Singla, Dinender

Degree

Doctor of Philosophy (Ph.D.)

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0005750

URL

http://purl.fcla.edu/fcla/etd/CFE0005750

Language

English

Release Date

August 2018

Length of Campus-only Access

3 years

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Medicine; Medicine -- Dissertations, Academic

Download

Share

COinS