Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair

Abstract

Stem cell therapy has emerged as a promising tool for the treatment of a variety of diseases. Previously, we have shown that Akt-modified mesenchymal stem cells mediate tissue repair through paracrine mechanisms. Using a comprehensive functional genomic strategy, we show that secreted frizzled related protein 2 (Sfrp2) is the key stem cell paracrine factor that mediates myocardial survival and repair after ischemic injury. Sfrp2 is known to modulate Wnt signaling, and we demonstrate that cardiomyocytes treated with secreted frizzled related protein increase cellular beta-catenin and up-regulate expression of antiapoptotic genes. These findings reveal the key role played by Sfrp2 in mediating the paracrine effects of Akt-mesenchymal stem cells on tissue repair and identify modulation of Wnt signaling as a therapeutic target for heart disease.

Fig. 1.

Sfrps are expressed in MSCs. (a) Levels of Sfrp1, Sfrp2, and Sfrp3 expression as estimated by microarray analysis show a nearly 10-fold up-regulation of Sfrp2 in Akt-MSCs compared with GFP-MSCs. (b) Real-time QPCR validation of mRNA expression levels demonstrates a 100-fold up-regulation of Sfrp2 gene expression in Akt-MSCs compared with GFP-MSCs.

Fig. 2.

Paracrine factors from Akt-MSCs mediate the survival signaling on cardiomyocytes. (a) Western blot analysis for Sfrp2 demonstrates presence of Sfrp2 protein in conditioned medium collected from Akt-MSCs or GFP-MSCs and inhibition of its accumulation in the medium in the presence of PI3-kinase inhibitor. (b) Relative reduction in mRNA levels of Sfrp2 in Akt-MSCs after knockdown of Sfrp2 with siRNA. (c) Effect of conditioned medium on apoptosis in ARVCs. Caspase activity of ARVCs after 24 h of hypoxia under different culture conditions (Ctr CM, control conditioned medium; Akt CM, Akt-conditioned medium; and Akt CM minus Sfrp2, Akt-conditioned medium after Sfrp2 knockdown) demonstrates reduction of caspase activity after Akt-conditioned medium treatment (Akt CM) and attenuation of this effect after treatment with Akt-conditioned medium after Sfrp2 knockdown (Akt CM minus Sfrp2).

Fig. 3.

Sfrp2 decreases caspase 3 activity in vitro. (a) Cleaved caspase 3 activity as measured by a fluorometric assay demonstrates decreased caspase activity in hypoxic cardiomyocytes after Sfrp2 treatment in a dose-dependent manner. The activity was calculated as fold changes with the same control. (b) Number of round-shaped cardiomyocytes was counted in six random high-power fields (×40) after 24 h of hypoxic exposure with/without Sfrp2 treatment and expressed as a percentage of the total number of cells present. Normoxia: 28.4 ± 2.7; hypoxia: 37.3 ± 0.75; hypoxia plus 300 pM Sfrp2: 26.4 ± 2.6; hypoxia plus 3,000 pM Sfrp2: 25.2 ± 1.0. (c) Representative high-power field photographs demonstrating decreased number of round-shaped cardiomyocytes after treatment with Sfrp2.

Fig. 4.

Akt-MSC-secreted Sfrp2 decreases cardiac infarct size. (Upper) Triphenyl tetrazolium chloride staining in three biventricular sections of similar thickness perpendicular to the long axis of the heart demonstrates decreased infarct size with Akt-conditioned medium and Sfrp2 and attenuation of reduction in infarct size with Akt-MSCs that did express reduced levels of Sfrp2 because of siRNA treatment. (Lower) Infarct size is expressed as a percentage of the total ventricular area. Rat hearts were treated with PBS as control. Akt, Akt-MSC-conditioned medium; Akt-Sfrp2, conditioned medium from Akt-MSCs that did express reduced levels of Sfrp2 because of siRNA treatment.

Fig. 5.

Hypoxic cardiomyocytes up-regulate Wnt3a expression, and Sfrp2 blocks proapoptotic effects of Wnt3a. (a) Wnt3a mRNA expression by RT-PCR is increased in hypoxic cardiomyocytes, whereas expression of Wnt5 remains unchanged. (b) Wnt3a (3 nM) increases caspase activity of cardiomyocytes undergoing hypoxia/reoxygenation injury; Sfrp2 at a similar concentration significantly attenuates Wnt3a-induced caspase activity. ∗, P < 0.05 versus normoxia; ∗∗, P < 0.05 versus Wnt plus hypoxia/reoxygenation; n = 6 per group.

Fig. 6.

Sfrp2 up-regulates Birc1b and β-catenin in hypoxic cardiomyocytes in vitro. (a) Microarray analysis demonstrates Sfrp2-mediated up-regulation of Birc1b gene expression in hypoxic cardiomyocytes. (b) RT-PCR confirmation of increased Birc1b expression in hypoxic cardiomyocytes after Sfrp2 treatment. (c) Western blot analysis for nuclear and total β-catenin expression in ARVCs demonstrates a reduction of β-catenin after hypoxia and up-regulation after treatment with Sfrp2.