Tissue-nonspecific alkaline phosphatase as a target of sFRP2 in cardiac fibroblasts

Recent studies of myocardial infarction in secreted Frizzled-related protein 2 (sFRP2) knockout mice and our hamster heart failure therapy based on sFRP2 blockade have established sFRP2 as a key profibrotic cytokine in the heart. The failing hamster heart is marked by prominent fibrosis and calcification with elevated expression of sFRP2. Noting the involvement of tissue-nonspecific alkaline phosphatase (TNAP) in bone mineralization and vascular calcification, we determined whether sFRP2 might be an upstream regulator of TNAP. Biochemical assays revealed an approximately twofold increase in the activity of TNAP and elevated levels of inorganic phosphate (Pi) in the failing heart compared with the normal heart. Neither was this change detected in the liver or hamstring muscle nor was it associated with systemic hyperphosphatemia. TNAP was readily cloned from the hamster heart and upon overexpression increased the level of extracellular but not intracellular Pi, which is consistent with the cell surface location of the ectoenzyme. In line with the previous demonstration that sFRP2 blockade attenuated fibrosis, we show here that the therapy downregulated TNAP. This in vivo finding is corroborated by the in vitro study showing that cultured cardiac fibroblasts treated with recombinant sFRP2 protein exhibited progressive increase in the expression and activity of TNAP, which was completely abrogated by cycloheximide or tunicamycin. Induction of TNAP by sFRP2 is restricted to cardiac fibroblasts among the multiple cell types examined, and was not observed with sFRP4. The current work indicates that sFRP2 may promote cardiac fibrocalcification through coordinate activation of tolloid-like metalloproteinases and TNAP.

Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention

Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. Although there is currently no therapy on the market that specifically targets fibrosis, the dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with 20 μg of sFRP2 antibody. Echocardiography, histology, and biochemical analyses were performed after 1 mo. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2 to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9 and 31 ± 3.2%, respectively. Functional improvement is associated with a ∼50% reduction in myocardial fibrosis, ∼65% decrease in apoptosis, and ∼75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured fibroblasts identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and hepatocyte growth factor (HGF) along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for antifibrotic therapy.

Intramuscular VEGF activates an SDF1-dependent progenitor cell cascade and an SDF1-independent muscle paracrine cascade for cardiac repair

The skeletal muscle is endowed with an impressive ability to regenerate after injury, and this ability is coupled to paracrine production of many trophic factors possessing cardiovascular benefits. Taking advantage of this humoral capacity of the muscle, we recently demonstrated an extracardiac therapeutic regimen based on intramuscular delivery of VEGF-A165 for repair of the failing hamster heart. This distal organ repair mechanism activates production from the injected hamstring of many trophic factors, among which stromal-derived factor-1 (SDF1) prominently mobilized multi-lineage progenitor cells expressing CXCR4 and their recruitment to the heart. The mobilized bone marrow progenitor cells express the cardiac transcription factors myocyte enhancer factor 2c and GATA4 and several major trophic factors, most notably IGF1 and VEGF. SDF1 blockade abrogated myocardial recruitment of CXCR4+ and c-kit+ progenitor cells with an insignificant effect on the hematopoietic progenitor lineage. The knockdown of cardiac progenitor cells led to deprivation of myocardial trophic factors, resulting in compromised cardiomyogenesis and angiogenesis. However, the VEGF-injected hamstring continued to synthesize cardioprotective factors, contributing to moderate myocardial tissue viability and function even in the presence of SDF1 blockade. These findings thus uncover two distinct but synergistic cardiac therapeutic mechanisms activated by intramuscular VEGF. Whereas the SDF1/CXCR4 axis activates the progenitor cell cascade and its trophic support of cardiomyogenesis intramuscularly, VEGF amplifies the skeletal muscle paracrine cascade capable of directly promoting myocardial survival independent of SDF1. Given that recent clinical trials of cardiac repair based on the use of marrow-mobilizing agents have been disappointing, the proposed dual therapeutic modality warrants further investigation.