We recently found that cardiac mast cells can induce fibrosis in the hypertensive heart, and that tryptase levels were increased in these hearts.1
We further demonstrated that tryptase was able to induce proliferation and collagen production by isolated adult cardiac fibroblasts. The stimulatory action of tryptase on fibroblast function is thought to be a PAR-2 mediated event;5
however, this had not been investigated in cardiac fibroblasts. The major finding of this study is that tryptase induces collagen production in cardiac fibroblasts via the activation of PAR-2 and subsequent selective induction of MAPKs.
PAR -1, -2, -3, and -4 are found in a variety of cells including many cell types throughout the cardiovascular system. PAR-1 has been shown to be expressed by cardiac fibroblasts and able to induce proliferation through the activation of ERK 1/2.12
However, while tryptase activates PAR-2, it is not a ligand for PAR-1, -3, and -4. To date, little is known about PAR-3 and PAR-4 activation in cardiac fibroblasts. This is the first study to examine PAR-2 activation in the cardiac fibroblast.
PAR-2 has been described in cardiac tissue; in fact, the expression of PAR-2 is present as early as E17 in mice and is constitutively expressed in the adult murine heart.13
Bohn et al. also reported PAR-2 transcript expression to be present in the adult human heart.14
While the location of this receptor was previously thought to be mainly on cardiomyocytes, we established herein that isolated adult cardiac fibroblasts also have PAR-2. We show that PAR-2 levels in isolated adult cardiac fibroblasts increase in the presence of tryptase; however, this increase may be due to proliferation of the cardiac fibroblasts caused by tryptase.1
Frungieri et al. reported that tryptase/PAR-2 activation, leading to phosphorylation of the MAPK ERK1/2, increased the fibrotic response in skin fibroblasts.6
However, it was unknown if tryptase activation of PAR-2 activates this same pathway in cardiac fibroblasts. The potential role of MAPKs in mediating tryptase-induced fibrosis in the heart had also previously been unexplored. MAPK signaling includes the activation of the serine/threonine kinases, ERK1/2, p38 isoforms, and SAPK/JNK, all of which have extensive homology to one another. Growth and differentiation factors that induce early activation of ERK do so by the sequential phosphorylation of the protein kinases Raf-1, MAP kinase kinase (MEK) 1 and 2 and ERK1/2 in most cells.15
We now show for the first time that there is an increase in ERK1/2 phosphorylation in isolated adult cardiac fibroblasts following treatment with tryptase. PAR-2 antagonism prevented ERK 1/2 phosphorylation and also prevented collagen production by cardiac fibroblasts. Furthermore, the MEK 1/2 inhibitor, PD98059 prevented collagen synthesis by cardiac fibroblasts stimulated with tryptase. This suggests that tryptase binding to PAR-2 receptor causes signaling events through ERK1/2 to elicit the fibrotic response, while p38 and SAPK/JNK do not appear to be involved ().
Schematic of Tryptase/PAR-2 Signaling. Tryptase activates PAR-2 in isolated adult cardiac fibroblast via ERK 1/2 to cause subsequent collagen production and fibrosis in the heart.
ERK1/2 has been shown to regulate procollagen gene expression in cardiac fibroblasts. ERK1/2 translocates to the nucleus to act directly on the α1
(I) procollagen gene. The transcription factors of Egr, Ets, and AP-1 families are downstream of ERK1/2.16-18
Specifically, the α1
(I) procollagen gene contains both AP-1 and ETS binding sites in the 5′UTR and first intron, and therefore these sequences may represent downstream targets of ERK1/2.19-21
Consequently, the α1
(I) collagen has been shown to be a biomarker for fibrosis in the hypertensive hearts of humans and rats.22-24
In order to determine if the same pattern of MAPK activation occurred in cardiac fibroblasts in the hypertensive heart, we isolated these cells from SHR and compared MAPK activation to that in fibroblasts from normotensive WKY hearts. We found that SHR cardiac fibroblasts had increased phosphorylation of ERK1/2 with no upregulation of p38 and SAPK/JNK, similar to what we observed in the isolated cultured fibroblasts treated with tryptase. This is consistent with our previous findings that tryptase is increased in SHR hearts.1
While together our data shows that tryptase stimulation of PAR-2 on isolated cardiac fibroblasts induces collagen synthesis, we wanted to know if PAR-2 was in fact important in hypertensive hearts in vivo. Therefore, we treated SHR with the PAR-2 antagonist peptide FSLLRY and found that fibrosis was prevented. However, this treatment did not prevent LV hypertrophy or hypertension itself. This result is identical to what we have shown previously with the mast cell stabilizer, nedocromil;1
strengthening the concept that mast cell tryptase is a major driver of fibrosis in the hypertensive heart. This increase in collagen is a result of increased synthesis of both collagen types I and III by myofibroblasts and an imbalance of collagens metabolism by matrix metalloproteinases in the SHR.25
Myofibroblasts’ ability to modulate collagen turnover contributes to tissue remodeling in the hypertensive heart, which ultimately leads to fibrosis.25
The phenotypic change of fibroblast to myofibroblast can be detected by the presence α-SMA and ED-A fibronectin in the fibroblast.26, 27
We found a trend towards increased α-SMA and a significant increase in ED-A fibronectin in isolated cardiac fibroblasts treated with tryptase. These markers are indicative of fibroblasts transitioning to a myofibroblastic phenotype, and suggest that tryptase and MAPK pathways can cause phenoconversion of the cardiac fibroblast to myofibroblast, which was prevented by blockade of PAR-2 and MAPK signaling pathway.
In summary, the results of this study reveal a previously unidentified pathway of cardiac fibroblast activation. This pathway is initiated by tryptase stimulation of PAR-2 and leads to induction of MAPK activation in the form of phosphorylation of ERK1/2. The phosphorylation of ERK leads to fibroblast conversion to a myofibroblastic phenotype, and ultimately collagen production and accumulation within the heart. It is concluded that mast cell tryptase plays a critical role in the fibrosis of the hypertensive heart.
Since tryptase is derived almost exclusively from mast cells, the results of this study reinforce the importance of mast cells in the development of fibrosis in the hypertensive heart which further extends our knowledge of how tryptase induces fibrosis in the heart. Our data now establishes tryptase activation of PAR-2 in cardiac fibroblasts and the subsequent selective activation of ERK1/2 as being involved in the transition of fibroblast to myofibroblastic phenotype and increasing collagen synthesis. Thus, this study represents another indicator of the importance of mast cells to myocardial remodeling by describing a previously unknown role for tryptase/PAR-2. Further, this study also highlights the importance of developing novel therapeutics that can target this receptor.