Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis.

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

Suppression of tumor metastasis by a RECK-activating small molecule.

RECK encodes a membrane-anchored protease-regulator which is often downregulated in a wide variety of cancers, and reduced RECK expression often correlates with poorer prognoses. In mouse models, forced expression of RECK in tumor xenografts results in suppression of tumor angiogenesis, invasion, and metastasis. RECK mutations, however, are rare in cancer genomes, suggesting that agents that re-activate dormant RECK may be of clinical value. We found a potent RECK-inducer, DSK638, that inhibits spontaneous lung metastasis in our mouse xenograft model. Induction of RECK expression involves SP1 sites in its promoter and may be mediated by KLF2. DSK638 also upregulates MXI1, an endogenous MYC-antagonist, and inhibition of metastasis by DSK638 is dependent on both RECK and MXI1. This study demonstrates the utility of our approach (using a simple reporter assay followed by multiple phenotypic assays) and DSK638 itself (as a reference compound) in finding potential metastasis-suppressing drugs.

Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

Fibrillins (FBNs) form mesh-like structures of microfibrils in various elastic tissues. RECK and FBN1 are co-expressed in many human tissues, suggesting a functional relationship. We found that dermal FBN1 fibers show atypical morphology in mice with reduced RECK expression (RECK-Hypo mice). Dermal FBN1 fibers in mice-lacking membrane-type 1-matrix metalloproteinase (MT1-MMP) show a similar atypical morphology, despite the current notion that MT1-MMP (a membrane-bound protease) and RECK (a membrane-bound protease inhibitor) have opposing functions. Our experiments using dermal fibroblasts indicated that RECK promotes pro-MT1-MMP activation, increases cell-associated gelatinase/collagenase activity, and decreases diffusible gelatinase/collagenase activity, while MT1-MMP stabilizes RECK in these cells. Experiments using purified proteins indicate that RECK and its binding partner ADAMTS10 keep the proteolytic activity of MT1-MMP within a certain range. These findings suggest that RECK, ADAMTS10, and MT1-MMP cooperate to support the formation of robust FBN1 fibers.

Abundant expression of the membrane-anchored protease-regulator RECK in the anterior pituitary gland and its implication in the growth hormone/insulin-like growth factor 1 axis in mice.

The tumor suppressor gene Reversion-inducing cysteine-rich protein with Kazal motifs (Reck) encodes a membrane-anchored protease regulator expressed in multiple tissues in mouse embryos and is essential for embryonic development. In postnatal mice, however, physiological roles for the RECK protein remain unclear. We found in this study that Reck is abundantly expressed in growth hormone (GH)-producing cells (somatotrophs) in the anterior pituitary gland (AP). We also found that two types of viable Reck mutant mice, one with reduced RECK expression (Hypo mice) and the other with induced Reck deficiency from 10 days after birth (iKO mice treated with tamoxifen), exhibit common phenotypes including decreases in body size and plasma levels of insulin-like growth factor-1 (IGF1). To gain insights into the function of RECK in the AP, we characterized several somatotroph-associated molecules in the AP of these mice. Immunoreactivity of GH was greatly reduced in tamoxifen-treated iKO mice; in these mice, two membrane receptors involved in the stimulation of GH secretion [growth hormone secretagogue receptor (GHSR) and growth hormone releasing hormone receptor (GHRHR)] were decreased, however, their mRNAs were increased. Decrease in GHSR immunoreactivity and concomitant increase in its mRNA were also found in the other mutant line, Hypo. Furthermore, reduced immunoreactivity of growth hormone receptor (GHR) and concomitant increase in its mRNA was also found in the liver of Hypo mice. These results raise the possibility that RECK supports proper functioning of the GH/IGF1 axis in mice, thereby affecting their growth and metabolism.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition.

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK-2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1ß, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation.

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1-100 µM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific Regulation of Wnt Bioavailability.

WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/ß-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/ß-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.

The RECK tumor-suppressor protein binds and stabilizes ADAMTS10.

The tumor suppressor protein RECK has been implicated in the regulation of matrix metalloproteinases (MMPs), NOTCH-signaling and WNT7-signaling. It remains unclear, however, how broad the spectrum of RECK targets extends. To find novel RECK binding partners, we took the unbiased approach of yeast two-hybrid screening. This approach detected ADAMTS10 as a RECK-interactor. ADAMTS10 has been characterized as a metalloproteinase involved in fibrillin-rich microfibril biogenesis, and its mutations have been implicated in the connective tissue disorder Weill-Marchesani syndrome. Experiments in vitro using recombinant proteins expressed in mammalian cells indicated that RECK indeed binds ADAMTS10 directly, that RECK protects ADAMTS10 from fragmentation following chemical activation and that ADAMTS10 interferes with the activity of RECK to inhibit MT1-MMP. In cultured cells, RECK increases the amount of ADAMTS10 associated with the cells. Hence, the present study has uncovered novel interactions between two molecules of known clinical importance, RECK and ADAMTS10.This article has an associated First Person interview with the first author of the paper.

Generation and characterization of a mouse line carrying Reck-CreERT2 knock-in allele.

Reck encodes a membrane-anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch-signaling, and Wnt7-signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, ReckCreERT2 (MGI symbol: Reck). This allele is defective in terms of Reck function but expected to induce loxP-mediated recombination in the cells committed to express Reck. Similarity in the expression patterns of the ReckCreERT2 transgene and the endogenous Reck gene was confirmed in five tissues. In the adult hippocampus, induction of Reck expression after transient cerebral ischemia could be demonstrated using this allele. These results indicate the utility of this Cre-driver allele in further studies.

Early treatment with tolvaptan improves diuretic response in acute heart failure with renal dysfunction.

BACKGROUND: Poor response to diuretics is associated with worse prognosis in patients with acute heart failure (AHF). We hypothesized that treatment with tolvaptan improves diuretic response in patients with AHF. METHODS: We performed a secondary analysis of the AQUAMARINE open-label randomized study in which a total of 217 AHF patients with renal impairment (eGFR < 60 mL/min/1.73 m2) were randomized to either tolvaptan or conventional treatment. We evaluated diuretic response to 40 mg furosemide or its equivalent based on two different parameters: change in body weight and net fluid loss within 48 h. RESULTS: The mean time from patient presentation to randomization was 2.9 h. Patients with a better diuretic response showed greater relief of dyspnea and less worsening of renal function. Tolvaptan patients showed a significantly better diuretic response measured by diuretic response based both body weight [-1.16 (IQR -3.00 to -0.57) kg/40 mg vs. -0.51 (IQR -1.13 to -0.20) kg/40 mg; P < 0.001] and net fluid loss [2125.0 (IQR 1370.0-3856.3) mL/40 mg vs. 1296.3 (IQR 725.2-1726.5) mL/40 mg; P < 0.001]. Higher diastolic blood pressure and use of tolvaptan were independent predictors of a better diuretic response. CONCLUSIONS: Better diuretic response was associated with greater dyspnea relief and less WRF. Early treatment with tolvaptan significantly improved diuretic response in AHF patients with renal dysfunction.

Efficacy of Tumor-Targeting Salmonella A1-R on a Melanoma Patient-Derived Orthotopic Xenograft (PDOX) Nude-Mouse Model.

Tumor-targeting Salmonella enterica serovar Typhimurium A1-R (Salmonella A1-R) had strong efficacy on a melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse model. GFP-expressing Salmonella A1-R highly and selectively colonized the PDOX melanoma and significantly suppressed tumor growth (p = 0.021). The combination of Salmonella A1-R and cisplatinum (CDDP), both at low-dose, also significantly suppressed the growth of the melanoma PDOX (P = 0.001). Salmonella A1-R has future clinical potential for combination chemotherapy with CDDP of melanoma, a highly-recalcitrant cancer.

Bioinformatic Studies to Predict MicroRNAs with the Potential of Uncoupling RECK Expression from Epithelial-Mesenchymal Transition in Cancer Cells.

RECK is downregulated in many tumors, and forced RECK expression in tumor cells often results in suppression of malignant phenotypes. Recent findings suggest that RECK is upregulated after epithelial-mesenchymal transition (EMT) in normal epithelium-derived cells but not in cancer cells. Since several microRNAs (miRs) are known to target RECK mRNA, we hypothesized that certain miR(s) may be involved in this suppression of RECK upregulation after EMT in cancer cells. To test this hypothesis, we used three approaches: (1) text mining to find miRs relevant to EMT in cancer cells, (2) predicting miR targets using four algorithms, and (3) comparing miR-seq data and RECK mRNA data using a novel non-parametric method. These approaches identified the miR-183-96-182 cluster as a strong candidate. We also looked for transcription factors and signaling molecules that may promote cancer EMT, miR-183-96-182 upregulation, and RECK downregulation. Here we describe our methods, findings, and a testable hypothesis on how RECK expression could be regulated in cancer cells after EMT.

Pattern of RECK CpG methylation as a potential marker for predicting breast cancer prognosis and drug-sensitivity.

The membrane-anchored glycoprotein RECK negatively regulates multiple metalloproteinases and is frequently downregulated in tumors. Forced RECK expression in cancer cells results in suppression of tumor angiogenesis, invasion, and metastasis in xenograft models. A previous methylome study on breast cancer tissues detected inverse correlation between RECK CpG methylation (in an intron-1 region) and relapse-free survival. In this study, we focused on another region of the RECK CpG island (a promoter/exon-1 region) and found an inverse correlation between its methylation and RECK-inducibility by an HDAC inhibitor, MS275, among a panel of breast cancer cell lines (n=15). In clinical samples (n=62), RECK intron-1 methylation was prevalent among luminal breast cancers as reported previously (26 of 38 cases; 68%) and particularly enriched in tumors of the ER+PR- subclass (10 of 10 cases) and of higher histological grades (Grade 2 and 3; 28 of 43 cases; P=0.006). In about a half of these cases, promoter/exon-1 methylation was absent, and hence, RECK may be inducible by certain drugs such as MS275. Our results indicate the value of combined use of two RECK methylation markers for predicting prognosis and drug-sensitivity of breast cancers.

The Wnt7's Tale: A story of an orphan who finds her tie to a famous family.

The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.

Critical roles for murine Reck in the regulation of vascular patterning and stabilization.

Extracellular matrix (ECM) is known to play several important roles in vascular development, although the molecular mechanisms behind these remain largely unknown. RECK, a tumor suppressor downregulated in a wide variety of cancers, encodes a membrane-anchored matrix-metalloproteinase-regulator. Mice lacking functional Reck die in utero, demonstrating its importance for mammalian embryogenesis; however, the underlying causes of mid-gestation lethality remain unclear. Using Reck conditional knockout mice, we have now demonstrated that the lack of Reck in vascular mural cells is largely responsible for mid-gestation lethality. Experiments using cultured aortic explants further revealed that Reck is essential for at least two events in sprouting angiogenesis; (1) correct association of mural and endothelial tip cells to the microvessels and (2) maintenance of fibronectin matrix surrounding the vessels. These findings demonstrate the importance of appropriate cell-cell interactions and ECM maintenance for angiogenesis and the involvement of Reck as a critical regulator of these events.

[Isolation and applications of transformation suppressor genes].

A common strategy to identify tumor suppressor genes has been positional cloning, taking advantages of chromosomal abnormalities, linkage to polymorphic markers, etc. We have been taking another approach, based on shotgun cloning with cDNA-expression library, to isolate genes suppressing an aspect of transformed phenotypes. Genes identified in such an artificial system, however, require subsequent validation for their clinical relevance through independent approaches, such as finding mutations and altered expression in human cancers. Whether the parental genes act as oncogenes or tumor suppressors needs to be deduced from available annotations and/or new experimental data. Once validated, such genes, being isolated by virtue of their biological activities, are likely to be useful in developing new strategies for tumor prognosis, tumor stratification, and drug discovery.

E-cadherin-downregulation and RECK-upregulation are coupled in the non-malignant epithelial cell line MCF10A but not in multiple carcinoma-derived cell lines.

Expression of a mesenchymal phenotype is often associated with invasive/metastatic behaviors of carcinoma cells. Acquisition of a mesenchymal phenotype by a carcinoma cell is known as epithelial-mesenchymal transition (EMT). The membrane-anchored matrix metalloproteinase-regulator RECK is abundant in normal mesenchymal cells. In aggressive carcinomas, however, RECK expression is often downregulated. This apparent paradox prompted us to clarify the relationship between EMT and RECK. We found that TGFß-induced E-cadherin downregulation, a hallmark of EMT, is accompanied by RECK-upregulation in a non-tumorigenic epithelial cell line (MCF10A). In contrast, the loss of E-cadherin expression is uncoupled from RECK-upregulation in carcinoma-derived cell lines (MCF7, MDA-MB-231, and A549). When RECK was artificially expressed in A549 cells, it showed little effect on EMT but elevated the level of integrin α5 and attenuated cell proliferation and migration. These findings implicate RECK in the regulation of proliferation and migration of normal epithelial cells after EMT and suggest how the uncoupling between EMT and RECK-upregulation impacts on the fates and behaviors of carcinoma cells.

Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration.

The omega-3 polyunsaturated fatty acids (ω-3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω-3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.

Acetylsalicylic acid inhibits IL-18-induced cardiac fibroblast migration through the induction of RECK.

The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS-dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a unique membrane-anchored MMP regulator, on IL-18-induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL-18-induced migration of primary mouse CF was dependent on both IKK/NF-κB- and JNK/AP-1-mediated MMP9 induction and Sp1-mediated RECK suppression, mechanisms that required Nox4-dependent H(2)O(2) generation. Notably, forced expression of RECK attenuated IL-18-induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL-18-induced H(2)O(2) generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL-18-induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms.