Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2. Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2. AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis.

Plasmin is shown to play a crucial role in many pathophysiologic processes primarily through its ability to degrade extracellular matrix (ECM) and/or mobilizing growth factors that are sequestered in the ECM. Cysteine-rich 61 (CCN1) is a matricellular protein of which expression is up-regulated in cancer and various vascular diseases. The present study was undertaken to investigate whether plasmin liberates CCN1 from the ECM and whether the released growth factor modulates endothelial cell migration. Treatment of breast carcinoma cells (MDA-MB-231) with plasmin released a truncated form of CCN1 (28 kDa) into the overlying medium. Experiments with recombinant CCN1 confirmed that plasmin effectively cleaves CCN1. Thrombin and other clotting/fibrinolytic proteases are ineffective in cleaving CCN1. Further studies revealed that the conditioned medium of plasmintreated carcinoma cells supports endothelial cell migration and that antibodies specific to CCN1 blocked this enhancing effect. These data were the first to show that plasmin can liberate a pluripotent matrix signaling protein, CCN1, from the ECM. Because both CCN1 and the components of the plasmin generation system are present in tumor cells and a variety of other cells, the proteolysis of CCN1 by plasmin may play a role in many pathophysiologic processes, including tumor cellmediated angiogenesis. (Cancer Res 2005; 65(21): 9705-11)

To cite this article: Mandal SK, Rao LVM, Tran TTT, Pendurthi UR. A novel mechanism of plasmin-induced mitogenesis in fibroblasts. J Thromb Haemost 2005; 3: 163-9.Summary. The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor-like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin-induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3 H-thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose-dependent manner. Protease-activated receptor-1 (PAR-1)-specific antibodies, but not PAR-2-specific antibodies, reduced the plasmin-induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR-1-deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin-induced DNA synthesis. Plasmin-induced increase in the DNA synthesis was completely abrogated by anti-Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrixassociated Cyr61 and the conditioned media from plasmintreated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR-1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.