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Introduction: Pathological cardiac fibrosis, through excessive extracellular matrix protein deposition from fibroblasts and pro-fibrotic immune responses and vascular stiffening is associated with most forms of cardiovascular disease. Pathological cardiac fibrosis and stiffening can lead to heart failure and arrythmias and vascular stiffening may lead to hypertension. ROCK, a serine/ threonine kinase downstream of the Rho-family of GTPases, may regulate many pro-fibrotic and pro-stiffening signaling pathways in numerous cell types. Areas covered:This article outlines the molecular mechanisms by which ROCK in fibroblasts, T helper cells, endothelial cells, vascular smooth muscle cells, and macrophages mediate fibrosis and stiffening. We speculate on how ROCK could be targeted to inhibit cardiovascular fibrosis and stiffening.Expert opinion: Critical gaps in knowledge must be addressed if ROCK inhibitors are to be used in the clinic. Numerous studies indicate that each ROCK isoform may play differential roles in regulating fibrosis and may have opposing roles in specific tissues. Future work needs to highlight the isoform-and tissue-specific contributions of ROCK in fibrosis, and how isoformspecific ROCK inhibitors in murine models and in clinical trials affect the pathophysiology of cardiac fibrosis and stiffening. This could progress knowledge regarding new treatments for heart failure, arrythmias and hypertension and the repair processes after myocardial infarction.

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5= splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5= splice site, but not the engineered strong 5= splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5= splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5= splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5= splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.A lternative splicing is a eukaryotic regulatory mechanism that allows for the generation of numerous protein isoforms with often diverse biological functions from a single gene (4,26,41). It begins with the spliceosome, which is assembled stepwise by the addition of discrete small nuclear ribonucleoprotein particles (snRNPs) and numerous accessory non-snRNP splicing factors (23, 33). The excision of introns followed by the joining of exons depends on the recognition and usage of 5= and 3= splice sites (5= ss and 3= ss, respectively) by the splicing machinery (19, 34). The initial splicing step is comprised of 5= ss recognition by U1 snRNP and binding of U2 auxiliary factor (U2AF) to the 3= ss. These factors and additional proteins form the E or commitment complex, which bridges the intron and brings the splice sites close together. U2AF then recruits U2 snRNP to form the A complex. Subsequent binding of the U4-U6-U5 tri-snRNP and many other factors result in a fully assembled spliceosome that supports a series of rearrangements via RNA-RNA and RNA-protein interactions and activates the catalytic steps of cleavage, exon joining, and intron release (4, 26).The splice site signals that define the 5= ss and 3= ss of an alternatively spliced exon are often weak. How and when they are used is believed to be modulated by a complex interplay of positive (splicing enhancers) and negative (splicing silencers) cis elements and trans-acting factors (4, 26). These form the basis for alternative splicing. Target prediction for specific splicing factors is difficult, largely due to the small size and degeneracy of splicing factorbinding motifs. An exception to this degeneracy is the hexanucleotide UGCAUG, which has been shown to be an important element for the splicing of several exons (3,5,14,16,20,24,...