The CNP pGC B cGMP pathway
The CNP/pGC-B/cGMP pathway has been reported to be potent inhibitor of fibrosis, from an intrinsic compensatory protective response as well as a therapeutic in cardiorenal injury and disease states [6,12,13,22,30]. Indeed, fibrosis is a progressive process leading to increased risk for morbidity and mortality  of which there are no FDA approved therapies targeting pGC-B and organ fibrosis. Hence, there is unmet clinical need for the discovery and development of novel pGC-B/cGMP potentiating therapeutics for fibrotic remodeling. In our study, we chose the heart and kidney as two target organs for assessing the anti-fibrotic actions of C53. Using HCFs and HRFs, which possess an abundance of pGC-B receptors [9,48], we observed that C53 significantly generated intracellular cGMP in vitro. Importantly, we mimicked a potential pathophysiological environment by stimulating HCFs and HRFs with TGFβ-1, which is an important driver of organ fibrosis and is activated in cardiorenal injury and disease states [, , ]. Herein, we observed that pharmacological treatment with C53 attenuated TGFβ-1 induced HCFs and HRFs proliferation over 72 hr. However pharmacological treatment with C53 suppressed the differentiation of HCFs and HRFs to myofibroblasts by TGFβ-1 for 24 hr. While our findings are the first to demonstrate the anti-fibrotic potential of C53 in the heart and kidney utilizing in vitro fibroblast studies, these results also suggest that C53 may have differential effects on two mechanisms of fibrosis (i.e. proliferation and differentiation) related to the development of fibrosis as seen in other studies . Therefore, additional studies are warranted to define C53's ability to target various fibrotic mechanisms with chronic therapy by employing in vivo models of fibrotic diseases in various organ systems as well as to investigate its effect on collagen BCECF-AM levels and turnover.
While the present study has potential therapeutic implications targeting fibrosis of the heart and kidney, it also has some limitations worth noting. Although our in vitro NEP assay provides indirect evidence of C53's resistance to NEP degradation, additional mass spectrometry-based studies are required to determine its proteolytic cleavage products. Further studies using direct receptor binding method such as radiometric ligand binding experiments or surface plasmon resonance (SPR) spectroscopy are warranted to determine the NPRC binding affinity of C53. Moreover, our in vivo studies were performed in normal rats. Thus, further studies are needed to define the biological actions of C53 under pathophysiological conditions that contribute to formation of cardiorenal fibrosis and to also investigate the therapeutic actions of C53 in vivo. Lastly, our acute infusion was administered intravenously and thus, other chronic delivery strategies such as subcutaneous administration, as successfully reported by Chen et al. , are warranted to demonstrate its long-term therapeutic effects in fibrotic disease models and to support its potential clinical development.
Sources of funding This work was supported by National Heart, Lung and Blood Institute (NHLBI) grants R01 HL132854 (Dr. Sangaralingham) and R01 HL36634 (Dr. Burnett), a National Institute on Aging (NIA) grant R01 AG056315 (Drs. Sangaralingham and Burnett), an American Heart Association - Scientist Development Award (13SDG16910051, Dr. Sangaralingham), an American Heart Association Predoctoral Fellowship (16PRE30770009. Dr. Y Chen), a Mayo Clinic Graduate School of Biomedical Sciences Fellowship (Dr. Y Chen) and the Mayo Foundation.
Introduction Quinones are a class of organic substances derived from aromatic structures and include both endogenous (ubiquinone) and xenobiotic (for example, thymoquinone and 1,4-benzoquinone) compounds. Ubiquinone (coenzyme Q10) is the ubiquitous component of the mitochondrial electron transport chain, which, upon activation, generates energy from adenosine triphosphate (ATP)