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The mechanisms of action of relaxin in the human cardiovascular system
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
posted on 22.02.2017, 04:02by Sarwar, Mohsin
In the recently completed RELAX-AHF phase III clinical trial, relaxin caused vasodilation and reduced mortality/adverse events in patients with acute heart failure (AHF). Despite its clinical potential, the mechanism of action of relaxin in humans is not well understood. This thesis aimed to elucidate some of the factors that underlie the cellular and molecular mechanism of action of relaxin in human primary vascular cells.
The first study examines the signal transduction mechanisms activated by relaxin and its effects on expression of key genes in human primary arterial (HUAEC) and venous (HUVEC) endothelial cells and smooth muscle cells (HUASMC, HUVSMC), and human cardiac fibroblasts (HCF). Acute relaxin stimulation (<1hr) increased cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and extracellular regulated kinase 1 and 2 (ERK1/2) signalling, while long-term relaxin stimulation (24-48hr) increased neuronal nitric oxide synthase (nNOS) and endothelin receptor B (ETB) expression, and matrix metalloproteinase 2 (MMP2) and MMP9 activity in RXFP1-expressing cells, but in a cell-dependent manner. Importantly, bell-shaped concentration response curves (CRCs) that are a hallmark of relaxin signalling in many systems were observed in venous cells involving Gαi/o associated with membrane lipid rafts. In contrast, normal sigmoidal CRCs were observed in arterial smooth muscle cells. The shape of the CRC was related to the types of G-proteins present, with bell-shaped CRCs associated with GαOB, and their location. This study highlighted differences in relaxin signalling in human arterial vs. venous cells with the latter contributing to its potential role as a venodilator.
The next study examined whether cross talk occurred between human endothelial (ECs) and smooth muscle cells (SMCs), and how this influenced relaxin signalling. Co-culture experiments showed that relaxin administration to endothelial cells (ECs: HUVEC and human coronary artery endothelial cells (HCAEC)) increased cGMP accumulation in smooth muscle cells (SMCs: HUASMC, HUVSMC) that were located in close proximity to ECs. This was dependent on RXFP1 expression in ECs and the size of the response in SMCs was magnified some 3-4 times by co-cultures compared with responses obtained in monocultures. Inhibition of NO production in ECs significantly inhibited cGMP accumulation in SMCs. Additionally in HCAECs but not in HUVECs, inhibition of prostanoid production in ECs significantly inhibited cGMP accumulation in SMCs. Inhibition of guanylyl cyclase in SMCs abolished cGMP accumulation in co-cultured SMCs. It was also demonstrated that relaxin stimulation of HCAECs but not HUVECs, increased cAMP accumulation in SMCs that was abolished by inhibition of prostanoid production in HCAECs. Taken together, the findings showed that relaxin promotes cellular cross talk via NO and prostanoids in human vascular cells.
In the final study, the mechanism of action of ML290, a small molecule agonist at RXFP1, was investigated. ML290 was found to be an allosteric biased agonist at RXFP1 that was able to recruit Gαs and GαOB but not Gαi. In HEK-RXFP1 cells, ML290 caused weak cAMP and p38 signalling but did not affect cGMP, ERK1/2 and c-Jun N-terminal kinases (JNK) signalling. In human vascular cells, ML290 was more potent for cGMP than cAMP accumulation, and did not induce ERK1/2 phosphorylation. ML290-mediated cAMP and cGMP accumulation was Gαs dependent, however, in smooth muscle cells but not endothelial cells, ML290-mediated cAMP and cGMP accumulation also involved βγ subunits and PI3K. Taken together, this information suggested that ML290 was an allosteric biased agonist for RXFP1.
In conclusion, this thesis has addressed some important knowledge gaps associated with the mechanism of action of relaxin in the human vasculature. Not only was the pattern of relaxin signal transduction specific for particular vascular-beds, the studies reported in this thesis identified its potential role as a venodilator. For the first time, prostanoids were shown to play a role in cellular crosstalk (between ECs and SMCs) that influences relaxin signalling. Finally, a small molecule agonist for RXFP1 (ML290) demonstrated signalling bias, which could have cGMP-mediated therapeutic benefit in humans.