We have identified a novel human relaxin gene, designated H3 relaxin, and an equivalent relaxin gene in the mouse from the Celera Genomics data base. Both genes encode a putative prohormone sequence incorporating the classic two-chain, three cysteine-bonded structure of the relaxin/insulin family and, importantly, contain the RXXXRXX(I/V) motif in the B-chain that is essential for relaxin receptor binding. A peptide derived from the likely proteolytic processing of the H3 relaxin prohormone sequence was synthesized and found to possess relaxin activity in bioassays utilizing the human monocytic cell line, THP-1, that expresses the relaxin receptor. The expression of this novel relaxin gene was studied in mouse tissues using RT-PCR, where transcripts were identified with a pattern of expression distinct from that of the previously characterized mouse relaxin. The highest levels of expression were found in the brain, whereas significant expression was also observed in the spleen, thymus, lung, and ovary. Northern blotting demonstrated an ϳ1.2-kb transcript present in mouse brain poly(A) RNA but not in other tissues. These data, together with the localization of transcripts in the pars ventromedialis of the dorsal tegmental nucleus of C57BLK6J mouse brain by in situ hybridization histochemistry, suggest a new role for relaxin in neuropeptide signaling processes. Together, these studies describe a third member of the human relaxin family and its equivalent in the mouse.Relaxin is a 6-kDa polypeptide hormone that is secreted by the ovary into the peripheral circulation in highest amounts during pregnancy and has a number of functions in mammals that are generally associated with female reproductive tract physiology (1). To date, only one relaxin gene has been characterized in most mammalian species, with the exception of the human where two separate genes have been described, designated H1 (2) and H2 (3) relaxin. The peptide encoded by the H2 gene is the major stored and circulating form in the human (4). H1 relaxin expression is restricted to the decidua, placenta, and prostate (5); however, the H1 peptide has similar biological activity to that of H2 relaxin in a rat atrial bioassay (6). The actions of relaxin include an ability to inhibit myometrial contractions, to stimulate remodeling of the connective tissue, and to induce softening of the tissues of the birth canal. Additionally, relaxin increases growth and differentiation of the mammary gland and nipple and induces the breakdown of collagen, one of the main components of connective tissue. Relaxin decreases collagen synthesis and increases the release of collagenases (7). These findings were recently confirmed by the establishment of the relaxin gene-knockout mouse (8), which exhibited a number of phenotypic properties associated with pregnancy. Female mice lacking a functionally active relaxin gene failed to relax and elongate the interpubic ligament of the pubic symphysis and could not suckle their pups, who in turn died within 24 h unless cross-fostered t...

Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.

Acute respiratory distress syndrome is characterized by loss of lung tissue as a result of inflammation and fibrosis. Augmenting tissue repair by the use of mesenchymal stem cells may be an important advance in treating this condition. We evaluated the role of term human umbilical cord cells derived from Wharton's jelly with a phenotype consistent with mesenchymal stem cells (uMSCs) in the treatment of a bleomycininduced mouse model of lung injury. uMSCs were administered systemically, and lungs were harvested at 7, 14, and 28 days post-bleomycin. Injected uMSCs were located in the lung 2 weeks later only in areas of inflammation and fibrosis but not in healthy lung tissue. The administration of uMSCs reduced inflammation and inhibited the expression of transforming growth factor-␤, interferon-␥, and the proinflammatory cytokines macrophage migratory inhibitory factor and tumor necrosis factor-␣. Collagen concentration in the lung was significantly reduced by uMSC treatment, which may have been a consequence of the simultaneous reduction in Smad2 phosphorylation (transforming growth factor-␤ activity). uMSCs also increased matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, favoring a pro-degradative milieu following collagen deposition. Notably, injected human lung fibroblasts did not influence either collagen or matrix metalloproteinase levels in the lung. The results of this study suggest that uMSCs have antifibrotic properties and may augment lung repair if used to treat acute respiratory distress syndrome. (Am J