Josh D. Silvertown

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

Our study reports a preliminary investigation into the role of human H2 relaxin in prostate tumor growth. A luciferase‐expressing human prostate cancer cell line, PC‐3, was generated and termed PC3‐Luc. PC3‐Luc cells were transduced with lentiviral vectors engineering the expression of either enhanced green fluorescent protein (eGFP) or both H2 relaxin and eGFP in a bicistronic format. These transduced cells were termed PC3‐Luc‐eGFP and PC3‐Luc‐H2/eGFP, respectively. To gauge effects, PC3‐Luc‐H2/eGFP and PC3‐Luc‐eGFP cells were injected into NOD/SCID mice and monitored over 6 weeks. PC‐3 tumor xenografts overexpressing H2 relaxin exhibited greater tumor volumes compared to control tumors. Circulating H2 relaxin levels in sera increased with the relative size of the tumor, with moderately elevated H2 relaxin levels in mice bearing PC3‐Luc‐H2/eGFP tumors compared to PC3‐Luc‐eGFP tumors. Zymographic analysis demonstrated that proMMP‐9 enzyme activity was significantly downregulated in H2 relaxin‐overexpressing tumors. An advanced angiogenic phenotype was observed in H2 relaxin‐overexpressing tumors indicated by greater intratumoral vascularization by immunohistochemical staining of endothelial cells with anti‐mouse CD31. Moreover, PC3‐Luc‐H2/eGFP tumors exhibited increased VEGF transcript by reverse‐transcription PCR, compared to basal levels in control animals. Taken together, our study provides the first account of a potential role of H2 relaxin in prostate tumor development.

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

In the post‐infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM‐1‐,VCAM‐positive). C2C12 myoblasts did not trans‐differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin‐producing C2C12 myoblasts displayed greater efficacy to engraft the post‐ischaemic scar and to induce extracellular matrix re‐modelling and angiogenesis as compared with the control cells. By echocardio‐graphy, C2C12‐engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post‐infarcted patients.

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (∆H2) impaired its biological function as measured by cAMP signaling. In a competition assay, ∆H2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP‐1 cells. In a flow cytometry‐based binding assay, ∆H2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC‐3 and LNCaP) were engineered to overexpress eGFP, wildtype (WT) H2, or ∆H2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing ∆H2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that ∆H2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of len‐ tivectors engineered to express ∆H2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.—Silvertown, J. D., Symes, J. C., Neschadim, A., Nonaka, T., Kao, J. C. H., Summerlee, A. J. S., Medin, J. A. Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth. FASEB J. 21, 754–765 (2007)

Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures.

The possibility that resident myocardial progenitor cells may be re-activated by transplantation of exogenous stem cells into the post-infarcted heart has been suggested as a possible mechanism to explain the hearts functional improvement after stem cell therapy. Here we studied whether differentiation of mouse neonatal immature cardiomyocytes in vitro was influenced by mouse skeletal myoblasts C2C12, wild type or engineered to secrete the cardiotropic hormone relaxin. The cultured cardiomyocytes formed spontaneously beating clusters and temporally exhibited cardiac immunophenotypical (cKit, atrial natriuretic peptide, troponin T, connexin-43, HCN4) and electrical features (inward voltage-dependent Na(+), T- and L-type Ca(2+) currents, outward and inward K(+) currents, I(f) pacemaker current). These clusters were functionally connected through nanotubular structures and undifferentiated cardiac cells in the form of flattened stripes, bridging the clusters through connexin-43-containing gap junctions. These findings suggested the existence of long distance cell-to-cell communications among the cardiomyocyte aggregates involved in the intercellular transfer of Ca(2+) signals and organelles, likely required for coordination of myocardial differentiation. Co-presence of the myoblasts greatly increased cardiomyocyte differentiation and the amount of intercellular connections. In fact, these cells formed a structural support guiding elongation of nanotubules and stripe-like cells. The secretion of relaxin by the engineered myoblasts accelerated and enhanced the cardiomyogenic potential of the co-culture. These findings underscore the possibility that grafted myoblasts and cardiotropic factors, such as relaxin, may influence regeneration of resident immature cardiac cells, thus adding a tile to the mosaic of mechanisms involved in the functional benefits of cell transplantation for cardiac repair.

Functional and histopathological improvement of the post-infarcted rat heart upon myoblast cell grafting and relaxin therapy

Although the myocardium contains progenitor cells potentially capable of regenerating tissue upon lethal ischaemic injury, their actual role in post‐infarction heart healing is negligible. Therefore, transplantation of extra‐cardiac stem cells is a promising therapeutic approach for post‐infarction heart dysfunction. Paracrine cardiotropic factors released by the grafted cells, such as the cardiotropic hormone relaxin (RLX), may beneficially influence remodelling of recipient hearts. The current study was designed to address whether grafting of mouse C2C12 myoblasts, genetically engineered to express green fluorescent protein (C2C12/GFP) or GFP and RLX (C2C12/RLX), are capable of improving long‐term heart remodelling in a rat model of surgically induced chronic myocardial infarction. One month after myocardial infarction, rats were treated with either culture medium (controls), or C2C12/GFP cells, or C2C12/RLX cells plus exogenous RLX, or exogenous RLX alone. The therapeutic effects were monitored for 2 further months. Cell transplantation and exogenous RLX improved the main echocardiographic parameters of cardiac function, increased myocardial viability (assessed by positron emission tomography), decreased cardiac sclerosis and myocardial cell apoptosis and increased microvascular density in the post‐infarction scar tissue. These effects were maximal upon treatment with C2C12/RLX plus exogenous RLX. These functional and histopathological findings provide further experimental evidence that myoblast cell grafting can improve myocardial performance and survival during post‐infarction heart remodelling and dysfunction. Further, this study provides a proof‐of‐principle to the novel concept that genetically engineered grafted cells can be effectively employed as cell‐based vehicles for the local delivery of therapeutic cardiotropic substances, such as RLX, capable of improving adverse heart remodelling.

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues.

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.

Central Effects of Long-Term Relaxin Expression in the Rat

Abstract: A recombinant adenovirus containing the human H2 preprorelaxin (hH2) cDNA and a reporter gene was coinjected with a transactivator virus (Ad‐tTA) into the lateral cerebral ventricles of female rats. Cardiovascular effects were measured over a 21‐day period. Circulating vasopressin in the periphery was significantly greater (P <.0001) in the relaxin‐treated group throughout the experimental period, compared with controls. There was a significant decrease in plasma osmolality (P <.05) by approximately 10 mmol/L in the treated group by day 14. Immunofluorescence for hH2 present in cryosections showed rAd transduction and hH2 expression from ependymal cells of the ventricular system. Adenovirus‐mediated delivery of hH2 to the brain is capable of producing bioactive relaxin that affects cardiovascular parameters.